Re: Isolation of buffy coat (WBC) from whole b lood (only 500 ľl) after removing the plasma
Dr Engelbert Buxbaum
engelbert_buxbaum at hotmail.com
Fri Mar 12 13:19:47 EST 2004
Elisabet W wrote:
> How can I remove white blood cells (WBC) from whole blood EDTA after
> pipetting off the plasma, when I only got 500 ľl whole blood? Should
> buffy coat get washed before storage? Do someone have a protocol?
Put the whole blood on a metrizamide gradient and ultracentrifuge, white
blood cells will band at a certain density, while erys are pelleted.
There is a standard protocol for isolating white blood cells this way,
check Medline for those terms.
Metrizamide is a compound originally developed as a contrast agents for
kidney X-ray, non-toxic and highly water soluble. It contains several
iodine atoms in each molecule. The resulting high molecular weight
allows the design of isotonic density gradients, in which living cells
can be separated isopycnically.
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