From: "April
Date: Fri Nov 22 1996 - 15:43:15 EST
We are not experiencing the background problems with either CB, marrow, or peripheral blood evaluation of CD34. Our backgrounds are usually 0-0.05% (10,000 total or 50,000 live gate acquisition) with FITC, PE, or PerCP conjugated flourochromes. Our assay volume is 150-200uL containing titrated range 10^5 - 10^6 cells and titrated CD34, diluent is PBS+1%(v/v)goat serum +0.1%(w/v)sodium azide, incubation 30min /wet ice/dark. We set up two negative controls : autofluorescence and protein concentration matched isotype control. We are using an amonium chloride lyse buffer, 3-4mL/15min/room temp/dark, sediment cells 800g/5min and wash with 3-4mL of the PBS solution. A source of the high non-specific binding may be related to the type of tube that your are using; we use minisorp tubes from Nunc Inter-Med Inc (Denmark) as they are a polyethylene specially treated to minimize protein adsorption onto the plastic and subsequent leaching during cell washing. April G. Durett Technology Transfer - Flow Cytometry Blood & Marrow Transplantation Univ Texas MD Anderson Cancer Center adurett@notes.mdacc.tmc.edu@internet To: cyto-inbox cc: From: yagrawal @ messi.uku.fi (Yashpal Agrawal) @ INTERNET Date: 11/21/96 03:52:12 PM Subject: staining of cord blood CD34+ cells Dear flowusers, Problem:I have been having problems with CD34 cell staining ofwhole Cord Blood(CB). Specifically the Isotype control (IgG1-PE from B.D) gives non-specific staining (0.2-0.6 % of mononuclear cells). This small amount of non-specific staining causes difficulty in interpretation when the CB stained with the specific HPCA-2-PE (also from BD) gives a positivity in 0.5 % of cells and subtracting the background gives 0% positivity. Do others have the same problem, any suggestions/advice is welcome ?. Methods used:We collect our CB in blood bags (CPDA) and the sample is stored at 4 C until analyzed which is 1-4 hrs. Staining is according to standard methods. 50 ul of CB + 20 ul antibody is incubated for 15 min in cold and in dark. After this 2 ml FACSlysis solution (BD) is added, incubated for 10 min and centrifuged for 5 min at 400 g. The cells are then washed twice in PBS and analyzed immediately. Thanking you all, Yash Pal Agrawal Kuopio, Finland yagrawal@messi.uku.fi
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