 |
 |
 |
 |
 |
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@samiot.co.za
|
|
|
|
|
|
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