Hello everyone. I have begun on analyzing platelets using a flow cytometry. As I have never done analysis of this cell type, can you please help me out.
For now I am using markers CD61 FITC/BV510, PAC-1 FITC, CD62P PE (10 micro liters each marker), currently ADP (10 micro liters) is going to be used as an activator, and whole blood (5 micro liters).
On my last analysis I used CD14 APC (monocytes), CD61 FITC (platelets) plus whole blood (no ADP) to see a clear population of platelets. This was achieved and a clear population was seen, however once ADP was introduced I was not able to see a clear population of activated platelets.
Should I use less ADP (perhaps 5 micro liters), less incubation (from 10 min to 5 min), increase the staining buffer ?
Thank you in advance
Completely agree with Waltraud C Schrottmaier comment.
The concentration of stimulant matter a lot. At higher concentration the stimulants become cytotoxic. You must titrate the amount of antibodies in different assays. Most of the antibodies works well in 1:10 to 1:50 dilution of recommended concentration.