From: "April
Date: Tue Nov 26 1996 - 08:16:04 EST
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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