Rick, Your problems are several layers deep here. First, Balb/c mice are NK1.1-, meaning they don't express the gene that leads to production of surface bound NK1.1, so no titration of PK136 will give you positive staining. Try using B6 mice instead. Second, if you are losing PerCP staining, you are either losing the antibody from the cells or destroying the fluorochrome. What kind of buffer are you storing those cells in? Have you checked the pH? I routinely store PerCP stained mouse cells 2-3 days with no problems. The formaldehyde solution you use (once it's in solution, it is formaldehyde, NOT paraformaldehyde) should have a pH between 7-7.4. Make sure you check this each time you make it. Also, if you are making fresh formaldehyde from paraformaldehyde polymer, it has to be made fresh each day, as this is very unstable and will turn to formic acid quite rapidly, leaving you with a non-fixing solution of pH 3-4. Third, looking for upregulation of CD95 will give you information about leukocyte activation, but tells you nothing about whether those cells are apoptotic or not. The events that lead to Fas mediated apoptosis are mostly intracellular, and related to phosphorylation and removal of the FLIP molecule from the intracellular domain of Fas. In fact, the amount of Fas on the surface of a lymphocyte has NO correlation with it's potential to undergo activation induced cell death. If I understand your project here, you are looking for apoptosis in NK cells. You need to be using markers that will identify NK cells and apoptotic cells in the strain of interest. I'd hit Pubmed and devise a better strategy before buying more reagents. Good luck. Kb ---------- Keith Bahjat Graduate Assistant University of Florida College of Medicine Gainesville, FL kbahjat@ufl.edu on 4/18/01 12:08 AM, Rick A. Bright at rbright@emory.edu wrote: > > 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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