Hello, I use CD138(BD, clone 281-2) to detect plasma cells in flowcytometry, but I could hardly find any positive cells(see details in the figure)? Was there something wrong or I just ignored?
I can detect GC formation and high specific antibody titer in serum 7d after immunization, but I just can't find plasma cells, it really puzzled me.
Any suggestions will be greatly appreciated.
I guess your gating is not in the right area. As expected, no B220 increase but there is increase in CD138 in immunized set. For calculation purpose you may set the gate in the area where a shift occurs on right side (after the arrow head of CD138, towards right: meaning, just shift your current gate towards left without changing Y-axis) and subtract the unimmunized cell number from immunized cell number within gate. However, for sorting purpose you need to be very stringent (which will usually be low in number, as the purity goes high).
Thanks, Goodwin, It may be helpful. In the figure, it showed much more dots in immuzied set, so in fact, the percentage or absolute number of CD138 positive cells has no difference between two groups. I have tried several Ab staining concentration, but it is no use, I'm really going crazy
Also I would recommend to have single staining to make sure CD138 positivity alone doesn't shift cells on Y-axis+X-axis. Some times it can happen. In such instances, you have plenty of cells. You need a CD138 alone control to make sure that.
Dear Zorro,
If you work with human plasma cells, my suggestion to you will be to test your anti-CD138 monoclonal antibody with any positive control specimen (plasma cell leukemia specimen or commercial plasma cell line). From my Laboratory experience, I could recommend to you anti-CD138-PE (Clone B-B4, isotype: mouse IgG1) produced by Miltenyi Biotec Inc. (order 130-081-301). It is the best of all anti-CD138 monoclonals which we used in our Laboratory. Other monoclonals which we used before (BD/Pharmingen CD138-PE, clone DL-101; BD CD138-PE, clone MI15) cannot compare to it.
Sincerely,
Nada Kraguljac Kurtović, BSc mol. biology, MSc
Specialist in Immunobiology
Laboratory for immunophenotyping and flow cytometry
Diagnostic Department, Clinic of Hematology, Clinical Center of Serbia
Belgrade, R. Serbia
Hello,Nada, I work with mice spleen samples, clone 281-2 are mostly used. Any way, thanks very much for your suggesstions, you are really kindhearted.
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