Problems with Fluo4- Direct
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Hello everyone,
We are trying to optimize the new Fluo4 direct (Invitrogen) for Cos-1 cells and we have found different problems either for the cell loading and the response to ionomicyn (5 uM). Do you have any experience on this probe?? Could you tell me which loading protocol you used? We have followed Invitrogen instructions. Please find below more details of different protocols and results we got. Thank you, any help is appreciated, 1) - Loading medium: Add an equal volume of 2X Fluo4 Direct reagent with/ or without Probenecid to cells containing culture medium (0%FCS) (1:1). - Incubate the cells for 30min at 37ºC - Observate the cells under the microscope without removing the medium PROBLEM: Cells were WELL charged with Fluo4 but DID NOT react to Ionomicin 2) - Loading medium: Add an equal volume of 2X Fluo4 Direct reagent without Probenecid to cells containing culture medium (0%FCS) (1:1). - Incubate the cells for 30min at 37ºC - Remove cell medium for fresh medium without FCS PROBLEM: Cells were NOT well charged with Fluo4 but DID react to Ionomicin 3) - Loading medium: Add an equal volume of 2X Fluo4 Direct reagent with Probenecid to cells containing culture medium (0%FCS) (1:1). - Incubate the cells for 30min at 37ºC - Remove loading medium for fresh medium without FCS PROBLEM: Cells were WELL charged with Flou4 but DID react to Ionomicin very SLOW Maria Calvo and Neus Abella -- ___________________________________ Dra. Maria Calvo Unitat de Microscòpia Confocal Serveis Cientificotècnics-C.Casanova Facultat de Medicina Universitat de Barcelona- IDIBAPS C/ Casanova 143 Barcelona 08036 Tel: 34 934037159/39930 Fax: 34 934039946 E-mail: [hidden email] ___________________________________ |
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Hi Maria and Neus,
I use FURA dyes, so I can't speak on the Fluo variant you're using. But, I have seen the problems you describe in 1) and 3) when the ionomycin used is old or has been in and out of -20degC many times (we like to store a concentrated stock in aliquots). Observation 2) seems to preclude the ionomycin, but if your problems are from a combination of issues, it's worth checking. Also, is the experimental protocol (cell type, cell passage, dye loading solution, solutions used during imaging, etc.) one that you've previously used successfully with other dyes? Best, Nate On Tue, Mar 2, 2010 at 10:57 AM, Maria Calvo <[hidden email]> wrote: Hello everyone, -- Nathan O'Connor Silver Laboratory Physiology and Biophysics Weill Cornell Medical College New York, NY |
 
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Lemasters, John J. |
Re: Problems with Fluo4- Direct
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Cells are very active in calcium homeostasis. Consequently, the
response of intracellular free calcium to ionophores like ionomycin is
sigmoidal. Lower doses of ionomycin produce relatively small changes because
the cells are so efficient at pumping the Ca back out. As ionomycin increases,
a point is reached where the cells can’t keep up. At this point, free Ca
increases greatly and cell death ensues due to mitochondrial injury, ATP
depletion, caspase activation, etc. For these reasons, graded responses to
ionomycin do not occur, although our experience is limited mostly to primary
cell cultures. -- John J. Lemasters, MD, PhD Professor and South Carolina COEE Endowed Chair Director, Center for Cell Death, Injury and Regeneration Departments of Pharmaceutical & Biomedical Sciences and
Biochemistry & Molecular Biology Medical University of South Carolina QF213 Quadrangle Building 280 Calhoun Street, MSC 140 Charleston, SC 29425 Office: 843-792-2153 Lab: 843-792-3530 Fax: 843-792-8436 Email: [hidden email] From: Confocal Microscopy
List [mailto:[hidden email]] On Behalf Of Nathan Hi Maria and Neus, On Tue, Mar 2, 2010 at 10:57 AM, Maria Calvo <[hidden email]> wrote: Hello everyone,
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Axel Kurt Preuss |
Re: Problems with Fluo4- Direct
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In reply to this post by Maria Calvo-2
Dear Maria
You ll probably have to lower the probenecid dose to maybe half or less, and play a little with its concentration. All you want is to prevent dye extrusion at MINIMUM physiological cost of probenecid to the cells. I suggest Loading medium: Add an equal volume of 2X Fluo4 Direct reagent without a quarter or fifth of the Probenecid you normally use to cells containing culture medium (0%FCS) (1:1). - Incubate the cells for 30min at 37ºC - Remove cell medium for fresh medium without (I would actually add a percent or half of FCS to give a sharper set inhibition of enzyme activity, give the sells some nutrient , and allow them to experience a a small Ca reversible Ca boost to "wake them up" before you start officially measuring Ca ). For measurements you can remove FCS again. Rationale: you have to get rid of the fluo 4 to prevent compartimentalisation (medium exchange after 30 min loading time OK), to get as little as possible fluo-4 as reactive as possible. Your experiment without probenecid was actually quite well. And that at minimum probenecid necessary. Alternatively, you could incubate a little longer with fluo 4 to raise a little its concentration (incubating 35 min), and add probenecid (fifth of your concentration used ) 10 min after you started loading your cells, and maybe keep it during Ca recording. I hope it helps Thanks Axel Astar IMCB Central Imaging , Singapore -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Maria Calvo Sent: Tuesday, March 02, 2010 11:58 PM To: [hidden email] Subject: Problems with Fluo4- Direct Hello everyone, We are trying to optimize the new Fluo4 direct (Invitrogen) for Cos-1 cells and we have found different problems either for the cell loading and the response to ionomicyn (5 uM). Do you have any experience on this probe?? Could you tell me which loading protocol you used? We have followed Invitrogen instructions. Please find below more details of different protocols and results we got. Thank you, any help is appreciated, 1) - Loading medium: Add an equal volume of 2X Fluo4 Direct reagent with/ or without Probenecid to cells containing culture medium (0%FCS) (1:1). - Incubate the cells for 30min at 37ºC - Observate the cells under the microscope without removing the medium PROBLEM: Cells were WELL charged with Fluo4 but DID NOT react to Ionomicin 2) - Loading medium: Add an equal volume of 2X Fluo4 Direct reagent without Probenecid to cells containing culture medium (0%FCS) (1:1). - Incubate the cells for 30min at 37ºC - Remove cell medium for fresh medium without FCS PROBLEM: Cells were NOT well charged with Fluo4 but DID react to Ionomicin 3) - Loading medium: Add an equal volume of 2X Fluo4 Direct reagent with Probenecid to cells containing culture medium (0%FCS) (1:1). - Incubate the cells for 30min at 37ºC - Remove loading medium for fresh medium without FCS PROBLEM: Cells were WELL charged with Flou4 but DID react to Ionomicin very SLOW Maria Calvo and Neus Abella -- ___________________________________ Dra. Maria Calvo Unitat de Microscòpia Confocal Serveis Cientificotècnics-C.Casanova Facultat de Medicina Universitat de Barcelona- IDIBAPS C/ Casanova 143 Barcelona 08036 Tel: 34 934037159/39930 Fax: 34 934039946 E-mail: [hidden email] ___________________________________ Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you. |
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Axel Kurt Preuss |
