I‘m interested to study the percentage of CD8:platelet aggregate in whole blood of patients collected in sodium citrate tube. Reason is that we want to study ‘real time’ interactions in the blood without disturbing them too much.
I’ve read papers online and the methods they use are either
1) isolating platelets and coculture with platelets or
2) direct staining in whole blood then lyse RBC.
My question is
1) is RBC lysis necessary (using lysing solution, no wash)? Can 0.2%PFA lyse RBC?
2) Do you use Fc block to block other interaction?
3) Please share protocols that would be useful
Thanks!
Not sure to fully capture the question about the Fc fragment but it may bind to many cells - from lymphocytes, basophils, ..., platelets. So you may have non specific or false positive signals when performing your flow cytometry. Best is to use blocking reagents (IgG, anti-Fc receptor antibodies) to saturate the receptors before staining your cells with labeled antibodies.
Red blood cells should be eliminated. Lysis would help to clear some confounding factors and to sort your flow cytometry deliverables.
Most of the time : Ice-cold ammonium chloride lysis buffer : NH4Cl, NaHCO3, EDTA, and washed several times along the centrifugation with PBS.
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