CD34-which antibody post bead selection?

Janssens, D, Denise, Mrs DJ at samiot.uct.ac.za
Mon Nov 25 12:05:53 EST 1996


Dear flow users,

We are currently selecting CD34+ cells from the non adherent, T-cell 
depleted mononuclear population of bone marrow and cord bloods and we 
are experiencing problems with the subsequent flow cytometric 
analysis.

Selection is performed using magnetic Dynabeads with the 
recommended standard procedures. The beads are coated with monoclonal 
antibody specific for a class III epitope on the CD34 antigen. Once 
selected, the Detachabead is employed to remove the beads from the 
CD34 positive cells.

The cells are then set up for flow cytometry analysis using the Dako 
CD34-FITC (conjugated), which is also a class III epitope antibody.

Procedure: 100ul cells + 10ul antibody
           Incubate 30 min R.T.
           Wash x 2 with tissue culture medium (TCM)
           Resuspend in 0,4ml TCM.
           
Problems:

1. No CD34 positivity being picked up at all
2. Background debris at the end of the scale (betw channel 100 and 
1000 right at the end), making the isotypic control impossible to 
set within acceptable limits.      

Is it possible that by using 2 antibodies directed at the same 
epitopes on the CD34 antigen, the second antigen-antibody reaction 
is not occurring due to the binding sites already having been used 
once?

If anyone has any ideas on this problem or on the origin of the 
background fluorescence, please e-mail to the following address:

dj at samiot.uct.co.za

Thankyou
Denise Janssens
Dept Haematology
UCT, Med Shool
Cape Town
South Africa






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