Re: Problems with Fluo4- Direct
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I replace the sentence
Rationale: you have to get rid of the fluo 4 to prevent compartimentalisation (medium exchange after 30 min loading time OK), to get as little as possible fluo-4 as reactive as possible. Your experiment without probenecid was actually quite well. And that at minimum probenecid necessary. With Rationale: you have to get rid of the fluo 4 to prevent compartimentalisation (medium exchange after 30 min loading time OK), to get as little as possible fluo-4 as reactive as possible. Your experiment without probenecid was actually quite well. Best to add some minute amount of Probenecid ( minimum probenecid necessary to keep fluo4 in) Thanks Axel Central Imaging (IMCB) 6-19B, cell +65 9271.5622 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Axel Kurt Preuss Sent: Wednesday, March 03, 2010 11:32 AM To: [hidden email] Subject: Re: Problems with Fluo4- Direct Dear Maria You ll probably have to lower the probenecid dose to maybe half or less, and play a little with its concentration. All you want is to prevent dye extrusion at MINIMUM physiological cost of probenecid to the cells. I suggest Loading medium: Add an equal volume of 2X Fluo4 Direct reagent without a quarter or fifth of the Probenecid you normally use to cells containing culture medium (0%FCS) (1:1). - Incubate the cells for 30min at 37ºC - Remove cell medium for fresh medium without (I would actually add a percent or half of FCS to give a sharper set inhibition of enzyme activity, give the sells some nutrient , and allow them to experience a a small Ca reversible Ca boost to "wake them up" before you start officially measuring Ca ). For measurements you can remove FCS again. Rationale: you have to get rid of the fluo 4 to prevent compartimentalisation (medium exchange after 30 min loading time OK), to get as little as possible fluo-4 as reactive as possible. Your experiment without probenecid was actually quite well. And that at minimum probenecid necessary. Alternatively, you could incubate a little longer with fluo 4 to raise a little its concentration (incubating 35 min), and add probenecid (fifth of your concentration used ) 10 min after you started loading your cells, and maybe keep it during Ca recording. I hope it helps Thanks Axel Astar IMCB Central Imaging , Singapore -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Maria Calvo Sent: Tuesday, March 02, 2010 11:58 PM To: [hidden email] Subject: Problems with Fluo4- Direct Hello everyone, We are trying to optimize the new Fluo4 direct (Invitrogen) for Cos-1 cells and we have found different problems either for the cell loading and the response to ionomicyn (5 uM). Do you have any experience on this probe?? Could you tell me which loading protocol you used? We have followed Invitrogen instructions. Please find below more details of different protocols and results we got. Thank you, any help is appreciated, 1) - Loading medium: Add an equal volume of 2X Fluo4 Direct reagent with/ or without Probenecid to cells containing culture medium (0%FCS) (1:1). - Incubate the cells for 30min at 37ºC - Observate the cells under the microscope without removing the medium PROBLEM: Cells were WELL charged with Fluo4 but DID NOT react to Ionomicin 2) - Loading medium: Add an equal volume of 2X Fluo4 Direct reagent without Probenecid to cells containing culture medium (0%FCS) (1:1). - Incubate the cells for 30min at 37ºC - Remove cell medium for fresh medium without FCS PROBLEM: Cells were NOT well charged with Fluo4 but DID react to Ionomicin 3) - Loading medium: Add an equal volume of 2X Fluo4 Direct reagent with Probenecid to cells containing culture medium (0%FCS) (1:1). - Incubate the cells for 30min at 37ºC - Remove loading medium for fresh medium without FCS PROBLEM: Cells were WELL charged with Flou4 but DID react to Ionomicin very SLOW Maria Calvo and Neus Abella -- ___________________________________ Dra. Maria Calvo Unitat de Microscòpia Confocal Serveis Cientificotècnics-C.Casanova Facultat de Medicina Universitat de Barcelona- IDIBAPS C/ Casanova 143 Barcelona 08036 Tel: 34 934037159/39930 Fax: 34 934039946 E-mail: [hidden email] ___________________________________ Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you. Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you. |
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Maria Calvo-2 |
Re: SOLVED Problems with Fluo4- Direct
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Dear All,
We tried again the Fluo4 Direct incubations with half of Probenecid and it worked fine. We will try more until we find the mininmum working concentration of Probenecid. Thank you for your help. Maria Calvo and Neus Abella Axel Kurt Preuss escribió: > I replace the sentence > Rationale: you have to get rid of the fluo 4 to prevent compartimentalisation (medium exchange after 30 min loading time OK), to get as little as possible fluo-4 as reactive as possible. Your experiment without probenecid was actually quite well. And that at minimum probenecid necessary. > > With > Rationale: you have to get rid of the fluo 4 to prevent compartimentalisation (medium exchange after 30 min loading time OK), to get as little as possible fluo-4 as reactive as possible. Your experiment without probenecid was actually quite well. > Best to add some minute amount of Probenecid ( minimum probenecid necessary to keep fluo4 in) > > Thanks > > Axel Central Imaging (IMCB) 6-19B, cell +65 9271.5622 > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Axel Kurt Preuss > Sent: Wednesday, March 03, 2010 11:32 AM > To: [hidden email] > Subject: Re: Problems with Fluo4- Direct > > Dear Maria > You ll probably have to lower the probenecid dose to maybe half or less, and play a little with its concentration. All you want is to prevent dye extrusion at MINIMUM physiological cost of probenecid to the cells. > > I suggest Loading medium: Add an equal volume of 2X Fluo4 Direct reagent > without a quarter or fifth of the Probenecid you normally use to cells containing culture medium (0%FCS) (1:1). > > - Incubate the cells for 30min at 37ºC > > - Remove cell medium for fresh medium without (I would actually add a percent or half of FCS to give a sharper set inhibition of enzyme activity, give the sells some nutrient , and allow them to experience a a small Ca reversible Ca boost to "wake them up" before you start officially measuring Ca ). For measurements you can remove FCS again. > > > Rationale: you have to get rid of the fluo 4 to prevent compartimentalisation (medium exchange after 30 min loading time OK), to get as little as possible fluo-4 as reactive as possible. Your experiment without probenecid was actually quite well. And that at minimum probenecid necessary. > > Alternatively, you could incubate a little longer with fluo 4 to raise a little its concentration (incubating 35 min), and add probenecid (fifth of your concentration used ) 10 min after you started loading your cells, and maybe keep it during Ca recording. > > > I hope it helps > > Thanks > > Axel Astar IMCB Central Imaging , Singapore > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Maria Calvo > Sent: Tuesday, March 02, 2010 11:58 PM > To: [hidden email] > Subject: Problems with Fluo4- Direct > > Hello everyone, > > We are trying to optimize the new Fluo4 direct (Invitrogen) for Cos-1 > cells and we have found different problems either for the cell loading > and the response to ionomicyn (5 uM). > > Do you have any experience