FRET using fixed cells
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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 all, I am a novice in the field. I use live cells transfected with tagged proteins for FRET and TIRF microscopy. My question is can one use fixed cells for FRET using AlexaFluor antibodies?? If yes, please guide me to some references and how to. Another question is has someone used TAT-fusion peptides labelled with AlexaFluor for TIRF and FRET?? Best Regards, Syed Khundmiri University of Louisville |
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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 Syed Khundmiri, The basic requirements for FRET are: 1) The biomolecules tagged with fluorophores are located in the nanometer range; 2) The acceptor absorption spectrum overlaps with the donor emission spectrum. There are some commonly used donor-acceptor pairs, including AlexaFluor dyes. TIRF is a high sensitivity fluorescence technique with which you can image cellular structures located in the range 0-150nm above the coverslip, so your cells should be flat and adherent to the coverslip. Generally, you can use FRET and TIRF for both live cells and fixed cells. Gary G. Li, Ph.D. Johns Hopkins School of Medicine Baltimore, MD 21224 On Fri, Dec 3, 2010 at 10:17 AM, Syed Khundmiri < [hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi all, > > I am a novice in the field. I use live cells transfected with tagged > proteins for > FRET and TIRF microscopy. My question is can one use fixed cells for FRET > using AlexaFluor antibodies?? If yes, please guide me to some references > and > how to. > > Another question is has someone used TAT-fusion peptides labelled with > AlexaFluor for TIRF and FRET?? > > Best Regards, > > Syed Khundmiri > University of Louisville > |
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In reply to this post by Syed J Khundmiri
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear Syed, If the fluorescent tag does not cause mis-localization or aggregation of a protein of interest, I recommend to use CeFP (Dave Piston) and the "late edition" of the standard EYFP (with the F64L substitution) as the fluorescence tags, and 440nm diode (or 427/10 nm Semrock excitation filter), 514 nm Argon line (504/12 nm exciter, Semrock) and 472/30nm and 542/27nm emission filters, also Semrock. TIRF or wide-field is the best optical systems for the sensitized emission FRET. Good luck, Vitaly 301-515-7833 ________________________________ From: Syed Khundmiri <[hidden email]> To: [hidden email] Sent: Fri, December 3, 2010 10:17:19 AM Subject: FRET using fixed cells ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi all, I am a novice in the field. I use live cells transfected with tagged proteins for FRET and TIRF microscopy. My question is can one use fixed cells for FRET using AlexaFluor antibodies?? If yes, please guide me to some references and how to. Another question is has someone used TAT-fusion peptides labelled with AlexaFluor for TIRF and FRET?? Best Regards, Syed Khundmiri University of Louisville |
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In reply to this post by Syed J Khundmiri
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** PS Live cells would give you better and reproducible data than the fixed material. Dear Syed, If the fluorescent tag does not cause mis-localization or aggregation of a protein of interest, I recommend to use CeFP (Dave Piston) and the "late edition" of the standard EYFP (with the F64L substitution) as the fluorescence tags, and 440nm diode (or 427/10 nm Semrock excitation filter), 514 nm Argon line (504/12 nm exciter, Semrock) and 472/30nm and 542/27nm emission filters, also Semrock. TIRF or wide-field is the best optical systems for the sensitized emission FRET. Good luck, Vitaly 301-515-7833 ________________________________ From: Syed Khundmiri <[hidden email]> To: [hidden email] Sent: Fri, December 3, 2010 10:17:19 AM Subject: FRET using fixed cells ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi all, I am a novice in the field. I use live cells transfected with tagged proteins for FRET and TIRF microscopy. My question is can one use fixed cells for FRET using AlexaFluor antibodies?? If yes, please guide me to some references and how to. Another question is has someone used TAT-fusion peptides labelled with AlexaFluor for TIRF and FRET?? Best Regards, Syed Khundmiri University of Louisville |
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In reply to this post by Syed J Khundmiri
