In that case, would using a nuclease be a problem? -Adrian ----- Original Message ----- From: "Hodge, Greg (HAEM)" <hodgeg@mail.wch.sa.gov.au> To: cyto-inbox Sent: Wednesday, October 30, 2002 3:02 PM Subject: RE: Lymph node preps > > > > > -----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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