Quenching fluorescent beads
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Renato A. Mortara |
Quenching fluorescent beads
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello, Is it possible to quench/kill the fluorescence of fluorescent beads ? The idea is to distinguish between internalized (intracellular) x extracellular ones in a phagocytosis assay. Many thanks for any input, Renato Renato A. Mortara Parasitology Division UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th floor São Paulo, SP 04023-062 Brazil Phone: 55 11 5579-8306 Fax: 55 11 5571-1095 email: [hidden email] home page: www.ecb.epm.br/~ramortara |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Renato - I suppose if the fluorophore is on the surface you can quench it. For example, orange-red fluorescence can be quenched by 5-10 mg/ml of acid blue 9 which doesn't harm the cells and doesn't penetrate intact membranes. Some fluorophores can be also be quenched by Trypan Blue. But if the beads are fluorescent inside, there isn't much you can do about it. Mike -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato Mortara Sent: Monday, November 29, 2010 2:19 PM To: [hidden email] Subject: Quenching fluorescent beads ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello, Is it possible to quench/kill the fluorescence of fluorescent beads ? The idea is to distinguish between internalized (intracellular) x extracellular ones in a phagocytosis assay. Many thanks for any input, Renato Renato A. Mortara Parasitology Division UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th floor São Paulo, SP 04023-062 Brazil Phone: 55 11 5579-8306 Fax: 55 11 5571-1095 email: [hidden email] home page: www.ecb.epm.br/~ramortara |
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Haller, Edward |
Re: Quenching fluorescent beads
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi, Renato, What about exploring this from a different approach. What about using a flurophore attached to the beads that only fluoresces in an acidic environment? Then, when it is taken into a phagosome, it will fluoresce. Ed Edward Haller, Lab Manager University of South Florida Integrative Biology Department Electron Microscopy Core SCA 110 4202 East Fowler Avenue Tampa, FL 33620 813-974-2676 [hidden email] Office: LSA 119 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato Mortara Sent: Monday, November 29, 2010 2:19 PM To: [hidden email] Subject: Quenching fluorescent beads ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello, Is it possible to quench/kill the fluorescence of fluorescent beads ? The idea is to distinguish between internalized (intracellular) x extracellular ones in a phagocytosis assay. Many thanks for any input, Renato Renato A. Mortara Parasitology Division UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th floor São Paulo, SP 04023-062 Brazil Phone: 55 11 5579-8306 Fax: 55 11 5571-1095 email: [hidden email] home page: www.ecb.epm.br/~ramortara |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** About ten years ago we did an assay where we had Cy5 and antibody coated beads that we incubated with the cells. Then we fixed the cells but did not permeabalize. We stained for the antibody with rhodamine. Thus, all beads were infrared but only beads not yet internalized were red. examples: http://www.einstein.yu.edu/aif/users/dcox/cups_3d/cups001202a.htm http://www.einstein.yu.edu/aif/users/dcox/cups_3d/cups001202.htm http://www.einstein.yu.edu/aif/users/dcox/cups_3d/cups0011.htm -Michael -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Haller, Edward Sent: Monday, November 29, 2010 3:14 PM To: [hidden email] Subject: Re: Quenching fluorescent beads ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi, Renato, What about exploring this from a different approach. What about using a flurophore attached to the beads that only fluoresces in an acidic environment? Then, when it is taken into a phagosome, it will fluoresce. Ed Edward Haller, Lab Manager University of South Florida Integrative Biology Department Electron Microscopy Core SCA 110 4202 East Fowler Avenue Tampa, FL 33620 813-974-2676 [hidden email] Office: LSA 119 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato Mortara Sent: Monday, November 29, 2010 2:19 PM To: [hidden email] Subject: Quenching fluorescent beads ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello, Is it possible to quench/kill the fluorescence of fluorescent beads ? The idea is to distinguish between internalized (intracellular) x extracellular ones in a phagocytosis assay. Many thanks for any input, Renato Renato A. Mortara Parasitology Division UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th floor São Paulo, SP 04023-062 Brazil Phone: 55 11 5579-8306 Fax: 55 11 5571-1095 email: [hidden email] home page: www.ecb.epm.br/~ramortara ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= |