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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 Syed, We used cells labeled with antibodies coupled to Alexas for FRET with success. The Alexa pairs we used were 488&555 and 568&633. Best regards, Laurent. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Syed Khundmiri Sent: vendredi 3 décembre 2010 16:17 To: [hidden email] Subject: FRET using fixed cells ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi all, I am a novice in the field. I use live cells transfected with tagged proteins for FRET and TIRF microscopy. My question is can one use fixed cells for FRET using AlexaFluor antibodies?? If yes, please guide me to some references and how to. Another question is has someone used TAT-fusion peptides labelled with AlexaFluor for TIRF and FRET?? Best Regards, Syed Khundmiri University of Louisville |
 
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Periasamy, Ammasi (ap3t) |
Re: FRET using fixed cells
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear All It has been widely used fixed cell for FRET. We recently found out that the E% decreases for the fixed cells versus live cell. We also found out that the fixative reduces the E%. you can clearly see the change in E% in the lifetime FRET imaging. We did not see much change in E% if you collect the data within few days after the cells are fixed. We used both formaldehyde and methanol as fixative. We are finalizing the data and plan to submit soon for a journal and I will post later as soon as it published. We also train the participants in our annual hands on training FRET workshop during March in the FLIM-FRET system live versus fixed cell. Not a good idea to use fixed cell for FRET or any fixed sample in the TIRF system. You are working with evanescent wave in the TIRF system with nanometer depth and it is require a alive specimen. Hope this helps Ammasi Prof. Ammasi Periasamy Director, Keck Center for Cellular Imaging (KCCI) 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/Contact/peri.php ************************ 10th Annual Workshop on FRET Microscopy, March 8-13, 2011 http://www.kcci.virginia.edu/workshop/workshop2011/ One Day Symposium to honor Prof. Theodor Förster's 100th Birth Day, March 10, 2011 http://www.kcci.virginia.edu/symposium/ ************************* -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gelman, Laurent Sent: Tuesday, December 07, 2010 6:51 AM To: [hidden email] Subject: Re: FRET using fixed cells ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Syed, We used cells labeled with antibodies coupled to Alexas for FRET with success. The Alexa pairs we used were 488&555 and 568&633. Best regards, Laurent. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Syed Khundmiri Sent: vendredi 3 décembre 2010 16:17 To: [hidden email] Subject: FRET using fixed cells ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi all, I am a novice in the field. I use live cells transfected with tagged proteins for FRET and TIRF microscopy. My question is can one use fixed cells for FRET using AlexaFluor antibodies?? If yes, please guide me to some references and how to. Another question is has someone used TAT-fusion peptides labelled with AlexaFluor for TIRF and FRET?? Best Regards, Syed Khundmiri University of Louisville |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** To add to the issues in comparing fixed and live cell results, unrelated to reagent choice, this November's Microscopy Today issue has an article on AFM imaging of live and fixed cells by A. Berquand et al, page 8-14. U2-OS cells shrunk from 14 microns in height to 5 microns after gluteraldehyde fixation. Not much change in lateral spacing, but a ~65% change (collapse) in Z needs to be considered in relating fixed specimen FRET results to live cell results. Mike Ignatius Molecular Probes/LifeTechnologies -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Periasamy, Ammasi (ap3t) Sent: Tuesday, December 07, 2010 6:46 AM To: [hidden email] Subject: Re: FRET using fixed cells ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear All It has been widely used fixed cell for FRET. We recently found out that the E% decreases for the fixed cells versus live cell. We also found out that the fixative reduces the E%. you can clearly see the change in E% in the lifetime FRET imaging. We did not see much change in E% if you collect the data within few days after the cells are fixed. We used both formaldehyde and methanol as fixative. We are finalizing the data and plan to submit soon for a journal and I will post later as soon as it published. We also train the participants in our annual hands on training FRET workshop during March in the FLIM-FRET system live versus fixed cell. Not a good idea to use fixed cell for FRET or any fixed sample in the TIRF system. You are working with evanescent wave in the TIRF system with nanometer depth and it is require a alive specimen. Hope this helps Ammasi Prof. Ammasi Periasamy Director, Keck Center for Cellular Imaging (KCCI) 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/Contact/peri.php ************************ 10th Annual Workshop on FRET Microscopy, March 8-13, 2011 http://www.kcci.virginia.edu/workshop/workshop2011/ One Day Symposium to honor Prof. Theodor Förster's 100th Birth Day, March 10, 2011 http://www.kcci.virginia.edu/symposium/ ************************* -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gelman, Laurent Sent: Tuesday, December 07, 2010 6:51 AM To: [hidden email] Subject: Re: FRET using fixed cells ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Syed, We used cells labeled with antibodies coupled to Alexas for FRET with success. The Alexa pairs we used were 488&555 and 568&633. Best regards, Laurent. