We work in V-bottom 96-well plates and wash by bringing the volume up to about 150 microliters per well, then spinning the plates at 1500 rpm for 1 min. in a benchtop centrifuge with plate carriers. This procedure gives a nice cell pellet concentrated at the bottom of the well, and it's easy to remove liquid with a multichannel pipette without disturbing the cells. We've also worked with round-bottom wells, but the cell pellet is more spread out and it's easier to remove cells by mistake. For most staining steps, we use 25 microliters of antibody solution and dilute this with PBS prior to the first spin, then do two washing steps with 150 microliters of PBS each. The force of the liquid coming out of the pipet is enough to resuspend the cells at each washing step. After removing the final wash buffer (but prior to adding the next antibody), we vortex the plate gently for a few seconds to loosen up the pellet and then add the antibody solution with a multichannel pipet, cover the plate, and vortex gently again. This is enough to resuspend the cells. We haven't tried aspirating with a multichannel vacuum system, but it should work with the V-bottom plate if the vacuum isn't too strong. Laird Bloom Phylos, Inc. 128 Spring St. Lexington, MA 02421 tel. (781) 862-6400 ext. 253 fax (781) 402-8813 www.phylos.com > ---------- > 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 > >
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