PLEASE! Do NOT reply to the List regarding this email! Reply to me directly. We have been struggling with a staining "artifact" for a while now, and I have run out of good ideas of how to proceed. I'd like to put this problem to the community, to see if we can solve it together. If you're not interested in a puzzle (which will be fairly lengthy), nor in immunophenotyping, then please skip this email. This is not a weighty problem, but it is perplexing and my hope is that the answer may ultimately teach us something interesting. Not only that, but it may also be important for laboratories who use FITC CD8 in their experiments! What I propose is to lay out the problem as we discovered it, and then summarize some of the ways in which we have tried to solve it. Then I would like to solicit suggestions for what is going on and how to answer the question... I will collect suggestions for a while, and, every so often, take the suggestions to the lab test the various ideas that come in. Think of it as doing experiments by email. Your brain, our hands. Your nutty ideas, our reagents. By the way, I would be grateful if anyone out there would reproduce our artifact. At least it would let me think that I haven't lost all my marbles. I will post the results of intermediate experiments (and/or the final answer), giving full credit to those who have made suggestions that were tested. Of course, it's also possible that I have missed something completely simple, and I will be inundated with emails pointing out an obvious solution... in which case this enigma will not be very interesting and will die a quiet death (as would my ego). Or maybe someone already knows the answer. Maybe it's even been published. But on the off-chance that there is an interesting story to this, I thought it might be fun to include the larger cytometry community in trying to track down an answer that has eluded us for several experiments. PLEASE! Do NOT reply to the List with your suggestions or comments to this problem! Let's not clog up this wonderful email listserv. Rather, I will summarize suggestions etc. and post them later in a single email. (Particularly since many people are likely to come up with similar suggestions.) Also, I want to encourage everyone who has an idea to send it to me--no matter how mundane you think it is. We've already tried some things that I would have never considered doing, simply out of frustration! OK... here's the problem. (If the figure below doesn't come out on your email, you can view most of this information at <http://www.drmr.com/CD8Enigma/CD8Story.htm>.) Our common compensation controls for years have been PBMC stained with anti-CD8 conjugated to every color that we use. Over the years, we have noticed at various times that the percentage of CD8 T cells that we get for any given PBMC sample varied according to the conjugate. Recently, we decided to explore this issue when we found a sample for which this was particularly bad. Specifically, we noticed that the fraction of CD8 cells revealed by FITC- or Alexa594-conjugated CD8 was much lower than that when stained with APC- or PE-conjugated of CD8. We did 2 or 3 experiments trying to understand where the cells went before checking the obvious in the FITC or Alexa-stained samples, a significant fraction of the CD8+ cells had "migrated" into the high-side scatter region, moving out of the lymphocyte gate. We hadn't lost the cells, we were simply gating them out. (See Figure below). The next experiment (co-staining with CD14) showed that these cells were not simply aggregating randomly or even with each other, but rather specifically with myeloid populations. (See Figure below). We have now done a few more experiments testing various conditions. An early experiment (before we knew about the "migration" out of the lymphocyte gate) tested the hypothesis that the cells were aggregating and were either lost during the centrifugation step, didn't enter the FACS tubing, or were lysed. So, we stained and immediately ran on the cytometery (no washing). We found there was little loss of cells. After we discovered the migration effect, we confirmed this finding by showing that only upon centrifugation did the cells appear in the monocyte gate. If you can do an experiment to confirm this, try the following. Stain different donor PBMC with FITC CD8 or PE CD8 (a two tube experiment!). Do you see CD8+ events in the high-side scatter domain with FITC CD8 but not PE CD8? The next experiment tested whether or not these cells were randomly aggregating. We costained with myeloid markers, and found that the migrated CD8+ cells were "apparently" CD14+; indicating an association with monocytes that survives the fluidics of the cytometer. It also indicated that this was no random association; it was specific with myeloid cells. We tested whether the association is dependent on temperatur was e of staining (ice vs. room temperature) or the presence of sodium azide. It is not. It does not appear to happen with antibodies to CD4 or CD19; however, these are not as bright so it is not definitive. CD4 is also expressed on monocytes, complicating this analysis. In any case, the association clearly did not occur using an antibody to CD8beta (conjugated to Alexa 594). Things we know about the "association" process (the CD8 cells co-mingling with large side-scattering cells). (1) It happens with unconjugated CD8, FITC CD8, Cascade Blue CD8, Alexa 594 CD8. It does not happen with PE CD8, Cy5PE CD8, APC CD8, Cy7APC CD8. (Note that it happens with small-molecule conjugates, and not with large-protein conjugates). (2) The effect is "dominant": i.e., costaining with subsaturating amounts of Alexa 594 CD8 and APC CD8 causes the association. (Even small amounts of Alexa 594 CD8 can cause it to happen, in a dose-dependent fashion.) (3) It happens with at least 3 clones of CD8 (OKT8, plus some commercial clones). It happens with commercial conjugates from multiple vendors and our own home-made conjugates. (4) It does not happen with antibodies to CD8beta nor with antibodies to CD4 or CD19. (5) It happens equally at room temperature and on ice and in the presence of azide. (6) It is minimal until cells are washed (by centrifugation); it does not appear to get much worse with multiple centrifugations. (7) Including excess unconjugated CD8 during the wash does not prevent it. (8) Most (but not all) of the high-side scatter CD8+ events are CD14+. (9) The extent of the effect is donor-dependent. In some donors, it is a relatively small fraction; in others, it can be a huge fraction of the CD8 T cells. In the figure below, approximately 30% of the CD8's "migrate." (10) Note that it still occurs to a small extent with APC-conjugated CD8. However, it is considerably less than with the small-molecule conjugates. (11) The effect is not seen with CD8-dull (CD8a/a) T cells, only with CD8-bright (CD8a/b) T cells. (Note, this is observation is impossible to discern from the graphs below--but it is in fact true). Figure: A single sample of PBMC was stained with FITC CD14 and either with Alexa594 CD8 (left panels) or APC CD8 (right oabeks). Top panels, ungated. Bottom panels, CD8+ gated.
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