Thank you all for responding so quickly to my issues. In summary, I have learned that I did not read the PharMingen catalog closely enough to know that the NK1.1 is NOT expressed in BALB/c mice; I should have used the DX5 Pan-NK mAB instead (it should be here today to use in the last timepoint of this expt.). It has also been recommended that I use BSA instead of FBS in my FACS Buffer due to the variability in FBS reactivity and between Lots of FBS. A few of you suggested that I use 2% paraformaldehyde in my fixing solution and to make it fresh daily. I have heard mixed reviews on the stability of this buffer. Some say that they make a lot, keep it in an amber bottle at 4degrees and it keeps for weeks. I have been making it fresh weekly and storing it at 4 degrees in an amber bottle. I still don't have a consensus on this matter. There are additional thoughts on the fact that I should not be using CD95 (Fas) and an indicator for cells undergoing apoptosis. I am looking more closely into this. Thank you for your great help. I received many rapid responses and they were in time to make a quick change and not lose valuable time and data. Rick -----Original Message----- From: Rick A. Bright [mailto:rbright@emory.edu] Sent: Wednesday, April 18, 2001 12:08 AM To: cyto-inbox Subject: Mouse NK1.1 cells in peripheral blood First, I want to thank the many of you who wrote to help me figure out my first FACS protocol. It is working very nicely and only took me one failed attempts (which wasn't a failure at all because I learned from the several mistakes), and has worked ever since. I am having a couple of problems overall that I hope someone can help me resolve. To remind everyone: I am collecting whole blood from female BALB/c mice (7-8 w.o.) and using 50ul to determine the blood cell count and look for an upregulation of an apoptotic marker, CD95 (Fas). My tubes contain (all PharMingen): 1) CD3-PerCP, CD4-PE, CD8-APC, CD95-FITC; 2)CD3-PerCP, CD19-PE, Ly-6G(Neuts)-APC, NK1.1-FITC; and 3) CD3-PerCp, CD19-PE, CD11b(Mac1)-APC, CD95-FITC I also use 5ul of Fc Block in each tube for 5 minutes prior to adding the antibodies and I use the BD FACSlyse buffer (2mL for 7-10min at rt). I keep everything in the dark at all times and besides the lyse step, all is done on ice. I have PBS, FBS and Azide in my FACS buffer and I fix the cells in a 500ul vol of 1% paraformaldehyde solution after 2 washes with FACS buffer (I do not wait o/n to read the cells, however, I read right away). First issue: I tend to lose the CD3-PerCp color rather quickly, especially on the double-positive with CD4-PE. Sometimes all of the CD4 are also CD3+ and sometimes they sit down on the PE axis. However, the CD3-PerCp/CD8-APC seem to be more stable as double-positives. Once, I read the cells immediately and all of the CD4+ were also CD3+, the next day, I checked again and the same #/% of CD4+ cells were collected, but they were not double pos for CD3 and were back down to the CD4 axis. Second issue: I cannot find any NK cells. I know that they should fall into the area that is slightly larger and slightly more granular than lymphocytes and I have read that the population can be as high as 5-10% of peripheral blood cells. Does this sound right? Is anyone currently looking at NK cells in mice? I am using the NK1.1 antibody from PharMingen at a 1:10 dilution, using 25 ul of the diluted antibodies in each tube with 50ul of whole blood and staining for 30min on ice. I check everywhere, even without any gates and see nothing pop up in the LR quad of my FL1 channel. Third issue (never say final, right?): When collecting events, I have taken off all thresholds to collect in the full window. However, as you know, I get many junk events that tends to quickly reduce the number of "good" events that I want. So, instead of setting thresholds (in case my cells change during apoptosis or other forms of death and are not rejects behind the threshold), I gated a region that goes all the way around the perimeter of the window and makes an inward loop over the xy point of axis. I still acquire and save all events, but have the count go up to 10000 events within the gated region (which omits that massive clump of debris at the axes. So, in a way, I am trying to collect 10K events, excluding the debris, but still collecting data on the debris. This brought my valid lymphocytes from a count of 800 to a count of thousands. Any thoughts on each or any of these questions would be most helpful and appreciated. I am very grateful for the expertise among this membership and will soon be in a better position to cross to the other side and give as much advise as I now borrow. Thank you. Rick
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