HeLa autofluorescence
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi we want to use adherent cells (ideally HeLa cells) for live imaging of viral particles traveling to the cell nucleus. We are having a lot of problems with autofluorescence in the Green/FITC channel. It is not simply diffuse autofluorescence but it has a clear morphology: green dots all over the cytoplasm. We are growing cells in phenol red free DMEM and it did not help all that much. Any suggestions? Thank you very much for your help. Caterina Strambio De Castillia Istitute of Research in Biomedicine Bellinzona Switzerland |
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Yes, We have experienced the same “phenotype” in HeLa cells – especially when they have been “marginally food deprived” or, more frequently, when using viral transfection systems that may adversly affect cell-health or pH homeostasis. These small particles have a peak absorption ~485nm and emit across a broad peak centered around 525-530nm. We found that dropping down to 477nm on our argon-ion line decreased this auto-fluorescence by a factor of 7. -Not enough to eliminate the signal, but enough to greatly attenuate it relative to the tag we were using. We also have another system that uses a 473nm laser (SS) for excitation that minimizes the green auto-fluorescence while still permitting use of a GFP/eGFP tag. I hope this helps. David Moore ------------------------- David S. Moore, M.S., Ph.D. Director Microscopy & Analytical Imaging Laboratory The University of Kansas 1045 Haworth Hall 1200 Sunnyside Avenue Lawrence, Kansas 66045 U.S.A Office: 785-864-4380 Lab: 785-864-4140 E-mail: [hidden email] Fax: 785-864-1815 From: Caterina Strambio <[hidden email]> Reply-To: Confocal Microscopy List <[hidden email]> Date: Fri, 26 Oct 2007 09:09:38 -0400 To: <[hidden email]> Subject: HeLa autofluorescence Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi we want to use adherent cells (ideally HeLa cells) for live imaging of viral particles traveling to the cell nucleus. We are having a lot of problems with autofluorescence in the Green/FITC channel. It is not simply diffuse autofluorescence but it has a clear morphology: green dots all over the cytoplasm. We are growing cells in phenol red free DMEM and it did not help all that much. Any suggestions? Thank you very much for your help. Caterina Strambio De Castillia Istitute of Research in Biomedicine Bellinzona Switzerland |
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In reply to this post by Caterina Strambio
Search the CONFOCAL archive at
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Hi. I'm not sure wether what I will say is relevant in your case, but I have had problem with autofluorescence in my cells while willing to use the FITC channel. This fluorescence is colocalised with mitochondria. I did a spectrum of it, and it matches with emission spectrum of FAD. This autofluorescence is greater in cells that have been in the dish for a while (probably due to oxidation, FAD is fluorescent in the oxidised state). I'm not sure that this case apply to your HeLa cells (I never worked with them), but I hope it will be of some help Best Sandrine Pouvreau Rush University Chicago, Il
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi we want to use adherent cells (ideally HeLa cells) for live imaging of viral particles traveling to the cell nucleus. We are having a lot of problems with autofluorescence in the Green/FITC channel. It is not simply diffuse autofluorescence but it has a clear morphology: green dots all over the cytoplasm. We are growing cells in phenol red free DMEM and it did not help all that much. Any suggestions? Thank you very much for your help. Caterina Strambio De Castillia Istitute of Research in Biomedicine Bellinzona Switzerland |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Thank you for you very useful comment.
I will keep your experience with FAD into close consideration as I think it is relevant with us. I have a lot of experience with imaging yeast and there too leaving cells on plates too long will increase autofluorescence. Thank you Caterina On 26-ott-07, at 17:07, Sandrine Pouvreau wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal |
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In reply to this post by Caterina Strambio
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Caterina, The autofluorescence you are observing is most likely flavin-related. Check out doi:10.1002/jemt.20497 Rajwa B et al. Single- and two-photon spectral imaging of intrinsic fluorescence of transformed human hepatocytes.Microsc Res Tech. 2007 Oct;70(10):869-79. I am afraid there is not much you can do, though. You may manipulate the redox conditions in the media environment, but it will most likely affect the overall viability of your cells. I do not agree with the previous posts stating that using the shorter wavelength helps. Actually, I was able to see more intense autofluorescence with 457 excitation than with 488 (for a given level of excitation power) for a wide range of cells. There were reports demonstrating that you can reduce the autofluorescence using media without riboflavin, for instance: Lowy RJ, Spring KR. Identification of riboflavin transport by MDCK cells using quantitative fluorescence video microscopy. J Membr Biol. 1990 Jul;117(1):91-9. However, I was unable to reproduce these results for HepG2. You may try with HeLa... Regards, Bartek Caterina Strambio wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi > we want to use adherent cells (ideally HeLa cells) for live imaging of viral > particles traveling to the cell nucleus. We are having a lot of problems > with autofluorescence in the Green/FITC channel. It is not simply diffuse > autofluorescence but it has a clear morphology: green dots all over the > cytoplasm. > > We are growing cells in phenol red free DMEM and it did not help all that much. > > Any suggestions? > > Thank you very much for your help. > > Caterina Strambio De Castillia > > Istitute of Research in Biomedicine > Bellinzona > Switzerland -- Bartlomiej Rajwa, PhD Purdue University Cytometry Laboratories Bindley Bioscience Center (BIND) 1203 W. State Street Purdue University, West Lafayette, IN 47907-2057 tel. (765) 494 0757 |
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