I've found some references that use 2200 to 2300 xg for 30 minutes? But then I've found some people on here asking about cell fractionation that have been recommended that 500xg is the max they should use. Is 2200xg overkill? Does 500xg just apply to separation and not packing of RBC?
Any help would be greatly appreciated.
Alan
Hi, Alan,
I don't think that it makes a difference using either Wintrobe tubes or other products for centrifugation. Therefore I will deal only with the g-forces and time of centrifugation one needs.
In my active benchwork, doing "buffy coat preparation for ultrastructural evaluation of white blood cells (using peripheral withdrawn blood and therefore separation into several liquid phase/cell layers by centrifugation/g-force ) as a routine standard I used suited (specially fabricated) glass vials for (refrigerated = 20-24°C) centrifugation by means of a swing rotor, at 1,000 g for 10 min. Notice: End of centrifugation: 'Brakes off'.
NB: For TEM-observation of lymphocytes the [plasma and the thrombocytes' [platelets'] layer was removed carefully by pipetting as deep as possible] white cell layer was harvested by most carefully overlaying with fixative. When the fixed WBC-layer was retrieved, processed, embedded into resin and cut as well as stained and examined usually the separation between the WBC-layer and RBC-layer, respectively was relatively a sharp line of demarcation. So it should be possible (no fixation!) to pipet away the 3 layers (plasma/serum, platelets, WBC's) and to collect at least 95-98% of RBC amount....(and if you find out that separation was not quite successful you can prolong the centrifugation time to 15 min and or increase also the g-force up to 1,200xg. Naturally your RBC's then will be tightly "packed"). It would be interesting to find out the protocol given in separation kits for RBC's by Ficoll-Hypaque...(e. g cf.: http://www.sciencedirect.com/science/article/pii/0022175974901094 )
Best of luck, W.M.
Thank you for your advice Wolfgang.
I've since come across several sources that use 2300 xg for 30 minutes to compact erythrocytes. This method has a strong positive correlation with methods such as cell counts so I have opted to use this in my study. I have tried methods similar to yours but have found the outcome to be inflated when compared to what would be expected from literature. If I find any other information I will be sure to re-post.
Alan
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