I need to extract PBMC from rat blood by Ficoll centrifugation. I found Ficoll solution for rat blood commercially available but it is quite expensive, and since I need to repeat this extraction several times I was considering producing Ficoll solution myself. I saw there exist specific solutions for human, mouse, and rat, so I guess they differ in something, concentration and density I guess. So, I do not want just to dissolve Ficoll but I want to make it in a concentration and density suitable to separate phases from rat blood. Does anybody has a preparation protocol? With references would be even better.
Thank you all for your contribution
Ficoll-Hypaque solution (density 1.077 g/liter)
64.0 g Ficoll (molecular weight 400,000; e.g., Sigma)
99.0 g sodium diatrizoate (e.g., Sigma)
0.7 g NaCl
Dissolve Ficoll and NaCl in 600 ml water using a magnetic stirrer at low speed. Add sodium diatrizoate. When all components are in solution, add water to 1 liter. Filter sterilize with a 0.22-μm filter unit. Store at 4° to 25° C and protect from direct light.
It is from Current Protocols (see link below).
In our experience we prepared Ficoll first and add sodium diatrizoate solution for i.v. infusions (contrast for roentgenologic investigations) till reach appropriate density at 20° C.
No idea about volume - it was more then 15 years ago. You have to mix properly and areometer is needed.
Good luck!
http://www.penningerlab.com/uploads/media/PBMC_isolation_from_human_blood_01.pdf
Generally density 1.077 g/liter work not bad for rat PBMC. But You have an opportunity to prepare 1.08 if you wish.
Have a nice experiments :)
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