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Syed Khundmiri Sent: vendredi 3 décembre 2010 16:17 To: [hidden email] Subject: FRET using fixed cells ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi all, I am a novice in the field. I use live cells transfected with tagged proteins for FRET and TIRF microscopy. My question is can one use fixed cells for FRET using AlexaFluor antibodies?? If yes, please guide me to some references and how to. Another question is has someone used TAT-fusion peptides labelled with AlexaFluor for TIRF and FRET?? Best Regards, Syed Khundmiri University of Louisville |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Thank you all for your valuable suggestions. We are using neutrophils for our studies. They are suspension cells and do not attach to the glass slides unless fixed with paraformaldehyde. However, if we incubate the cells for an hour they will settle down and attach loosely to collagen coated plates. They do not survive more than few hours and therefore we cannot do any transfections. However, we know that they take up antibodies. If I let them settle down and do not fix them with paraformaldehyde, then is it possible to do FRET/TIRF in these cells using fluorescent antibodies??? Syed Syed Khundmiri, Ph.D. University of Louisville >>> "Ignatius, Mike" <[hidden email]> 12/7/2010 12:50 PM >>> ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** To add to the issues in comparing fixed and live cell results, unrelated to reagent choice, this November's Microscopy Today issue has an article on AFM imaging of live and fixed cells by A. Berquand et al, page 8-14. U2-OS cells shrunk from 14 microns in height to 5 microns after gluteraldehyde fixation. Not much change in lateral spacing, but a ~65% change (collapse) in Z needs to be considered in relating fixed specimen FRET results to live cell results. Mike Ignatius Molecular Probes/LifeTechnologies -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Periasamy, Ammasi (ap3t) Sent: Tuesday, December 07, 2010 6:46 AM To: [hidden email] Subject: Re: FRET using fixed cells ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear All It has been widely used fixed cell for FRET. We recently found out that the E% decreases for the fixed cells versus live cell. We also found out that the fixative reduces the E%. you can clearly see the change in E% in the lifetime FRET imaging. We did not see much change in E% if you collect the data within few days after the cells are fixed. We used both formaldehyde and methanol as fixative. We are finalizing the data and plan to submit soon for a journal and I will post later as soon as it published. We also train the participants in our annual hands on training FRET workshop during March in the FLIM-FRET system live versus fixed cell. Not a good idea to use fixed cell for FRET or any fixed sample in the TIRF system. You are working with evanescent wave in the TIRF system with nanometer depth and it is require a alive specimen. Hope this helps Ammasi Prof. Ammasi Periasamy Director, Keck Center for Cellular Imaging (KCCI) 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/Contact/peri.php ************************ 10th Annual Workshop on FRET Microscopy, March 8-13, 2011 http://www.kcci.virginia.edu/workshop/workshop2011/ One Day Symposium to honor Prof. Theodor Förster's 100th Birth Day, March 10, 2011 http://www.kcci.virginia.edu/symposium/ ************************* -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gelman, Laurent Sent: Tuesday, December 07, 2010 6:51 AM To: [hidden email] Subject: Re: FRET using fixed cells ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Syed, We used cells labeled with antibodies coupled to Alexas for FRET with success. The Alexa pairs we used were 488&555 and 568&633. Best regards, Laurent. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Syed Khundmiri Sent: vendredi 3 décembre 2010 16:17 To: [hidden email] Subject: FRET using fixed cells ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi all, I am a novice in the field. I use live cells transfected with tagged proteins for FRET and TIRF microscopy. My question is can one use fixed cells for FRET using AlexaFluor antibodies?? If yes, please guide me to some references and how to. Another question is has someone used TAT-fusion peptides labelled with AlexaFluor for TIRF and FRET?? Best Regards, Syed Khundmiri University of Louisville |
