I am going to isolate prp from whole blood and I will be using the absolute basic essentials. A centrifuge, and ficol 1077. I will be using 18ccs of blood. Can someone please tell me the best way? I have searched and always find protocols using a particular system or product rather than the basics. How much ficol and how long to centrifuge? Thank you!
You don't need Ficoll. Just a single centrifugation step is fine. I assume you're talking about platelet-rich plasma, right? I'll look up the exact centrifugation speed and time tomorrow, when I'm in the lab. If you need to do it right now, I would bet 1400xG, 15 minutes. Take supernatant. Do not aspire any white blood cells, but make sure to get close to them. The majority of platelets in close to the white cells.
Best wishes,
Coen
Okay. I checked. Its a lot lower speed. 160xG. 15 minutes. The rest stays the same.
Good luck!
Coen
PS If you would like to have washed platelets, further down the road:
1. Korporaal SJA, Van Eck M, Adelmeijer J, Ijsseldijk M, Out R, Lisman T, Lenting PJ, Van Berkel TJC, Akkerman J-WN. Platelet activation by oxidized low density lipoprotein is mediated by CD36 and scavenger receptor-A. Arterioscler. Thromb. Vasc. Biol. 2007;27:2476–2483.
"Blood was collected from healthy volunteers (with informed consent) into 0.1 volume of 130 mmol/L trisodium citrate. The donors claimed not to have taken any medication 10 days prior to blood collection. Platelet-rich plasma (PRP) was prepared by centrifugation (156 x g, 15 min, 20°C). Then, 0.1 volume of ACD (2.5% trisodium citrate, 1.5% citric acid and 2% D-glucose) was added to lower the pH to 6.5. Platelets were further purified by centrifugation (330 x g, 15 min, 20°C). The platelet pellet was resuspended in Hepes-Tyrode buffer (145 mmol/L NaCl, 5 mmol/L KCl, 0.5 mmol/L Na2HPO4, 1 mmol/L MgSO4, 10 mmol/L Hepes, 5 mmol/L D- glucose, pH 6.5). Prostacyclin (PGI2) was added to a final concentration of 10 ng/mL and the washing procedure was repeated once. The platelet pellet was resuspended in Hepes-Tyrode buffer, pH 7.2. Platelet count was adjusted to 2.0 x 1011 platelets/L, unless stated otherwise"
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