on this probe?? Could you tell me which > loading protocol you used? We have followed Invitrogen instructions. > > Please find below more details of different protocols and results we got. > > Thank you, any help is appreciated, > > 1) > > - Loading medium: Add an equal volume of 2X Fluo4 Direct reagent > with/ or without Probenecid to cells containing culture medium (0%FCS) > (1:1). > > - Incubate the cells for 30min at 37ºC > > - Observate the cells under the microscope without removing the > medium > > PROBLEM: Cells were WELL charged with Fluo4 but DID NOT react to Ionomicin > > > 2) > > - Loading medium: Add an equal volume of 2X Fluo4 Direct reagent > without Probenecid to cells containing culture medium (0%FCS) (1:1). > > - Incubate the cells for 30min at 37ºC > > - Remove cell medium for fresh medium without FCS > > PROBLEM: Cells were NOT well charged with Fluo4 but DID react to Ionomicin > > 3) > > - Loading medium: Add an equal volume of 2X Fluo4 Direct reagent > with Probenecid to cells containing culture medium (0%FCS) (1:1). > > - Incubate the cells for 30min at 37ºC > > - Remove loading medium for fresh medium without FCS > > PROBLEM: Cells were WELL charged with Flou4 but DID react to Ionomicin > very SLOW > > > > Maria Calvo and Neus Abella > > -- > > > ___________________________________ > > Dra. Maria Calvo > > Unitat de Microscòpia Confocal > Serveis Cientificotècnics-C.Casanova > Facultat de Medicina > Universitat de Barcelona- IDIBAPS > C/ Casanova 143 > Barcelona 08036 > > Tel: 34 934037159/39930 > Fax: 34 934039946 > E-mail: [hidden email] > ___________________________________ > > Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you. > > Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you. > > -- ___________________________________ Dra. Maria Calvo Unitat de Microscòpia Confocal Serveis Cientificotècnics-C.Casanova Facultat de Medicina Universitat de Barcelona- IDIBAPS C/ Casanova 143 Barcelona 08036 Tel: 34 934037159/39930 Fax: 34 934039946 E-mail: [hidden email] ___________________________________ |
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Louis Villeneuve |
96-well plates good for high NA lens
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Bonjour à tous, I am looking to buy some 96 well plates that can be imaged with high NA lens like a 63x plan-apochromat. Any suggestion of brand/providers? Thanks, Louis Louis Villeneuve Research Associate- Confocal Microscopy Montreal Heart Institute- Research Center 5000 East Belanger Montreal (Qc), Canada H1T 1C8 514-376-3330 ext 3511 514-376-1355 (Fax) [hidden email] |
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Shaw, Andrew-2 |
Re: 96-well plates good for high NA lens
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Try: MatTek Corp. 200 Homer Ave. Ashland, MA 01721 No commercial interest. Andrew Shaw Research Lab Coordinator Image Analysis Core Morehouse School of Medicine Atlanta, GA 30310 From: Confocal Microscopy
List [mailto:[hidden email]] On Behalf Of [hidden email]
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Anda Cornea |
Re: 96-well plates good for high NA lens
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In reply to this post by Louis Villeneuve
I use greiner bio-one www.gbo.com. Number on pack, catalogue number I
presume, is 655090. Anda Cornea, Ph.D. Director of the Imaging Core Oregon National Primate Research Center Oregon Heath & Science University 503-690-5293 From: Confocal Microscopy
List [mailto:[hidden email]] On Behalf Of [hidden email]
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Julio Vazquez |
Re: 96-well plates good for high NA lens
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In reply to this post by Louis Villeneuve
Hi Louis,
Below is a summary of plate info we have for our customers. The BD Falcon and Greiner glass bottom plates should be perfect. The Nunc Optiplate, BD Falcon Thin, and Greiner Bio-one plastic should also work. Glass bottom plates are generally better, but more expensive. Thin/Clear optical plastic bottom plates are typically OK for low NA objectives and objectives such as 10x/0.45 or 20x/0.75 PlanApos. For oil immersion, it may be best to use the glass bottom (0.17 mm), or ask for a sample of the thin plastic bottom plates from vendor (or colleague) and test them to make sure they will work for you.... their thickness may be greater than 0.17 mm (e.g. 0.25 mm or so), and therefore you may have working distance issues, although in principle they should be OK. I have plate specs for some of the models which I can email you if needed.
-- Julio Vazquez Fred Hutchinson Cancer Research Center 1100 Fairview Ave. N., mailstop DE-512 Seattle, WA 98109-1024 === The plates below (Nunc Optiplate, BG Falcon Clear Bottom, and Greiner) work well on the Cellomics ArrayScan. Other plates may work, but we haven't tested them. Beware that any given vendor may have dozens of different types of plates, so be sure to purchase optical grade plates for imaging. Standard cell culture plates will give poor results, and/or may not be compatible with the instrument or objective. Plates for plate readers or other plate assays will typically not be good either. Some vendors will provide samples (check web sites). Falcon BD BioCoat Thin (imaging grade plastic bottom): 96-well clear bottom and 384 well thin bottom, BD Cat # 353219 (Fisher cat # 08-772-225) Falcon BD BioCoat 384-well: BD bioscience cat 354667 (collagen coated) and similar BD Falcon glass bottom plates: # 357311 (96-well) / 357312 (384-well) BD Falcon plates can be "Tissue Culture Treated", Collagen Coated, etc..., and therefore may have different catalog numbers. Just make sure to get the BioCoat Imaging type of plates similar to the ones above. Greiner Bio-One plates: 96 well culture plate with flat bottom; black or white, Cat # 655083/655086 and similar Greiner have plates with coverglass or optical plastic bottom. Nunc Optiplate: Nunc Cat # 165306; Sigma-Aldrich Cat # P8866; Fisher Scientific catalog # 12-566-35 and 12-566-37 (Nunc plates may now be sold by Thermo-Fisher) Perkin Elmer Packard View plates: (Cat # ?) Should work, but have not used them. Covers (seals) for plates: Use clear or dark seals. Do not use foil seals as they create reflections that impair image quality. Plate seals can be obtained from Island Scientific/Genesee Scientific (ThermalSeal film, Cat # 12-168). from Sigma-Aldrich, Nunc sealing tape for multiwell plates, Cat # T9696, T9571, and similar: From Fisher (Fisherbrand adhesive plate seals, Cat # 08-408-240) On May 3, 2010, at 10:15 AM, [hidden email] wrote:
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Knecht, David |
Re: 96-well plates good for high NA lens
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I am wondering how you plan to use these plates. I have thought of using a motorized stage to do a well scan, but could never come up with a solution to the problem of keeping oil on the entire plate. I guess if you use a few wells at a time it would work, but I would not expect to get more than 3-4 wells. before running out of oil. Dave
