Rick, Instead of worrying about isolating cells of interest, why don't you utilize your cytometer as it was designed; to analyze large numbers of cells rapidly. I don't mean that in a derogatory sense, but just to awaken you to what the hardware can do. We've all been lulled into thinking that 5000-10000 events per sample is a maximum, and we have to perform enrichment steps prior to using the cytometer to make sure this holds true. The only caveats are time and reagents needed (since you'll likely have a larger volume of cell suspension, you'll need more antibody). Prep that sample and acquire 1x10^6 events. Acquire 5x10^6 events. Just let it run till you're sure you have the necessary data. It takes less time to do the correct experiment once than to do the wrong experiment multiple times. I would suggest filtering your suspension after your collagenase digestion, then using an anti-CD45 antibody to resolve the leukocytes from the surrounding tissues. And as another note, some component(s) of commercial collagenase preparations is quite efficient at cleaving certain antigens from the cell surface (it's not trypsin). We found almost everything was affected to some degree, but CD4 appeared to be the worst. Limiting the collagenase digest to 30 minutes and transferring immediately to ice cold, EDTA containing buffer gave the best results in our hands. I list that as an example of how any handling or processing of the cell suspension has the potential for altering antigen expression. This is a big reason why I like to make a single cell suspension and analyze the whole thing. Good luck. Kb -- Science is built with facts as a house is with stones--but a collection of facts is no more a science than a heap of stones is a house. -Jules Henry Poincare (1854-1912) Keith Bahjat, PhD Applications Engineering Manager Cytomation, Inc Fort Collins, CO (970) 226-2200 x223 keithb@cytomation.com on 4/2/02 7:52 AM, Stojan Dimitrov at stoianski_fl@yahoo.com wrote: > > Dear Rick, > Why dont you try using the MACS system. > http://www.miltenyibiotec.com/index.php?site=products-human > Using anti-CD45 magnetic MicroBeads (for leucocytes) > you might separate all cells of hematopoietic origin > except erythrocytes, platelets and their precursor > cells. > Yours, > Stoyan > > --- Rick Arthur Bright <rbright@emory.edu> wrote: >> >> I am trying to analyze lymphocytes, monocytes, >> granulocytes, etc from >> brain and lung tissues. After collagenase digestion >> I, of course, have a >> lot of junk...stuff I don't want to run through the >> instrument. >> >> What is the best way of separating the cells from >> the junk? I tried >> Ficoll-paque, which nets me the lymphocytes, but I >> lose other cells types >> that might be important. >> >> Would a Percoll gradient be a better route to go? >> What gradients would be >> most appropriate. I am digging in the literature as >> well, finding many >> reference to percoll gradients, but few of them say >> what the gradients are >> and where the cells of interest will settle. >> >> Any quick thoughts would be helpful. I, hopefully, >> will stumble upon that >> magic reference sometime tonight, but if you know >> already I would >> appreciate your help. >> >> Thanks >> >> Rick >> >> >> > > > __________________________________________________ > Do You Yahoo!? > Yahoo! Tax Center - online filing with TurboTax > http://http://taxes.yahoo.com/ >
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