Hi Liliana,

1. Make your titration with activated platelets, normally you will not see any positive result for CD62P on unactivated platelets. but because you are using PRP there will be low level of activation due to centrifugation and preparation phase.

2. Your dilutions seemed quite high to me for antibodies to be used on flow, are you using a primary antibody? In this case, our platelets will be even more stimulated while you are preparing for secondary antibody. You can decide through the amount that does not effect the staining index, best is to make a lineer graph to see where you obtain the same results (mean/median channel) with suggested quantity.

3. To use geometric mean or median depends on the cytometry you are measuring, but as long as you are using the same parameter for all your data in this research you will be fine. The best way to present your data would be following MiCyt Guidelines available at below links:

• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2773297/
• http://onlinelibrary.wiley.com/doi/10.1002/0471142956.cy1018s61/full

With best wishes,

Hi Gulderen Yanikkaya Demirel thank you so much for your answer.

So the optimal antibody dilution for activated platelets can be used for unstimulated cells?

To select the optimal dilutions, I 've been told that I should look for the maximal separation between specific fluorescence of unstimulated and stimulated. But I don't understand what parameter I should be comparing. Is it the separation index, the staining index or the fluorescence intensity?

This is my first time working with platelets. I will test a narrower range with dilutions between 1:480 and 1:640 (or lower). I am only staining my cells with the primary antibody CD62P-PE from Thermo fisher.

Hi Liliana,

You can use separation index. There is a well written article published in Cytometry for Platelet Analysis. You may find most of the answers to your questions in this article.

Benjamin E.J. Spurgeon and Khalid M. Naseem Platelet Flow Cytometry: Instrument Setup, Controls, and Panel Performance Cytometry Part B (Clinical Cytometry) 98B:19–27 (2020).

I also suggest to look up Alan Michelson articles (recent and old ones), and protocols in Current Protocols-Flow Cytometry, they have been very useful for me.

Best wishes,