On May 3, 2010, at 2:01 PM, Julio Vazquez wrote: > Hi Louis, > > Below is a summary of plate info we have for our customers. The BD Falcon and Greiner glass bottom plates should be perfect. The Nunc Optiplate, BD Falcon Thin, and Greiner Bio-one plastic should also work. Glass bottom plates are generally better, but more expensive. Thin/Clear optical plastic bottom plates are typically OK for low NA objectives and objectives such as 10x/0.45 or 20x/0.75 PlanApos. For oil immersion, it may be best to use the glass bottom (0.17 mm), or ask for a sample of the thin plastic bottom plates from vendor (or colleague) and test them to make sure they will work for you.... their thickness may be greater than 0.17 mm (e.g. 0.25 mm or so), and therefore you may have working distance issues, although in principle they should be OK. I have plate specs for some of the models which I can email you if needed. > -- > Julio Vazquez > Fred Hutchinson Cancer Research Center > 1100 Fairview Ave. N., mailstop DE-512 > Seattle, WA 98109-1024 > > > http://www.fhcrc.org/ > > > === > > > The plates below (Nunc Optiplate, BG Falcon Clear Bottom, and Greiner) work well on the Cellomics ArrayScan. Other plates may work, but we haven't tested them. Beware that any given vendor may have dozens of different types of plates, so be sure to purchase optical grade plates for imaging. Standard cell culture plates will give poor results, and/or may not be compatible with the instrument or objective. Plates for plate readers or other plate assays will typically not be good either. Some vendors will provide samples (check web sites). > > > > > Falcon BD BioCoat Thin (imaging grade plastic bottom): 96-well clear bottom and 384 well thin bottom, BD Cat # 353219 (Fisher cat # 08-772-225) > > Falcon BD BioCoat 384-well: BD bioscience cat 354667 (collagen coated) and similar > > BD Falcon glass bottom plates: # 357311 (96-well) / 357312 (384-well) > > BD Falcon plates can be "Tissue Culture Treated", Collagen Coated, etc..., and therefore may have different catalog numbers. Just make sure to get the BioCoat Imaging type of plates similar to the ones above. > > > Greiner Bio-One plates: 96 well culture plate with flat bottom; black or white, Cat # 655083/655086 and similar > > http://www.greinerbioone.com/en/usa/articles/catalogue/article/491_10/18026/ > > > Greiner have plates with coverglass or optical plastic bottom. > > > > Nunc Optiplate: Nunc Cat # 165306; Sigma-Aldrich Cat # P8866; Fisher Scientific catalog # 12-566-35 and 12-566-37 > > (Nunc plates may now be sold by Thermo-Fisher) > > > > Perkin Elmer Packard View plates: (Cat # ?) Should work, but have not used them. > > > Covers (seals) for plates: > > Use clear or dark seals. Do not use foil seals as they create reflections that impair image quality. > > Plate seals can be obtained from Island Scientific/Genesee Scientific (ThermalSeal film, Cat # 12-168). > > https://www.geneseesci.com/ > > from Sigma-Aldrich, Nunc sealing tape for multiwell plates, Cat # T9696, T9571, and similar: > > http://www.sigmaaldrich.com/labware/labware-products.html?TablePage=17200082 > > From Fisher (Fisherbrand adhesive plate seals, Cat # 08-408-240) > > https://www.fishersci.com/wps/portal/PRODUCTDETAIL?LBCID=45488914&productId=3743760&catalogId=29102&pos=23&catCode=SA_SC&fromCat=yes&keepSessionSearchOutPut=true&brCategoryId=71102&hlpi=false&fromSearch= > > > > > On May 3, 2010, at 10:15 AM, [hidden email] wrote: > >> >> Bonjour à tous, >> >> I am looking to buy some 96 well plates that can be imaged with high NA lens like a 63x plan-apochromat. Any suggestion of brand/providers? >> >> Thanks, >> >> Louis >> >> Louis Villeneuve >> Research Associate- Confocal Microscopy >> Montreal Heart Institute- Research Center >> 5000 East Belanger >> Montreal (Qc), Canada >> H1T 1C8 >> >> 514-376-3330 ext 3511 >> 514-376-1355 (Fax) >> >> [hidden email] > Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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Boswell, Carl A - (cboswell) |
Re: 96-well plates good for high NA lens
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Hi David,
I would imagine that a mini-drop of oil on the bottom of each well would suffice to replenish the amount lost moving from one to the next. The bottom would become smeared, but it shouldn't be so much as to drench the objective. It might take some experience to learn how much was good enough. c Carl A. Boswell, Ph.D. Molecular and Cellular Biology University of Arizona 520-954-7053 FAX 520-621-3709 ----- Original Message ----- From: "David Knecht" <[hidden email]> To: <[hidden email]> Sent: Monday, May 03, 2010 3:11 PM Subject: Re: 96-well plates good for high NA lens I am wondering how you plan to use these plates. I have thought of using a motorized stage to do a well scan, but could never come up with a solution to the problem of keeping oil on the entire plate. I guess if you use a few wells at a time it would work, but I would not expect to get more than 3-4 wells. before running out of oil. Dave On May 3, 2010, at 2:01 PM, Julio Vazquez wrote: > Hi Louis, > > Below is a summary of plate info we have for our customers. The BD Falcon > and Greiner glass bottom plates should be perfect. The Nunc Optiplate, BD > Falcon Thin, and Greiner Bio-one plastic should also work. Glass bottom > plates are generally better, but more expensive. Thin/Clear optical > plastic bottom plates are typically OK for low NA objectives and > objectives such as 10x/0.45 or 20x/0.75 PlanApos. For oil immersion, it > may be best to use the glass bottom (0.17 mm), or ask for a sample of the > thin plastic bottom plates from vendor (or colleague) and test them to > make sure they will work for you.... their thickness may be greater than > 0.17 mm (e.g. 0.25 mm or so), and therefore you may have working distance > issues, although in principle they should be OK. I have plate specs for > some of the models which I can email you if needed. > -- > Julio Vazquez > Fred Hutchinson Cancer Research Center > 1100 Fairview Ave. N., mailstop DE-512 > Seattle, WA 98109-1024 > > > http://www.fhcrc.org/ > > > === > > > The plates below (Nunc Optiplate, BG Falcon Clear Bottom, and Greiner) > work well on the Cellomics ArrayScan. Other plates may work, but we > haven't tested them. Beware that any given vendor may have dozens of > different types of plates, so be sure to purchase optical grade plates for > imaging. Standard cell culture plates will give poor results, and/or may > not be compatible with the instrument or objective. Plates for plate > readers or other plate assays will typically not be good either. Some > vendors will provide samples (check web sites). > > > > > Falcon BD BioCoat Thin (imaging grade plastic bottom): 96-well clear > bottom and 384 well thin bottom, BD Cat # 353219 (Fisher cat # 08-772-225) > > Falcon BD BioCoat 384-well: BD bioscience cat 354667 (collagen coated) and > similar > > BD Falcon glass bottom plates: # 357311 (96-well) / 357312 (384-well) > > BD Falcon plates can be "Tissue Culture Treated", Collagen Coated, etc..., > and therefore may have different catalog numbers. Just make sure to get > the BioCoat Imaging type of plates similar to the ones above. > > > Greiner Bio-One plates: 96 well culture plate with flat bottom; black or > white, Cat # 655083/655086 and similar > > http://www.greinerbioone.com/en/usa/articles/catalogue/article/491_10/18026/ > > > Greiner have plates with coverglass or optical plastic bottom. > > > > Nunc Optiplate: Nunc Cat # 165306; Sigma-Aldrich Cat # P8866; Fisher > Scientific catalog # 12-566-35 and 12-566-37 > > (Nunc plates may now be sold by Thermo-Fisher) > > > > Perkin Elmer Packard View plates: (Cat # ?) Should work, but have not used > them. > > > Covers (seals) for plates: > > Use clear or dark seals. Do not use foil seals as they create reflections > that impair image quality. > > Plate seals can be obtained from Island Scientific/Genesee Scientific > (ThermalSeal film, Cat # 12-168). > > https://www.geneseesci.com/ > > from Sigma-Aldrich, Nunc sealing tape for multiwell plates, Cat # T9696, > T9571, and similar: > > http://www.sigmaaldrich.com/labware/labware-products.html?TablePage=17200082 > > From Fisher (Fisherbrand adhesive plate seals, Cat # 08-408-240) > > https://www.fishersci.com/wps/portal/PRODUCTDETAIL?LBCID=45488914&productId=3743760&catalogId=29102&pos=23&catCode=SA_SC&fromCat=yes&keepSessionSearchOutPut=true&brCategoryId=71102&hlpi=false&fromSearch= > > > > > On May 3, 2010, at 10:15 AM, [hidden email] wrote: > >> >> Bonjour à tous, >> >> I am looking to buy some 96 well plates that can be imaged with high NA >> lens like a 63x plan-apochromat. Any suggestion of brand/providers? >> >> Thanks, >> >> Louis >> >> Louis Villeneuve >> Research Associate- Confocal Microscopy >> Montreal Heart Institute- Research Center >> 5000 East Belanger >> Montreal (Qc), Canada >> H1T 1C8 >> >> 514-376-3330 ext 3511 >> 514-376-1355 (Fax) >> >> [hidden email] > Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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Julio Vazquez |
