Re: CD34-which antibody post bead selection?
(no name) ((no email))
26 Nov 96 9:16:04 EDT
We were involved in a clinical trial using the Dynabeads and the Baxter Isolex
selection system for bone marrow and peripheral blood and found no problem with
identifying the CD34+ cells using a second class III antibody (8G12) from
Becton Dickinson, FITC conjugated (for other studies we use PE and PerCP
conjugated with similar results).
1. If not all of the beads are removed the CD34+ cells will fall outside the
'normal' light scatter region and show up more in the monocyte region.
2. The high background you are experiencing may be attributable to the use of
a tissue culture medium as a wash solution and diluent, depending on the
components (ie, flavins, phenol red) could be increasing the autofluoresce. We
use PBS (w/o phenol red, divalent ions) supplemented with !% (v/v)goat serum
and 0.1%(w/v) sodium azide.
3. You only mentioned volumes of cells and antibody, have you titrated the
antibody that you are using against a cell concentration? Using the BD
8G12-FITC, we are using 500ng mAb against 10^5-10^6 nCells.
4. If you are not using sodium azide and incubating the reaction at room
temperature, capping is possibly occurring and will decrease the expression of
the CD34 as it is no longer distributed evenly along the membrane. We use
0.1%(w/v)sodium azide and incubate the reaction in wet ice or 4oC for 30min.
5. We also set up two negative controls : autofluorescence and a protein
concentration fluorochrome matched isotype. Our backgrounds are within the
first log of fluorescence and the FITC positive cells have a mean fluorescence
of 100 (FITC, PerCP) and 750 (PE).
April G. Durett
Technology Transfer - Flow Cytometry
Blood & Marrow Transplantation
University of Texas MD Anderson Cancer Center
adurett@notes.mdacc.tmc.edu
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