Hello dear

do you add RBCs lysis solution previously.,

this solution will ensure lyse all RBCs..

No i didn't. Is always need to add RBC lysis solution? Will it activate the platelet?

We use ACK Lysing Buffer from Thermo Fisher Scientific all the time to remove RBCs from the cell suspensions. Works very well, although you will have to test yourself if it has any effects on cell population of your interest. 

Hi Samarjit

I suggest you increase the g force of the first centrifugation by a little bit – say 250-300g.  This will help to settle the RBCs more firmly.

Also turn the brake off, or at most have a low deceleration setting on the centrifuge so as to avoid stirring up the RBC layer.

Do not have a brake or at most have a very low deceleration setting on the centrifuge so as to avoid stirring up the RBC layer.

Be careful not to let your pipette tip touch the sides of the tube and only collect the PRP in the top two-thirds of the plasma layer.

I would avoid using lysis solutions with platelets.

Hope these suggestions are useful.

Regards, Rosemary

As Rosmary writes, leave about half a cm above the RBC layer when collecting your PRP, this way you get rid of most RBCs. After the second centrifugation step it is still common to have a few RBC in the platelet pellet. These are usually in the bottom of the pellet so avoid resuspending the RBCs when you resuspend the platelets and you will be fine.

Rosemary has given good advise, that I recommend you follow. However, I would try isolating without increasing the centrifugation speed first. If you follow the other advise and still get contaminations this might be a good option, but otherwise it's wise to try to keep the speed down.  

If you want your platelets to be in a good, resting condition after isolation lysis of the RBC sounds like a terrible idea.

One way to get rbc's out of the way is to use HESPAN (hydroxy-ethyl-starch) a volume expander. It causes the RBC's to form rouleaux and sink to the bottom leaving a clear interface.  Now, for platelet preparation, one should have little if any RBC's present. Review your procedure for preparation.

Sergio

What is the working concentration of PGI2 that should be used while pelleting down platelets?

-Thank you in advance.

Mushtak T. S. Al-Ouqaili Kushal Prajapati Sorry for the very late response, but I would like to ask when you use RBC lysis buffer, how many g (RCF) do you use for the centrifugation step and removal of the red blood cell debris?

Thank you in advance.