To the Monocyte Mavens out there: I'm looking for advice on working w/monocytes. We are trying to culture PBMC's (RPMI,10%NCS) w/various hormones Tx's. I'm harvesting cells @ 24/48/72 hr post culture for flow analysis. The lymphocytes are not much of a problem -but the monocytes have proven a real challenge. I'm using an EDTA-based removal media. I rinse the wells (24well plates) with Ca/Mg-free buffer and add500ul "cell-stripper" (Mediatech), wait a few minutes and collect the cells. Wash in Ca/Mg-free PBS/2%NCS/0.02% w/azide, then stain on ice (20min). Wash again, fix (0.5% EM-grade formaldehyde), run flow analysis anywhere from 1hr to 36hr post collection/staining. The Ab mixes are CD14/11b/80/86 (for the mono's) and CD3/4/8/40L(lymphs). The lymphs look great (on the cytometer) so I'm assuming the incubation timing,temp, washing,etc is fine. Ok,so these are the problems: 1) Although I can see a fairly tight lymph population (SSC/FSC), the rest of the cells are one Big Galaxy. Are monocytes cultured over 24hr typically very spread out? I cannot seem to find any area to gate via scatter. When backgating on CD3 I also notice the lymphs have a subset of cells that are also spread all over the window- so I have mono's and lymphs sharing the same scatter area. 2) I tried gating CD14 vs SSC and CD14 vs FSC. The CD14 staining is much dimmer than I'm used to in lysed preps. (I've read that CD14 can be decreased on stim'd monos). Since some lymphs pick up a bit of anti-CD14, this is not very clean either. (Is there a more appropriate monocyte marker that does NOT stain lymph's?) To muddy the water even more, the CD14+ cells are very similiar to the lymphs in FSC. I can get a slight separation via 14/SSC, but it's not much to crow about. 3) I've been combing Pubmed/google/ Current protocols/everything except the phone book for info on culturing mono's. I can't find any "hints" for this type of culture. (Most cultures seems to be around 6hr for cytokines analysis, or >7days with IL4 and such added; and seem to be mostly interested in culture sup's not the cells ). So for 24-72hr do these cells REQUIRE cytokines & such to be happy? (We'd like to observe the effect of the hormones and were hoping extra supplements would not be required; but if the cells aren't happy this may not be the way to go) And is the EDTA method best to harvest mono's you want to flow analyze? 4) It also seems to be true that I'm recovering very few monocytes. (although hard to say since I'm not sure WHAT's coming off). Going into culture the PBMC prep is about 75-85% CD3+ (depends on patient and TX), coming out the cells suspension is >90% CD3+. I monitor the cell removal via microscope-no cells left in the well. Can anyone pass along any hints/trade secrets/ advice of any type on how to troubleshoot this mess? I'm really losing what's left of my sanity here.... Thanks bunny Bunny Cotleur, M.S. Sr. Technologist Cleveland Clinic Foundation Neurosciences NC30 9500 Euclid Avenue Cleveland, OH 44195 cotleur@ccf.org (216) 444-1164 fax (216) 444-7197
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