> -----Original Message----- > From: Paul M. Kuon [SMTP:pmk3351@louisiana.edu] > Sent: Wednesday, 30 October 2002 2:58 AM > To: Cytometry Mailing List > Subject: Lymph node preps > > Paul, try syringing the cell suspension several times through a fine gauge > needle in RPMI 10% FCS. Wash several times (to remove DNA and other cell > debris that cause clumping) but always resuspend using syringing > technique. Hope this helps. > > Greg Hodge PhD > Senior Medical Scientist, > Flow Cytometry Laboratory > Haematology Department, > Women's and Children's Hospital > 72 King William Rd, > North Adelaide, > South Australia 5006 > > -------------------------------------------------------------------------- > ---------------- > > > > > We are attempting to isolate lymphocytes for Rhesus lymph nodes, but > have a > clumping problem. > > Currently, we grind the nodes in a sterile tissue grinder, filter > the cell > suspension to remove garbage, and then wash. Most of the cells clump after > the first centrifugation. We have tried several washing solutions (PBS > with > and without albumin, with and without EDTA, RPMI with and without albumin > etc) and multiple RCF and the clumping always occurs. The procedure > requires > the washing steps so we are left in a bind. > > Any help or recommendations will be greatly appreciated. > > Paul Kuon MT(ASCP) > Research Associate > University of Louisiana Lafayette NIRC
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