Re: 96-well plates good for high NA lens
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In reply to this post by Knecht, David
We have not used plates in combination with high NA oil lenses. Leica however recently came up with a water delivery system to be used with water immersion lenses specifically for this purpose (Google: Leica water dispenser)
The Perkin Elmer/Evotec Opera HCS system also uses a similar system. No commercial interest with either company. -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 On May 3, 2010, at 3:11 PM, David Knecht wrote:
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Nico Stuurman |
Re: 96-well plates good for high NA lens
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In reply to this post by Louis Villeneuve
I am looking to buy some 96 well plates that can be imaged with high NA lens like a 63x plan-apochromat. Any suggestion of brand/providers? We had great success with glass bottom dishes from Matrical ( http://www.matrical.com/Clear_Bottom_Microplates.php ). The wells have a large viewing area and the plates have a very low skirt which is important to get your bulky high na objective at the outer rows. They were also by far the most affordable glass bottom plates at the time, and the company even made us plates made with piranha-washed glass on request. No commercial interest but a happy customer. Best, Nico |
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Mancini, Michael A |
Re: 96-well plates good for high NA lens
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1. regarding oil immersion, if you ‘spot’ a tiny bit of oil on each (or every other) well of a coverglass-bottom multiwell plate, you can indeed scan all 96 wells. Yes, it’s messy; but it works. 2. regarding glass bottom dishes/plates in general, be aware that some manufacturers provide a product that will leach estrogenic activity from the well bottom adhesive and/or plastic itself. If you’re using full serum in your cells, it probably won’t matter, but if your biology is sensitive to estrogen (and perhaps other hormones), your experiments may be hard to interpret. We’ve seen intrinsic estrogen activity in some plates that is equivalent to adding 10 nM 17-beta estradiol. Given the wide range of biologies sensitive to steroid or steroid-like activity, I’m surprised vendors don’t seem to be paying attention to this issue... Mike Mancini Baylor College of Medicine mancini@... On 5/3/10 10:38 PM, "Nico Stuurman" <nico@...> wrote: I am looking to buy some 96 well plates that can be imaged with high NA lens like a 63x plan-apochromat. Any suggestion of brand/providers? |
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Louis Villeneuve |
Re: 96-well plates good for high NA lens
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In reply to this post by Knecht, David
Hi David, For the moment, it will be on a regular confocal (not high troughtput) so adding a little oil will not be difficult (for a test of 1 or 2 slides). But I will keep n mind your advice. those little things are not written in textbook!!!! Louis
I am wondering how you plan to use these plates. I have thought of using a motorized stage to do a well scan, but could never come up with a solution to the problem of keeping oil on the entire plate. I guess if you use a few wells at a time it would work, but I would not expect to get more than 3-4 wells. before running out of oil. Dave On May 3, 2010, at 2:01 PM, Julio Vazquez wrote: > Hi Louis, > > Below is a summary of plate info we have for our customers. The BD Falcon and Greiner glass bottom plates should be perfect. The Nunc Optiplate, BD Falcon Thin, and Greiner Bio-one plastic should also work. Glass bottom plates are generally better, but more expensive. Thin/Clear optical plastic bottom plates are typically OK for low NA objectives and objectives such as 10x/0.45 or 20x/0.75 PlanApos. For oil immersion, it may be best to use the glass bottom (0.17 mm), or ask for a sample of the thin plastic bottom plates from vendor (or colleague) and test them to make sure they will work for you.... their thickness may be greater than 0.17 mm (e.g. 0.25 mm or so), and therefore you may have working distance issues, although in principle they should be OK. I have plate specs for some of the models which I can email you if needed. > -- > Julio Vazquez > Fred Hutchinson Cancer Research Center > 1100 Fairview Ave. N., mailstop DE-512 > Seattle, WA 98109-1024 > > > http://www.fhcrc.org/ > > > === > > > The plates below (Nunc Optiplate, BG Falcon Clear Bottom, and Greiner) work well on the Cellomics ArrayScan. Other plates may work, but we haven't tested them. Beware that any given vendor may have dozens of different types of plates, so be sure to purchase optical grade plates for imaging. Standard cell culture plates will give poor results, and/or may not be compatible with the instrument or objective. Plates for plate readers or other plate assays will typically not be good either. Some vendors will provide samples (check web sites). > > > > > Falcon BD BioCoat Thin (imaging grade plastic bottom): 96-well clear bottom and 384 well thin bottom, BD Cat # 353219 (Fisher cat # 08-772-225) > > Falcon BD BioCoat 384-well: BD bioscience cat 354667 (collagen coated) and similar > > BD Falcon glass bottom plates: # 357311 (96-well) / 357312 (384-well) > > BD Falcon plates can be "Tissue Culture Treated", Collagen Coated, etc..., and therefore may have different catalog numbers. Just make sure to get the BioCoat Imaging type of plates similar to the ones above. > > > Greiner Bio-One plates: 96 well culture plate with flat bottom; black or white, Cat # 655083/655086 and similar > > http://www.greinerbioone.com/en/usa/articles/catalogue/article/491_10/18026/ > > > Greiner have plates with coverglass or optical plastic bottom. > > > > Nunc Optiplate: Nunc Cat # 165306; Sigma-Aldrich Cat # P8866; Fisher Scientific catalog # 12-566-35 and 12-566-37 > > (Nunc plates may now be sold by Thermo-Fisher) > > > > Perkin Elmer Packard View plates: (Cat # ?) Should work, but have not used them. > > > Covers (seals) for plates: > > Use clear or dark seals. Do not use foil seals as they create reflections that impair image quality. > > Plate seals can be obtained from Island Scientific/Genesee Scientific (ThermalSeal film, Cat # 12-168). > > https://www.geneseesci.com/ > > from Sigma-Aldrich, Nunc sealing tape for multiwell plates, Cat # T9696, T9571, and similar: > > http://www.sigmaaldrich.com/labware/labware-products.html?TablePage=17200082 > > From Fisher (Fisherbrand adhesive plate seals, Cat # 08-408-240) > > https://www.fishersci.com/wps/portal/PRODUCTDETAIL?LBCID=45488914&productId=3743760&catalogId=29102&pos=23&catCode=SA_SC&fromCat=yes&keepSessionSearchOutPut=true&brCategoryId=71102&hlpi=false&fromSearch= > > > > > On May 3, 2010, at 10:15 AM, [hidden email] wrote: > >> >> Bonjour à tous, >> >> I am looking to buy some 96 well plates that can be imaged with high NA lens like a 63x plan-apochromat. Any suggestion of brand/providers? >> >> Thanks, >> >> Louis >> >> Louis Villeneuve >> Research Associate- Confocal Microscopy >> Montreal Heart Institute- Research Center >> 5000 East Belanger >> Montreal (Qc), Canada >> H1T 1C8 >> >> 514-376-3330 ext 3511 >> 514-376-1355 (Fax) >> >> [hidden email] > Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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Knecht, David |
