Re: staining of cord blood CD34+ cells

(no name) ((no email))
22 Nov 96 16:43:15 EDT

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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