Hi all, I'm having some problems with a protocol design to phenotype CD8+ specific populations. Briefly, frozen PBMC were thawed, washed and incubated with a specific pentamer. After washing, the cells were stained with the following antibody cocktail: - CD3-FITC - Fluorotag-PE (for labelling the pentamers) - CCR7-PECy7 - CD8-AmCyan - CD45RA-Pacific Blue After washing twice, cells were then fixed with freshly prepared PBS + 1% FCS + 2.5% PFA (pH adjusted to 7.45). The analysis was performed within 2 hours, initially in a CyAn ADP and also in a FACSAria. The problem is that I cannot detect any positive signal with the AmCyan- and the Pacific Blue-conjugated antibodies! In single stained control tubes, even after adjusting the voltage and gain, there was no difference from the unstained control. Interestingly, with the CD8-AmCyan fluorochrome, I could detect a positive signal in the fluorescence channel used for FITC, but no signal at all in the proper channel for this fluorochrome. I don't have any idea why this isn't working, and due to the very small time delay from staining and analysing the samples, it's somewhat unlikely that this effect is due to the fixation step - not essential for this particular antibody panel, but really important for my forthcoming experiments. I would really appreciate any information that could help me to overcome these issues. Regards, André. ------------------------------------------ André Oliveira, MD, MSc Centre for Research in Infectious Diseases University College Dublin Belfield, Dublin 5 Ireland P: 353 1 716 1229 F: 353 1 716 1239 andre.oliveira@ucd.ieReceived on Wed Jul 25 12:58:00 2007
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