What's the best way to resuspend cells??

Mr. P.A. Sansom psansom at hgmp.mrc.ac.uk
Tue Jan 24 14:13:32 EST 1995


What's the best way to resuspend cells??

I have monocyte and lymphocyte cell lines that I use in assay at a standard
concentration of 10^7 per ml. This involves harvesting by centrifugation.

I centrifuge at 200g for 5 minutes, resuspend and wash the cells once with
PBS at 37'C and resuspend (after a second centrifugation) at 10^7 per ml.
Resuspension is performed with a 'whirlimix' or 'vortex'.

I have assayed the PBS wash for my proteolytic activity of interest and
found a substantial amount to be present. I am uncertain as to whether this
is due to loss of the enzyme from the cell surface during resuspension or
leakage of the enzyme from inside the cell.

Whole cell lysates degraded my substrate very rapidly, but the profile is
dissimilar to that obtained with whole cells or the PBS wash, suggesting
that the enzyme is indeed a cell surface protein.

I would like to know if anyone has any suggestions for a gentler way of
concentrating my cells (short of letting them settle overnight!) or a way
of determining that the enzyme is indeed an external protein. I am about
to try trypsinisation to release it, but this doesn't get around my 
leakage question.

All tips, comments and suggestions gratefully received!

Thanks in advance

Paul

psansom at hgmp.mrc.ac.uk

Paul A Sansom                                  tel: 081-748-9966 x4208
Department of Biochemistry                     fax: 081-748-5090
The Kennedy Institute of Rheumatology
6 Bute Gardens
Hammersmith
London W6 7DW



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