Confusion with staining protocol

S Newton S.Newton at sheffield.ac.uk
Mon Jan 21 10:20:53 EST 2008


Dear all

A user in the facility is trying to reproduce some work from a paper, however I
am confused as to why there are certain steps in the protocol.

Basically tumours are mashed up and then the proportion of IgG(-FITC) positive
and F480(-FITC) cells determined by flow. 

1) Tumour cell suspnsions obtained.
2) Cells fixedin 70% ethanol then washed off
3) Incubation with either IgG FITC or F480 FITC washed off
4) PI and RNase are added

However no mention is made in the paper of cell cycle analysis, which has made
me ponder if the fixation and PI addition are to distinguish cells from debris,
however no mention of the PI staining is mentioned in the results.

The user has repeated this staining BUT the IgG and F480 antibodies are binding
to all the cells following fixation. (They are extracellular antigens we are
interested...hence the fixation confusion). 

We have looked at live cells and can see a population of IgG positive cells and
have also determined their viability. I personally think that we should
continue looking at live tumour cells, but am reluctant to send this 1st year
PhD student on a wild goose chase. 

I have searched the literature but am not having any joy.....

Any thoughts?
Thanks
Sue
-- 
Sue Newton
Flow Cytometry Technician
DU27. D Floor
Medical School
University of Sheffield
Beech Hill Road
Sheffield
S10 2RX

Phone number: 0114 271 3023



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