Hi
I was trying to separate my eosinophil population from neutrophil by using a BD CD193 (CCR3)-BV510. But it showing positive expression for all of my granulocytes. What could be the reason for this? I am using this with other antibodies such as CD11c, CD11b, CD88, CD54, CD16 and CD62L. Last time I used this with only CD16 it worked very well and was showing response in only eosinophils and not on neutrophils but now with all other antibodies it showing positive response for all granulocyte population.
I am attaching the flowcytometry dot plot of unstained cells and CCR3 positive cells.
Regards
Ravi
CD 16 may be specific to eosinophils but others markers you have used may not be specific to eosinophils. Can you try CDs specific to eosinophil only?
CCR3 I tried is specific for eosinophil only but the issue is it showing positive response for neutrophils also, I want to know the reason why it is showing positive for neutrophils?
Hi Ravi,
I have worked with Eosinophil and flow cytometry for about 10 years.
Have never seen anything similar. Could it be unspecific bindkng? Have you stained whole blood or Granulocytes? Gated on SSC high cells?
Neutrophils don't have CCR3. Only Eosinophil, Basophils and a small population of Lymphocytes. I usually stain wole blood and gate Eosinophil as high SSC/CCR3+. Activated Eosinophil may up regulate CD16, but Neutrophils have much higher expression.
Hi Kerstin
I used granulocyte population that I isolated from whole blood and then stained them, I am also not able to understand that what is the reason for this, the antibody is CCR3-BV510, but last time I recorded in FACS Canto 2 in AmCyan channel it worked very well only showing positive for eosinophils but after moving my experiment to BD fortessa I got BV510 channel which is specific for the above fluorochrome and it was then that things started getting wrong, may be I try again in AmCYan channel.