Re: 96-well plates good for high NA lens
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I have not seen anyone mention the ibidi 96 well plate (http://www.ibidi.de/products/disposables/P_8962X_Plate96well.html). I have not used them but I have used their other plastic dishes and they have worked well for us. The plastic is compatible with DIC and oil immersion objectives and there is no issue of attachment of glass to plastic or durability. The only thing that has been problematic is that the bottom does not seem to be compatible with Perfect Focus type systems, but I don't know why yet. Dave
On May 4, 2010, at 8:50 AM, [hidden email] wrote: > > Hi David, > > For the moment, it will be on a regular confocal (not high troughtput) so adding a little oil will not be difficult (for a test of 1 or 2 slides). But I will keep n mind your advice. those little things are not written in textbook!!!! > > Louis > > > David Knecht <[hidden email]>@LISTS.UMN.EDU > Envoyé par : Confocal Microscopy List <[hidden email]> > 2010-05-03 18:11 > Veuillez répondre à > Confocal Microscopy List <[hidden email]> > > A > [hidden email] > cc > Objet > Re: 96-well plates good for high NA lens > > > > > > > > I am wondering how you plan to use these plates. I have thought of using a motorized stage to do a well scan, but could never come up with a solution to the problem of keeping oil on the entire plate. I guess if you use a few wells at a time it would work, but I would not expect to get more than 3-4 wells. before running out of oil. Dave > > On May 3, 2010, at 2:01 PM, Julio Vazquez wrote: > > > Hi Louis, > > > > Below is a summary of plate info we have for our customers. The BD Falcon and Greiner glass bottom plates should be perfect. The Nunc Optiplate, BD Falcon Thin, and Greiner Bio-one plastic should also work. Glass bottom plates are generally better, but more expensive. Thin/Clear optical plastic bottom plates are typically OK for low NA objectives and objectives such as 10x/0.45 or 20x/0.75 PlanApos. For oil immersion, it may be best to use the glass bottom (0.17 mm), or ask for a sample of the thin plastic bottom plates from vendor (or colleague) and test them to make sure they will work for you.... their thickness may be greater than 0.17 mm (e.g. 0.25 mm or so), and therefore you may have working distance issues, although in principle they should be OK. I have plate specs for some of the models which I can email you if needed. > > -- > > Julio Vazquez > > Fred Hutchinson Cancer Research Center > > 1100 Fairview Ave. N., mailstop DE-512 > > Seattle, WA 98109-1024 > > > > > > http://www.fhcrc.org/ > > > > > > === > > > > > > The plates below (Nunc Optiplate, BG Falcon Clear Bottom, and Greiner) work well on the Cellomics ArrayScan. Other plates may work, but we haven't tested them. Beware that any given vendor may have dozens of different types of plates, so be sure to purchase optical grade plates for imaging. Standard cell culture plates will give poor results, and/or may not be compatible with the instrument or objective. Plates for plate readers or other plate assays will typically not be good either. Some vendors will provide samples (check web sites). > > > > > > > > > > Falcon BD BioCoat Thin (imaging grade plastic bottom): 96-well clear bottom and 384 well thin bottom, BD Cat # 353219 (Fisher cat # 08-772-225) > > > > Falcon BD BioCoat 384-well: BD bioscience cat 354667 (collagen coated) and similar > > > > BD Falcon glass bottom plates: # 357311 (96-well) / 357312 (384-well) > > > > BD Falcon plates can be "Tissue Culture Treated", Collagen Coated, etc..., and therefore may have different catalog numbers. Just make sure to get the BioCoat Imaging type of plates similar to the ones above. > > > > > > Greiner Bio-One plates: 96 well culture plate with flat bottom; black or white, Cat # 655083/655086 and similar > > > > http://www.greinerbioone.com/en/usa/articles/catalogue/article/491_10/18026/ > > > > > > Greiner have plates with coverglass or optical plastic bottom. > > > > > > > > Nunc Optiplate: Nunc Cat # 165306; Sigma-Aldrich Cat # P8866; Fisher Scientific catalog # 12-566-35 and 12-566-37 > > > > (Nunc plates may now be sold by Thermo-Fisher) > > > > > > > > Perkin Elmer Packard View plates: (Cat # ?) Should work, but have not used them. > > > > > > Covers (seals) for plates: > > > > Use clear or dark seals. Do not use foil seals as they create reflections that impair image quality. > > > > Plate seals can be obtained from Island Scientific/Genesee Scientific (ThermalSeal film, Cat # 12-168). > > > > https://www.geneseesci.com/ > > > > from Sigma-Aldrich, Nunc sealing tape for multiwell plates, Cat # T9696, T9571, and similar: > > > > http://www.sigmaaldrich.com/labware/labware-products.html?TablePage=17200082 > > > > From Fisher (Fisherbrand adhesive plate seals, Cat # 08-408-240) > > > > https://www.fishersci.com/wps/portal/PRODUCTDETAIL?LBCID=45488914&productId=3743760&catalogId=29102&pos=23&catCode=SA_SC&fromCat=yes&keepSessionSearchOutPut=true&brCategoryId=71102&hlpi=false&fromSearch= > > > > > > > > > > On May 3, 2010, at 10:15 AM, [hidden email] wrote: > > > >> > >> Bonjour à tous, > >> > >> I am looking to buy some 96 well plates that can be imaged with high NA lens like a 63x plan-apochromat. Any suggestion of brand/providers? > >> > >> Thanks, > >> > >> Louis > >> > >> Louis Villeneuve > >> Research Associate- Confocal Microscopy > >> Montreal Heart Institute- Research Center > >> 5000 East Belanger > >> Montreal (Qc), Canada > >> H1T 1C8 > >> > >> 514-376-3330 ext 3511 > >> 514-376-1355 (Fax) > >> > >> [hidden email] > > > > Dr. David Knecht > Department of Molecular and Cell Biology > Co-head Flow Cytometry and Confocal Microscopy Facility > U-3125 > 91 N. Eagleville Rd. > University of Connecticut > Storrs, CT 06269 > 860-486-2200 > 860-486-4331 (fax) > > > Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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B. Prabhakar Pandian |
Fluorescent labeling of bacterial spores
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Hello Everybody,
I was emailing to see if anybody has any method for labeling of bacterial spores, especially bacillus subtilis. Thanks, -Prabhakar |
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Coutu, Cathy |
Re: Fluorescent labeling of bacterial spores
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Hi Prabhakar,
