Hi

sometimes ACD is not enough when platelets undergo extensive manipulation, so I would add a little bit more ACD before the first spin. Also not sure why let the blood sit after the first spin.

Hi Andree

PBS is not the best choice of buffer to use for the isolation of resting platelets. A buffer such as HEPES-Tyrode (see recipe below) is recommended.  Also, it is recommended to keep the number of manipulations to an absolute minimum - usually a single wash centrifugation step is sufficient.

Hepes-Tyrode buffer pH 7.4 (134 mM sodium chloride, 12 mM sodium bicarbonate, 2.9 mM potasium chloride, 0.34 mM sodium phosphate monobasic, 1 mM magnesium chloride (avoid for adhesion assay), 5 mM Hepes, 5 mM glucose,  with or without 1 % BSA).

Regards, Rosemary

I absolutely agree with Rosemary.

So you don't have to make up fresh 1X buffer every time, there is a recipe for making Tyrode's stock buffers in this reference:

Preparation of washed platelet suspensions from human and rodent blood.

Cazenave JP, Ohlmann P, Cassel D, Eckly A, Hechler B, Gachet C.

Methods Mol Biol. (2004) 272:13-28.

(In fact this is the first of a very good set of platelet methods in 3 volumes of the journal and worth having if you're in platelet research for the long haul even if they are >10 years old. The Michelson book is a must also.)

The glucose grows bugs quickly so I'd make up a 1X solution once a week and take care to refrigerate it but DO warm it up to room temp prior to use. Platelets hate the cold and clump.

Addition of additional platelet inhibitors in each wash step is recommended, fickle beasts they are. I used 0.8 mM EGTA additionally.

Best of luck in your research.

Thanks for the replies, much appreciated. I'm still confused as to why the platelets isolated from the ACD tubes clump when the ones in the EDTA tubes don't with the same method? All the searches I've done suggest using ACD tubes when clumping is a problem and to avoid EDTA. Should I be including PGI2 with the whole blood when using ACD tubes? I don't seem to need to when I use EDTA tubes.

Hi Andree

Yes, you can include a platelet inhibitor when using ACD-anticoagulated whole blood. 

EDTA causes irreversible damage to platelets, which includes preventing their ability to clump.      

Regards, Rosemary

You could try using CTAD tubes which also have theophylline in them and/or adding it to your wash buffer.

Regards,

Barry.

Just thought of another point. Do control the acceleration and deceleration of your centrifuge steps to avoid sheer stress.

I would strongly advise against EDTA when isolatiung platelets or doing anything to monitor platelet function. EDTA leads to platelet shinkage and was once widely reported as the cause of the so called pseudothrombocytopenia. ACD is pretty good as an anticoagulant, although - the same as for 3.2% citrate, you may find the opinions that it chelkates Ca2+, and therefore leads to altered natural millieu of platelets. And this is right, the conditions are not natural, however, with ACD and citrate-anticoagulated blood you may much more easily use isolated platelets in further functional assays. Some researchers add apyrase/PGE1 to prevent undesirable artefactual spontaneous platelet activation. You have to be aware, however, that when measuring the extent of platelet activation and/or reactivity with the use of some techniques, like flow cytometry, the anticoagulant used matters a lot and you cannot pool the outcomes collected with various anticoagulants for one characteristics of a group. Still, some other anticoagulants are often used by researchers, like hirudin (see f.i. the papers by Heptinstall and coworkers) or low mol wt heparin, like for lab rodents. For isolation, we have the best experience with Tyrode's buffer, preferably with BSA and PGE1, if possible. Take care of the temperature, avoid too cold buffers, etc. Some people regard the use of vacuum syringes for blood withdrawal the high risk factor, but we have not confirmed that. Finally, you have to accept that when you withdraw blood from individuals in whom platelets in a circulating blood are highly 'primed', the risk of undesired clumping rises a lot in a course of blood preparation (diabetes, atherothrombotic, hyperlipidemic, etc.). While you can still consider such an increased formation of microaggregates in the course of preparation a hallmark of increased platelet priming in a circulating blood, you cannot of course proceed with the cells in functional assays.   

hi again, thanks for your additional replies. they have been really helpful.

cheers, Andree

Try 150g.

 Hi, may I know if the commerical BD vacutainer ACD-A tubes will work? the amount of citrate seems to be on the lower side for the commerical tubes.