I am working with whole blood and conjugating the WBC with CD4 FITC. I am using the PBS buffer containing 2mM EDTA (pH 7.2).
However, I m not getting the result. I found that CD4FITC works well at pH 9. So, I want to know is it possible to work at pH 9 with WBC in whole blood sample?
If, it have effect on RBC and Platelets, then it will not affect my experiment goal.
The anti CD4 FITC should work well at neutral pH also. How are you trying to observe labeling? There are a lot of rbc there so it might be hard to even find the wbc you are looking for. We routinely stain CD4+ T cells using PBS, 2 mM EDTA, pH 7.2 so it isn't a pH problem.
Dear Anne lodge
I am staining the CD4 with antiCD4 FITC antibody but not getting the good fluorescense. Actually i am working On whole blood. First I am invubating 10 ul of blood with. RBC lysis buffer for 5 min and then adding the antibody and make up the volume with PBS 2 mM EDTA in such a way that 200 ul contains 1 ul of whole bllod. However at natural pH 7.2 I am not getting the number of fluorescense.
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