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Kilgore, Jason-2 |
Re: Quenching fluorescent beads - **vendor reply**
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In reply to this post by Haller, Edward
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** ** VENDOR REPLY ** This could be achieved using pHrodo SE labeling of an amine-coated bead. pHrodo is a dye which is non-fluorescent in neutral pH, but bright red fluorescent (similar in wavelength to Texas Red) upon introduction in an acidic environment. It was developed primarily for phagocytosis and endocytosis assays. Invitrogen sells pHrodo SE as catalog P36600, as well as amine-coated beads of various sizes. Cheers, Jason Jason A. Kilgore Sr. Assoc. Technical Application Scientist Molecular Probes Labeling and Detection Technologies Cells Systems Division T 1 800 955 6288 x2 then x4 or 541 335 0353 . F 541 335 0238 29851 Willow Creek Rd . Eugene . OR . 97402-9132 . United States www.invitrogen.com/technicalsupport Click below to visit the new social network for scientific collaboration: -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Haller, Edward Sent: Monday, November 29, 2010 12:14 PM To: [hidden email] Subject: Re: Quenching fluorescent beads ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi, Renato, What about exploring this from a different approach. What about using a flurophore attached to the beads that only fluoresces in an acidic environment? Then, when it is taken into a phagosome, it will fluoresce. Ed Edward Haller, Lab Manager University of South Florida Integrative Biology Department Electron Microscopy Core SCA 110 4202 East Fowler Avenue Tampa, FL 33620 813-974-2676 [hidden email] Office: LSA 119 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato Mortara Sent: Monday, November 29, 2010 2:19 PM To: [hidden email] Subject: Quenching fluorescent beads ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello, Is it possible to quench/kill the fluorescence of fluorescent beads ? The idea is to distinguish between internalized (intracellular) x extracellular ones in a phagocytosis assay. Many thanks for any input, Renato Renato A. Mortara Parasitology Division UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th floor São Paulo, SP 04023-062 Brazil Phone: 55 11 5579-8306 Fax: 55 11 5571-1095 email: [hidden email] home page: www.ecb.epm.br/~ramortara |
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Renato A. Mortara |
RES: Quenching fluorescent beads - **vendor reply**
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Thanks everyone for the input ! Best regards to all, Renato -----Mensagem original----- De: Confocal Microscopy List [mailto:[hidden email]] Em nome de Kilgore, Jason Enviada em: segunda-feira, 29 de novembro de 2010 19:01 Para: [hidden email] Assunto: Re: Quenching fluorescent beads - **vendor reply** ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** ** VENDOR REPLY ** This could be achieved using pHrodo SE labeling of an amine-coated bead. pHrodo is a dye which is non-fluorescent in neutral pH, but bright red fluorescent (similar in wavelength to Texas Red) upon introduction in an acidic environment. It was developed primarily for phagocytosis and endocytosis assays. Invitrogen sells pHrodo SE as catalog P36600, as well as amine-coated beads of various sizes. Cheers, Jason Jason A. Kilgore Sr. Assoc. Technical Application Scientist Molecular Probes Labeling and Detection Technologies Cells Systems Division T 1 800 955 6288 x2 then x4 or 541 335 0353 . F 541 335 0238 29851 Willow Creek Rd . Eugene . OR . 97402-9132 . United States www.invitrogen.com/technicalsupport Click below to visit the new social network for scientific collaboration: -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Haller, Edward Sent: Monday, November 29, 2010 12:14 PM To: [hidden email] Subject: Re: Quenching fluorescent beads ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi, Renato, What about exploring this from a different approach. What about using a flurophore attached to the beads that only fluoresces in an acidic environment? Then, when it is taken into a phagosome, it will fluoresce. Ed Edward Haller, Lab Manager University of South Florida Integrative Biology Department Electron Microscopy Core SCA 110 4202 East Fowler Avenue Tampa, FL 33620 813-974-2676 [hidden email] Office: LSA 119 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato Mortara Sent: Monday, November 29, 2010 2:19 PM To: [hidden email] Subject: Quenching fluorescent beads ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello, Is it possible to quench/kill the fluorescence of fluorescent beads ? The idea is to distinguish between internalized (intracellular) x extracellular ones in a phagocytosis assay. Many thanks for any input, Renato Renato A. Mortara Parasitology Division UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th floor São Paulo, SP 04023-062 Brazil Phone: 55 11 5579-8306 Fax: 55 11 5571-1095 email: [hidden email] home page: www.ecb.epm.br/~ramortara |
