Doug- I stain in 96well plates routinely. I use Fisherbrand polypro/u-bottom that holds 300ul. I wash using 280uL, spin at 500g for 5min, then "flick" the supe into the sink. The one step I experimented with (alot!) was centrifuging: how fast+how long= best recovery. (This step you'll need to work out for your centrifuge). In my hands, I have much better luck flicking than aspirating. I experience very little cell loss. (*I compared to staining the same volume in a 1.7ml microfuge tube/aspirating sup; and found I have similiar cell recovery). Typically I stain 95uL whole blood/well; and my lysed/fixed sample is resuspended in 200ul for analysis. Good luck! ---------- > From: Reed, Doug S Dr USAMRIID > Sent: Monday, December 10, 2001 3:53 PM > To: Cytometry Mailing List > Subject: multiwell autosampler - washing > > We've just gotten in our multiwell autosampler and I'm looking for some > advice from those who have been using them for a while. I don't (at this > point) need any help with the system itself, but what I am interested in > is how people are washing the cells in the plates. Do you do it manually > or are you washing with an ELISA washer. And how do you get rid of the > supernatant between washes? > > Thanks! > > Doug > > Douglas S. Reed, Ph.D. > Principal Investigator > Respiratory and Mucosal Immunity > Department of Aerobiology & Product Evaluation > Division of Toxinology & Aerobiology > U.S. Army Medical Research Institute of Infectious Diseases > 1425 Porter St. Ft. Detrick > Frederick, MD 21702-5011 > 301-619-6728 > 301-619-6911 (Fax) > doug.reed@det.amedd.army.mil -- Bunny Cotleur Cleveland Clinic Foundation Neurosciences NC30 9500 Euclid Avenue Cleveland, OH 44195 (216) 444-1164 cotleua@ccf.org
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