We are performing an experiment with NK cells and platelet cytotoxicity but we have troubles with platelet separation. Any recommendations?
Thank you in advance
Dear Antonio, what do you mean "troubles with platelet separation"? I usually spun citrted blood to separate PRP - platelet reach plasma (150 G for 15 min) using a horizontal rotor.
Please see this paper by Merolla et al. where different centrifugation conditions are discussed:
Dear Ewa, Thanks for your response. We going to focus our experiment in this way.
Dear Antonio
you can get PRP (platelet reach plasma) easily by the way that Ewa had told you. but if you want to see the cytotixic effect of plateles, then i think you should use washed platelet, as there are hundreds of protein are present in the PRP. for washing purpose you can use Sepharose 2B coloumn, and complete Tyrod's buffer.
Many thanks!
Whats its the best median for platelet? We want to estimulate NK cells and see the cytotoxicity effect on platelet expressing several HLA class I.
Have you experience in this? Its the first time that we work with platelet.
Hi Antonio. A question that you are not the sole to try to solve apparently; abstract in ESCCA meeting last sept. in Lisbon (P)
[TECH-02] AN ALTERNATIVE APPROACH TO ISOLATING PLATELETS BY FLUORESCENCE-ACTIVATED CELL SORTING
Claudia Andrade 1, Cláudia Bispo2, Rui Gardner3, Daniel Gala4, Alexandre Salvador5
Instituto Gulbenkian de Ciência, Flow Cytometry Unit , Oeiras, Portugal
I can make the link with those researchers if you wish.
One suggestion: add PGE1 (or PGI2) and even also apyrase (anti-ADP enzyme) in your blood sample when creating the PRP by centrifugation or separating platelets using Sepharose chromatography. This willl help preventing platelet activation and is reversible (aafter less than 30 min).
Thank philippe. That´s exactly what a was looking for. I going to search the abstract and try to contact with the author.
Many thanks
I informed her on LinkedIn that she should have a look to RG.
If ever you need ad hoc reagents (e.g. CD41, CD62P, CD61 etc... FITC or PE-MAbs) have a look to 3w.biocytex.fr (although as R&T director, I am not here to sell anything, I want the people to get the best possible reagents and I am proud to say that BioCytex has good stuff to share in the platelet field).
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