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Kavita Aswani-2 |
FW: Quenching fluorescent beads
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In reply to this post by Renato A. Mortara
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Renato, I have in the past successfully quenched FITC-labeled bacteria with Trypan blue. Please contact me if you would like the reference. Cheers Kavita Dr. Kavita Aswani, PhD Senior Product Manager- Life Sciences LUMEN DYNAMICS (formerly EXFO Life Sciences and Industrial Division) 2260 Argentia Road, Mississauga, Ontario, Canada, L5N 6H7 www.LDGI.com Tel: 1 905 812-3342 Cell: 1 647 290-3506 Toll Free (USA and Canada): 1 800 668-8752 Fax: 1 905 821-2055 Email: [hidden email] -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of MODEL, MICHAEL Sent: November 29, 2010 2:50 PM To: [hidden email] Subject: Re: Quenching fluorescent beads ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Renato - I suppose if the fluorophore is on the surface you can quench it. For example, orange-red fluorescence can be quenched by 5-10 mg/ml of acid blue 9 which doesn't harm the cells and doesn't penetrate intact membranes. Some fluorophores can be also be quenched by Trypan Blue. But if the beads are fluorescent inside, there isn't much you can do about it. Mike -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato Mortara Sent: Monday, November 29, 2010 2:19 PM To: [hidden email] Subject: Quenching fluorescent beads ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello, Is it possible to quench/kill the fluorescence of fluorescent beads ? The idea is to distinguish between internalized (intracellular) x extracellular ones in a phagocytosis assay. Many thanks for any input, Renato Renato A. Mortara Parasitology Division UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th floor São Paulo, SP 04023-062 Brazil Phone: 55 11 5579-8306 Fax: 55 11 5571-1095 email: [hidden email] home page: www.ecb.epm.br/~ramortara |
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Knecht, David |
Re: Quenching fluorescent beads
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** In my experience, Trypan blue quenching only works with things like yeast and bacteria that absorb the dye at high concentration. A bead suspended in Trypan blue will be quenched no differently inside or outside the cell because the decrease in fluorescence is due only to absorption of the excitation and emission signal. Dave On Nov 30, 2010, at 9:33 AM, Kavita Aswani wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi Renato, > I have in the past successfully quenched FITC-labeled bacteria with Trypan blue. Please contact me if you would like the reference. > Cheers > Kavita > > > Dr. Kavita Aswani, PhD > Senior Product Manager- Life Sciences > LUMEN DYNAMICS > (formerly EXFO Life Sciences and Industrial Division) > > 2260 Argentia Road, Mississauga, > Ontario, Canada, L5N 6H7 > www.LDGI.com > Tel: 1 905 812-3342 > Cell: 1 647 290-3506 > Toll Free (USA and Canada): 1 800 668-8752 > Fax: 1 905 821-2055 > Email: [hidden email] > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of MODEL, MICHAEL > Sent: November 29, 2010 2:50 PM > To: [hidden email] > Subject: Re: Quenching fluorescent beads > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi Renato - I suppose if the fluorophore is on the surface you can quench it. For example, orange-red fluorescence can be quenched by 5-10 mg/ml of acid blue 9 which doesn't harm the cells and doesn't penetrate intact membranes. Some fluorophores can be also be quenched by Trypan Blue. But if the beads are fluorescent inside, there isn't much you can do about it. > > Mike > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato Mortara > Sent: Monday, November 29, 2010 2:19 PM > To: [hidden email] > Subject: Quenching fluorescent beads > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hello, > > Is it possible to quench/kill the fluorescence of fluorescent beads ? The > idea is to distinguish between internalized (intracellular) x extracellular > ones in a phagocytosis assay. > > Many thanks for any input, > > Renato > > Renato A. Mortara > Parasitology Division > UNIFESP - Escola Paulista de Medicina > Rua Botucatu, 862, 6th floor > São Paulo, SP > 04023-062 > Brazil > Phone: 55 11 5579-8306 > Fax: 55 11 5571-1095 > email: [hidden email] > home page: www.ecb.epm.br/~ramortara 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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