Hello, I am investigating the activation of platelets with the use of flow cytometry. I am specifically looking at their activation when agonists such as ADP, collagen, and thrombin are introduced in whole blood. So far I have been staining the cells first with various markers then agonists are introduced and last step is to fix them with formaldehyde.
I am wondering what would happen if i were to fix the whole blood, then stain and put agonists?
Alternatively I can stain first, then fix the whole blood then put agonists.
Or even put agonists, then fix the whole blood and later on stain?
Can you please give me your thoughts as well as pros and cons on that matter?
Please bear in mind that I am looking at the activation of platelets.
Thank you very much
Hi Aleksandar Dishkelov . Fixation generally inactivates cell surface proteins so that is a non-starter before any activation.
Adding agonist first would work if the activated platelets don't clump together and be useless for flow-cy. Also, having the fixation step last ensures that the antibodies are fixed to the platelet surface.
Considering the normal physiology of activation of platelets, agonists activate platelets through a cascade of reactions, involving a number of cell surface receptors. As you want to see the activation of platelets, fixation should definitely be the last step.
Hello, as I am new to flow cytometry and have to set up a similar protocol, I would like to ask if you include any dilution or wash steps after blood drawing and after fixation with PBS.
Thanks in advance!
Theano Dermintzoglou Hello Theano, the protocol I am currently using is in a fast manner as I am trying to observed the activation of platelets. Whole blood is taken with a syringe and placed in a anti coagulation tube / heparin to prevent any clots. Prior to this, a fixation solution is prepared (to stop any further activity of platelets). Here comes the fun important part about PBS, it is used only for the creation of fixation solution. To dilute the WB ( I am using 5 microlitres so that I don't clog the flow cytometer) I am using only staining solution. Also PBS is used in a combination of antibodies markers and WB itself for a proper mixture to occur. I am still working on my protocol, but once I have completed it I will provide it for you. If you any more questions please do ask me.
Thank you very much! So, if I unterstood well, you are using the whole blood, then stain and fix or fix and stain? I will probably follow fixation after staining. What did you mean by diluting with staining solution?