I'm also about to start an experiment involving bacillus, where I'll be visualizing bacillus interacting with fungi and plant roots. I haven't done this before, and would appreciate any suggestions the group has. I've done a quick literature search, and found three methods to visualize the bacillus: 1. Use GFP-transformed lines (Bloemberg, 2007). 2. Use in situ hybridization (Aspray et al, 2006). 3. Use a live/dead stain (Bais et al, 2004). I don't know which method is best. I don't know if GFP is expressed in the bacillus spores (as opposed to active bacteria). But that's what I have to offer. I'd be interested in any other suggestions or hints folks have. Cathy Bloemberg, G. V. (2007). "Microscopic analysis of plant-bacterium interactions using auto fluorescent proteins." European Journal of Plant Pathology 119(3): 301-309. Plant growth promoting rhizobacteria (PGPR) include bacteria that fix nitrogen (e.g., Rhizobiaceae, Herbaspirillum, Azoarcus), produce phytohormones (e.g., Azospirillum) and provide protection against fungal and/or bacterial pathogens (e.g., Pseudomonas, Bacillus, Streptomyces). Interactions between PGPR and plants can be divided into different steps which include initial attraction, attachment, proliferation and colonization e.g., of roots, stem, leaves and flowers. At the genetic level the expression of many bacterial genes are altered during these processes. In addition to the interaction with the plant, PGPR interact and compete with the endogenous microflora, consisting of other bacteria, fungi and/or mycorrhizal fungi. In the case of biocontrol bacterial strains, a direct interaction with the pathogen is often required to suppress the disease. Microscopic analyses of plant growth promoting rhizobacteria (PGPR) in their natural environment and in specific during their interaction(s) with the host plant(s) and/or their target organism(s) is essential for the elucidation of their functioning and the successful application of commercial inoculants. With the discovery and development of auto fluorescent proteins (AFPs) as markers and the development of highly sophisticated fluorescence microscopes such as confocal laser scanning microscopes, a new dimension has been created for studying PGPR in their natural environment. This paper will give a short overview on available tools, the application of AFPs in PGPR research and some future perspectives. Several recent reviews will give the reader an option for further reading (Bloemberg and Lugtenberg 2004; Chalfie and Kain 2005; Larrainzar et al. 2005; Rediers et al. 2005; Bloemberg and Camacho 2006). © 2007 KNPV. Aspray, T. J., E. Eirian Jones, et al. (2006). "Importance of mycorrhization helper bacteria cell density and metabolite localization for the Pinus sylvestris-Lactarius rufus symbiosis." FEMS Microbiology Ecology 56(1): 25-33. Mycorrhization helper bacteria, Paenibacillus sp. EJP73 and Burkholderia sp. EJP67, were used to study the importance of bacterial inoculum dose and bacterial derived soluble and volatile metabolites localization for enhancing mycorrhiza formation in the Pinus sylvestris-Lactarius rufus symbiosis, using a laboratory based microcosm. EJP73 and EJP67 produced different responses in relation to the inoculum dose; EJP73 significantly enhanced mycorrhiza formation to the same degree at all doses tested (10<sup>5</sup>, 10<sup>7</sup>, 10 <sup>9</sup> and 10<sup>10</sup> CFU mL<sup>-1</sup>), whereas, EJP67 only stimulated mycorrhiza formation within a narrow range of inoculum densities (10<sup>7</sup> and 10<sup>9</sup> CFU mL<sup>-1</sup>). The importance of soluble bacterial metabolites was assessed by applying spent broth derived from exponential and stationary phase bacterial cultures to microcosms. No spent broth enhanced mycorrhiza formation over the control. As EJP73 produced the helper effect over a wide range of inoculum doses, this bacterium was chosen for further study. Physical separation of EJP73 from the fungal and plant symbiosis partners was carried out, in order to determine the contribution of constitutively produced bacterial volatile metabolites to the mycorrhization helper bacteria effect. When EJP73 was physically separated from the symbiosis, it had a significant negative effect on mycorrhiza formation. These results suggest that close proximity, or indeed cell contact, is required for the helper effect. Therefore, fluorescent in situ hybridization in conjunction with cryosectioning was used to determine the localization of EJP73 in mycorrhizal tissue. The cells were found to occur as rows or clusters (∼10 cells) within the mycorrhizal mantle, both at the root tip and along the length of the mycorrhizal short roots. © 2005 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved. Bais, H. P., R. Fall, et al. (2004). "Biocontrol of Bacillus subtilis against Infection of Arabidopsis Roots by Pseudomonas syringae Is Facilitated by Biofilm Formation and Surfactin Production." Plant Physiology 134(1): 307-319. Relatively little is known about the exact mechanisms used by Bacillus subtilis in its behavior as a biocontrol agent on plants. Here, we report the development of a sensitive plant infection model demonstrating that the bacterial pathogen Pseudomonas syringae pv tomato DC3000 is capable of infecting Arabidopsis roots both in vitro and in soil. Using this infection model, we demonstrated the biocontrol ability of a wild-type B. subtilis strain 6051 against P. syringae. Arabidopsis root surfaces treated with B. subtilis were analyzed with confocal scanning laser microscopy to reveal a three-dimensional B. subtilis biofilm. It is known that formation of biofilms by B. subtilis is a complex process that includes secretion of surfactin, a lipopeptide antimicrobial agent. To determine the role of surfactin in biocontrol by B. subtilis, we tested a mutant strain, M1, with a deletion in a surfactin synthase gene and, thus, deficient in surfactin production. B. subtilis M1 was ineffective as a biocontrol agent against P. syringae infectivity in Arabidopsis and also failed to form robust biofilms on either roots or inert surfaces. The antibacterial activity of surfactin against P. syringae was determined in both broth and agar cultures and also by live-dead staining methods. Although the minimum inhibitory concentrations determined were relatively high (25 μg mL<sup>-1</sup>), the levels of the lipopeptide in roots colonized by B. subtilis are likely to be sufficient to kill P. syringae. Our results collectively indicate that upon root colonization, B. subtilis 6051 forms a stable, extensive biofilm and secretes surfactin, which act together to protect plants against attack by pathogenic bacteria. Cathy Coutu, M. Sc. Technician / Technicienne Genomics, Bioinformatics, and other Bioinformation / Génomique, Bioinformatique et Bioinformation Agriculture and Agri-Food Canada/Agriculture et Agroalimentaire Canada 107 Science place / 107 Place Science Saskatoon, Saskatchewan / Saskatoon (Saskatchewan) S7N 0X2 [hidden email] Telephone/Téléphone: 306-956-2801 Facsimile/Télécopieur: 306-956-7247 Teletypewriter | Téléimprimeur 613-759-7470 Government of Canada | Gouvernement du Canada -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of B. Prabhakar Pandian Sent: Thursday, June 03, 2010 11:48 AM To: [hidden email] Subject: Fluorescent labeling of bacterial spores Hello Everybody, I was emailing to see if anybody has any method for labeling of bacterial spores, especially bacillus subtilis. Thanks, -Prabhakar |
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Mike Buchin |
Re: Fluorescent labeling of bacterial spores
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This is a commercial response.
