Ca-measurement in glandular tissue
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Cselenyak Attila |
Ca-measurement in glandular tissue
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Dear List members,
we would like measure Ca(2+) in glandular tissue with Fluo3 using confocal mircoscopy. We tried to load the tissue pieces with different concentrations of the dye for 1,5 hours in 37C, washing after that with PBS, and the tissue was put in a perfusion system. But we could not see any changes in the fluorescence intensity when giving either ATP or A23187 ionophore to the perfusion system. Do you have any experience what would be an optimal loading protocol or Fluo3 is suitable for this kind of measurements? Thanks is advence! Best regards, Attila -- Attila Cselenyak PhD student Semmelweis University Basic Medical Science Center Institute Of Human Physiology And Clinical Experimental Research H-1094, Budapest Tűzoltó utca 37-47. Phone: +36-1-4591500 ext.60342 Mobile: +36-70-3874931 |
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Axel Kurt Preuss |
Re: Ca-measurement in glandular tissue
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Dear Attila
1.5 hrs is far too long I would try 40 min, maybe even only 30 min Then washing in HBSS. The phosphate in PBS can be a problem (catching your Ca away) I would clamp Ca down to 1 mM , maybe even 300 micro M during washing But the most critical step is what cells you want to label. If you soak the whole tissue in fluo 4 you might get abundant fluorescence making cells indiscriminable or buffer too much Ca Cheers Axel Axel K Preuss PhD, A*Star IMCB-Central Imaging Facility 6-19B Sent from. +65 9271 5622 On May 11, 2010, at 5:50 PM, "Cselenyak Attila" <[hidden email]> wrote: > Dear List members, > > we would like measure Ca(2+) in glandular tissue with Fluo3 using > confocal mircoscopy. We tried to load the tissue pieces with different > concentrations of the dye for 1,5 hours in 37C, washing after that > with PBS, and the tissue was put in a perfusion system. But we could > not see any changes in the fluorescence intensity when giving either > ATP or A23187 ionophore to the perfusion system. > > Do you have any experience what would be an optimal loading protocol > or Fluo3 is suitable for this kind of measurements? > > Thanks is advence! > > Best regards, > > Attila > > -- > Attila Cselenyak > PhD student > > Semmelweis University > Basic Medical Science Center > Institute Of Human Physiology And Clinical Experimental Research > H-1094, Budapest > Tűzoltó utca 37-47. > Phone: +36-1-4591500 ext.60342 > Mobile: +36-70-3874931 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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Louis Villeneuve |
RE Ca-measurement in glandular tissue
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In reply to this post by Cselenyak Attila
Hi Attila, Do you see any fluorescence? Does fluo3 etting inside the tissue? Maybe adding some pluronic acid can help to have a better signal! Have a nice day, Louisssssssss
Dear List members, we would like measure Ca(2+) in glandular tissue with Fluo3 using confocal mircoscopy. We tried to load the tissue pieces with different concentrations of the dye for 1,5 hours in 37C, washing after that with PBS, and the tissue was put in a perfusion system. But we could not see any changes in the fluorescence intensity when giving either ATP or A23187 ionophore to the perfusion system. Do you have any experience what would be an optimal loading protocol or Fluo3 is suitable for this kind of measurements? Thanks is advence! Best regards, Attila -- Attila Cselenyak PhD student Semmelweis University Basic Medical Science Center Institute Of Human Physiology And Clinical Experimental Research H-1094, Budapest Tűzoltó utca 37-47. Phone: +36-1-4591500 ext.60342 Mobile: +36-70-3874931 |
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Axel Kurt Preuss |
Re: RE Ca-measurement in glandular tissue
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He is compartmentalising his fluo, so whether he sees fluorescence or notay be secondary
After 1.5 hrs loading time the poor fluo 4 molecules are probably gasping in pain and can't take it anymore ( with it I mean the Ca) (scientifically actually they probably could "take" the Ca but without signal increase Best to load cells the old fashioned way per injection. But If it has to be AM best to try different concentrations in incubator for not more than 40 minutes Yes, with so many cells in the tissue competing for fluo it could be fluo concentration is not sufficient. But he HAS to try different concentrations, as overloading could also be the case, and MUST avoid compartmentalisation And I would stay away from PBS It is aost hopeless to try to "stain" tissue fragments with Ca dye (AM) Bbbwouldb probably only work if there are cells extruding and not touching other tissue. Such exposed cells might take up fluo the right way Best to use a method to selectively target a few cells. This means injecting the free acid or experimenting with electrophoresis (dextran coupled dyes) or local perfusion with AM Or to digest the tissue to the point of discrete cells My text may be garbled due to compartmentilizing iPhone keyboard Thanks Axel Axel K Preuss PhD, A*Star IMCB-Central Imaging Facility 6-19B Sent from. +65 9271 5622 On May 11, 2010, at 8:38 PM, "[hidden email]<mailto:[hidden email]>" <[hidden email]<mailto:[hidden email]>> wrote: Hi Attila, Do you see any fluorescence? Does fluo3 etting inside the tissue? Maybe adding some pluronic acid can help to have a better signal! Have a nice day, Louisssssssss Cselenyak Attila <[hidden email]<mailto:[hidden email]>>@LISTS.UMN.EDU Envoyé par : Confocal Microscopy List <[hidden email]<mailto:[hidden email]>> 2010-05-11 05:41 Veuillez répondre à Confocal Microscopy List <[hidden email]<mailto:[hidden email]>> A [hidden email]<mailto:[hidden email]> cc Objet Ca-measurement in glandular tissue Dear List members, we would like measure Ca(2+) in glandular tissue with Fluo3 using confocal mircoscopy. We tried to load the tissue pieces with different concentrations of the dye for 1,5 hours in 37C, washing after that with PBS, and the tissue was put in a perfusion system. But we could not see any changes in the fluorescence intensity when giving either ATP or A23187 ionophore to the perfusion system. Do you have any experience what would be an optimal loading protocol or Fluo3 is suitable for this kind of measurements? Thanks is advence! Best regards, Attila -- Attila Cselenyak PhD student Semmelweis University Basic Medical Science Center Institute Of Human Physiology And Clinical Experimental Research H-1094, Budapest Tűzoltó utca 37-47. Phone: +36-1-4591500 ext.60342 Mobile: +36-70-3874931 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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Periasamy, Ammasi (ap3t) |
Re: RE Ca-measurement in glandular tissue
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In reply to this post by Louis Villeneuve
We did collect calcium signal in the tissue using calcium green,
but we injected the dye. We are getting pretty good signals. Hope this helps. Ammasi Periasamy, Ph.D. Director, Keck Center for Cellular Imaging (KCCI) Professor of Biology and Biomedical Engineering Biology, Gilmer Hall (064), 485 McCormick Rd University of Virginia Charlottesville, VA 22904 Voice: 434-243-7602 (Office); 982-4869 (lab) Fax:434-982-5210; Email:[hidden email] http://www.kcci.virginia.edu ************************ 10th Annual Workshop on FRET Microscopy, March 8-12, 2011 http://www.kcci.virginia.edu/workshop/workshop2011/index.php ************************* From: Confocal Microscopy
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