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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:
Thankyou
Denise Janssens
Dept Haematology
UCT, Med Shool
Cape Town
South Africa
Yes,
The detachabead procedure removes the *bead from the antibody* - leaving the
antibody still attached to the antigen on the cell. This first antibody will then
effectively block your second antibody from binding thus giving you a false
negative result.
Peter Chapple
Senior Scientist - Pathology
Peter MacCallum Cancer Institute
Melbourne AUSTRALIA
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web