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B. Prabhakar Pandian |
Fluorescent bacteriophage
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In reply to this post by Ignatius, Mike-2
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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 Everybody, Does anyone have an idea of where I can purchase fluorescent bacteriophages. Thanks, -Prabhakar |
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In reply to this post by Syed J Khundmiri
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** It is possible to do TIRF with fixed cells as long as you keep them in PBS so that the refractive index difference at the interface with the glass is appropriate for TIRF. Don't know about TIRF-FRET with fixed cells. I defer to Peri on that. Kate |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Excellent. Ed ******************************************************************** PLEASE NOTE: WHEN TRAVELING I USE OTHER ELECTRONIC MAIL ACCOUNTS. PLEASE SEND ALL YOUR REPLYS TO: [hidden email]. Mailing Address: Eduardo Rosa-Molinar, Ph.D. Group Leader, Biological Imaging Group (BIG) University of Puerto Rico-Rio Piedras P.O. Box 21809 UPR Station San Juan, Puerto Rico 00931-1809 Telephone: 787-764-0000; extension 1-3551 (voice mail only) Facsimile: 888-762-7577 Electronic Mail: mailto:[hidden email]@hpcf.upr.edu Website: http://pisces.cnnet.clu.edu/erm-lab Shipping Address: University of Puerto Rico-Rio Piedras Biological Imaging Group (BIG) Attention: Eduardo Rosa-Molinar, Ph.D. Group Leader, Biological Imaging Group (BIG) Esquina Barbosa y Gandara Avenue Julio Garcia Bldg; Rm 118 Rio Piedras, Puerto Rico 00931 Telephone: 787-764-0000; extension 1-3551 (voice mail only) Facsimile: 888-762-7577 Electronic Mail: mailto:[hidden email]@hpcf.upr.edu Website: http://pisces.cnnet.clu.edu/erm-lab Note: This email and any files transmitted with it may contain information that is PRIVILEDGE, CONFIDENTIAL, and exempt from disclosure under applicable law. It is intended only for the individual(s) or entity named above. If you are not an intended recipient of this e-mail, you are hereby notified that any unauthorized use, dissemination or copying of this e-mail or the information contained in it or attached to it is strictly prohibited. If you have received this email in error, please delete it and immediately notify the person named above by reply email. ******************************************************************** -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kate Luby-Phelps Sent: Wednesday, December 08, 2010 9:35 AM To: [hidden email] Subject: Re: FRET using fixed cells ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** It is possible to do TIRF with fixed cells as long as you keep them in PBS so that the refractive index difference at the interface with the glass is appropriate for TIRF. Don't know about TIRF-FRET with fixed cells. I defer to Peri on that. Kate |
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In reply to this post by lgelman
Suspension cells could be also transfected while in suspensio
***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hy Syed, Suspension cells could be also transfected while in suspension state and immobilized on the coated coverglass immediately before imaging. Yes, antibody based FRET works but it is much harder to get impressive and accurate quantitative data with the antibody based FRET, as the FRET signal is usually weak. However, if you would be happy with the 'YES FRET' and 'NO FRET' readout, then the antibody based approach could be OK. Cheers, Vitaly +1-301-515-7833 ________________________________ From: "Gelman, Laurent" <[hidden email]> To: [hidden email] Sent: Tue, December 7, 2010 6:51:26 AM Subject: Re: FRET using fixed cells ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Syed, We used cells labeled with antibodies coupled to Alexas for FRET with success. The Alexa pairs we used were 488&555 and 568&633. Best regards, Laurent. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Syed Khundmiri Sent: vendredi 3 décembre 2010 16:17 To: [hidden email] Subject: FRET using fixed cells ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi all, I am a novice in the field. I use live cells transfected with tagged proteins for FRET and TIRF microscopy. My question is can one use fixed cells for FRET using AlexaFluor antibodies?? If yes, please guide me to some references and how to. Another question is has someone used TAT-fusion peptides labelled with AlexaFluor for TIRF and FRET?? Best Regards, Syed Khundmiri University of Louisville |
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