resuspending cells

[Eric T.]

In article <3g3jgt$t51 at mserv1.dl.ac.uk>
psansom at hgmp.mrc.ac.uk (Mr. P.A. Sansom) writes:

> 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

DO NOT vortex your cells you will break the membrane...  You should try
to resuspend your cells using a pipet

Best luck!