I usually use flow-check or flow-set beads from Beckman-Coulter ( they are at 1 X 10.6/mL). I put 10 uL in 1mL of cell suspension, I stop the analysis on a 1000 beads ( equivalent to 100 uL of cell suspension), I just have to multiply by 10 the number of cells ( in a specific gate) and I have the cell concentration per mL. You just have to be carefull for cell aggregats, that is the same problem for any counting techniques Put aside a vial of beads for that specific purpose, vortex it well before using and pay attention to tiny air bubbles when pipetting ( specially following the vortexing ). Lucie Bourget Flowcytometry Biotechnology Research Institute National Research Council Canada Montréal, QC, H4P 2R2 Tel: office 514-496-6274 lab 514-496-6330 Fax: 514-496-5143 -----Original Message----- From: Arnold Pizzey [mailto:a.pizzey@ucl.ac.uk] Sent: Thursday, May 11, 2000 4:31 AM To: Cytometry Mailing List Subject: True count substitute At 09:21 09/05/00 -0400, you wrote: > > > >Dear Flowers, > >BD does not make the TruCount tubes available without buying it with their kits >(that we do not need). Does anyone know a similar product for cell counting on a >flow cytometer? > >Thanks, > >Akos Szilvasi >Biogen, Cambridge, MA > > > > > Greetings Akos, I have been using the following method to obtain absolute counts by flow; 1) centrifuge 5ml of Beckman-Coulter Flow-check Fluorospheres 400g 10mins. 2) Discard supernatent and resuspend in 5ml fetal bovine serum 3) centrifuge as above and resuspend in 5ml fetal bovine serum 4) Count beads by haemocytometer (the hard part - I count around 1000 beads) 5) Add beads to sample and analyse by flow. regards, Arnold _/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/ Arnold Richard Pizzey Department of Haematology Royal Free and University College London Medical School 98 Chenies Mews London WC1E 6HX U.K voice: +44 020-7679-6234 Fax: +44 020-7679-6222 email: a.pizzey@ucl.ac.uk _/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/
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