We interact with sites using luciferase (non-confocal) for imaging plants (seedlings/root growth), bacteria and fungi. This may be way off base, but if interested I can forward a few contacts for follow up. Mike Michael Buchin Stanford Photonics, Inc. Ph: 650-969-5991 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Coutu, Cathy Sent: Friday, June 04, 2010 10:43 AM To: [hidden email] Subject: Re: Fluorescent labeling of bacterial spores Hi Prabhakar, I'm also about to start an experiment involving bacillus, where I'll be visualizing bacillus interacting with fungi and plant roots. I haven't done this before, and would appreciate any suggestions the group has. I've done a quick literature search, and found three methods to visualize the bacillus: 1. Use GFP-transformed lines (Bloemberg, 2007). 2. Use in situ hybridization (Aspray et al, 2006). 3. Use a live/dead stain (Bais et al, 2004). I don't know which method is best. I don't know if GFP is expressed in the bacillus spores (as opposed to active bacteria). But that's what I have to offer. I'd be interested in any other suggestions or hints folks have. Cathy Bloemberg, G. V. (2007). "Microscopic analysis of plant-bacterium interactions using auto fluorescent proteins." European Journal of Plant Pathology 119(3): 301-309. Plant growth promoting rhizobacteria (PGPR) include bacteria that fix nitrogen (e.g., Rhizobiaceae, Herbaspirillum, Azoarcus), produce phytohormones (e.g., Azospirillum) and provide protection against fungal and/or bacterial pathogens (e.g., Pseudomonas, Bacillus, Streptomyces). Interactions between PGPR and plants can be divided into different steps which include initial attraction, attachment, proliferation and colonization e.g., of roots, stem, leaves and flowers. At the genetic level the expression of many bacterial genes are altered during these processes. In addition to the interaction with the plant, PGPR interact and compete with the endogenous microflora, consisting of other bacteria, fungi and/or mycorrhizal fungi. In the case of biocontrol bacterial strains, a direct interaction with the pathogen is often required to suppress the disease. Microscopic analyses of plant growth promoting rhizobacteria (PGPR) in their natural environment and in specific during their interaction(s) with the host plant(s) and/or their target organism(s) is essential for the elucidation of their functioning and the successful application of commercial inoculants. With the discovery and development of auto fluorescent proteins (AFPs) as markers and the development of highly sophisticated fluorescence microscopes such as confocal laser scanning microscopes, a new dimension has been created for studying PGPR in their natural environment. This paper will give a short overview on available tools, the application of AFPs in PGPR research and some future perspectives. Several recent reviews will give the reader an option for further reading (Bloemberg and Lugtenberg 2004; Chalfie and Kain 2005; Larrainzar et al. 2005; Rediers et al. 2005; Bloemberg and Camacho 2006). © 2007 KNPV. Aspray, T. J., E. Eirian Jones, et al. (2006). "Importance of mycorrhization helper bacteria cell density and metabolite localization for the Pinus sylvestris-Lactarius rufus symbiosis." FEMS Microbiology Ecology 56(1): 25-33. Mycorrhization helper bacteria, Paenibacillus sp. EJP73 and Burkholderia sp. EJP67, were used to study the importance of bacterial inoculum dose and bacterial derived soluble and volatile metabolites localization for enhancing mycorrhiza formation in the Pinus sylvestris-Lactarius rufus symbiosis, using a laboratory based microcosm. EJP73 and EJP67 produced different responses in relation to the inoculum dose; EJP73 significantly enhanced mycorrhiza formation to the same degree at all doses tested (10<sup>5</sup>, 10<sup>7</sup>, 10 <sup>9</sup> and 10<sup>10</sup> CFU mL<sup>-1</sup>), whereas, EJP67 only stimulated mycorrhiza formation within a narrow range of inoculum densities (10<sup>7</sup> and 10<sup>9</sup> CFU mL<sup>-1</sup>). The importance of soluble bacterial metabolites was assessed by applying spent broth derived from exponential and stationary phase bacterial cultures to microcosms. No spent broth enhanced mycorrhiza formation over the control. As EJP73 produced the helper effect over a wide range of inoculum doses, this bacterium was chosen for further study. Physical separation of EJP73 from the fungal and plant symbiosis partners was carried out, in order to determine the contribution of constitutively produced bacterial volatile metabolites to the mycorrhization helper bacteria effect. When EJP73 was physically separated from the symbiosis, it had a significant negative effect on mycorrhiza formation. These results suggest that close proximity, or indeed cell contact, is required for the helper effect. Therefore, fluorescent in situ hybridization in conjunction with cryosectioning was used to determine the localization of EJP73 in mycorrhizal tissue. The cells were found to occur as rows or clusters (∼10 cells) within the mycorrhizal mantle, both at the root tip and along the length of the mycorrhizal short roots. © 2005 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved. Bais, H. P., R. Fall, et al. (2004). "Biocontrol of Bacillus subtilis against Infection of Arabidopsis Roots by Pseudomonas syringae Is Facilitated by Biofilm Formation and Surfactin Production." Plant Physiology 134(1): 307-319. Relatively little is known about the exact mechanisms used by Bacillus subtilis in its behavior as a biocontrol agent on plants. Here, we report the development of a sensitive plant infection model demonstrating that the bacterial pathogen Pseudomonas syringae pv tomato DC3000 is capable of infecting Arabidopsis roots both in vitro and in soil. Using this infection model, we demonstrated the biocontrol ability of a wild-type B. subtilis strain 6051 against P. syringae. Arabidopsis root surfaces treated with B. subtilis were analyzed with confocal scanning laser microscopy to reveal a three-dimensional B. subtilis biofilm. It is known that formation of biofilms by B. subtilis is a complex process that includes secretion of surfactin, a lipopeptide antimicrobial agent. To determine the role of surfactin in biocontrol by B. subtilis, we tested a mutant strain, M1, with a deletion in a surfactin synthase gene and, thus, deficient in surfactin production. B. subtilis M1 was ineffective as a biocontrol agent against P. syringae infectivity in Arabidopsis and also failed to form robust biofilms on either roots or inert surfaces. The antibacterial activity of surfactin against P. syringae was determined in both broth and agar cultures and also by live-dead staining methods. Although the minimum inhibitory concentrations determined were relatively high (25 μg mL<sup>-1</sup>), the levels of the lipopeptide in roots colonized by B. subtilis are likely to be sufficient to kill P. syringae. Our results collectively indicate that upon root colonization, B. subtilis 6051 forms a stable, extensive biofilm and secretes surfactin, which act together to protect plants against attack by pathogenic bacteria. Cathy Coutu, M. Sc. Technician / Technicienne Genomics, Bioinformatics, and other Bioinformation / Génomique, Bioinformatique et Bioinformation Agriculture and Agri-Food Canada/Agriculture et Agroalimentaire Canada 107 Science place / 107 Place Science Saskatoon, Saskatchewan / Saskatoon (Saskatchewan) S7N 0X2 [hidden email] Telephone/Téléphone: 306-956-2801 Facsimile/Télécopieur: 306-956-7247 Teletypewriter | Téléimprimeur 613-759-7470 Government of Canada | Gouvernement du Canada -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of B. Prabhakar Pandian Sent: Thursday, June 03, 2010 11:48 AM To: [hidden email] Subject: Fluorescent labeling of bacterial spores Hello Everybody, I was emailing to see if anybody has any method for labeling of bacterial spores, especially bacillus subtilis. Thanks, -Prabhakar |
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