Re: Fix & Perm delicate cells

Uriel,

I haven't seen a reply to your email, but for what its worth,  Intraprep is
a commercial kit, which we buy from Beckman Coulter. It seems to be a good
permeabilising reagent, which we find copes well with fragile cells. We use
it on blast cells and occasionally on lymphoma cells, and the FSxSS
properties change very little compared to an ammonium chloride based lysing
reagent used for surface staining. The FSxSS properties of the monocytes we
see in clinical specimens changes very little after treatment.
Caltag fix and perm is also another reliable reagent for intracellular
staining. (I haven't used it for a number of years (10!)so I can't comment
on FSxSS changes)

Regards,

Michelle Petrasich


Michelle Petrasich
Techncial Specialist
Immunophenotyping and Transplantion Section
LabPlus, Auckland City Hospital
Auckland, NZ

----- Original Message ----- 
From: "Uriel TK" <utk1@013.net>
To: cyto-inbox
Sent: Tuesday, November 30, 2004 3:24 AM
Subject: Fix & Perm delicate cells


> Dear Friends:
>
> I need to do intracellular stains on dendritic cells and monocytes, but
> these cells are "fragile" and the couple of protocols I have tried (one
> commercial, one in-house) to fix and perm the cells are a little too
harsh.
> We need to conserve FSC-SSC parameters as much as possible. I have learned
> that I should use a crosslinking agent (ie aldehyde) and not methanol
since
> it tends to distort the morphology more, and I also learned that the
crucial
> step is the fixation, since then I can use a very mild triton or saponin
> solution and just incubate a little longer  - is it true?. There are many
> fix & perm protocols out there, but I am asking for suggestions for fix &
> perm protocols that are specially delicate, with emphasis on conserving
> FSC-SSC characteristics as much as possible, if something like that exists
> (I acquire the cells immediately after the staining in a FACScan).
>
> Your help is greatly appreciated!
>
> Sincerely,
> Uriel.
>
>