CD34-which antibody to use post bead selection?

Bowers, D, Desiree, Mrs DB at samiot.uct.ac.za
Tue Nov 26 09:03:01 EST 1996


Dear flow users,

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

Selection is performed using the magnetic Dynabeads using the 
recommended procedures. The beads are coated wirtyh monoclonal 
antibody specific for the class III epitope of the CD34 antigen. Once 
selected, the Detachabead is employed to remove tyhe 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 antobody.

Procedure : 100ul cells + 10ul antibody
            Incubate 30 min R.T
            Wash x 2 with tissue culture medium (TCM)
            Resuspend in 0.4 ml TCM
            Analyse within 2 hours
            
Problems:

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

Is it possible that by using 2 antibodies directed at the same 
epoitopes on the Cd34 antigen, the second antigen-antibody reaction 
is not occurring due to the binding sites already having been used 
once?

Does anyone have any ideas on this problem or on the origin of the 
background fluorescence? Any suggestions would be most welcome.

Thanking you,
Desiree Bowers

e-mail: db at samiot.co.za       
                    








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