Intracellular cytokine detection

Brian A Telfer btelfer at FS1.PA.MAN.AC.UK
Thu Mar 14 09:41:59 EST 1996


Just a quick word. I have been involved with both human and murine cell stimulation and we (de caestecker MP) found that inhuman lymph cells the best stimulator was Lipopolysaccharide (LPS) and in rat/mouse Conconavalin A.
I found that stimulation with PMA/ionomycin did not produce IFN gamma probably because the store of IFNg was rapidly depleted i.e. it had all gone by the time incubation and permeabilisation was completed. PHA was another mitogen that I looked at with slighly better results,but my main worry was the effect of using the DB-1 antibody in the first place and the effect on it by the permeabilising agent Saponin.
Later PCR results on rat cytokines showed that IFNg was present and probably either barely 'activated'or that I was detecting remnants of an already depleted cell. I may publish this later however our human work appears in European Journal of Immunological Methods 1994.> Re: intracellular cytokine labelling
> 
> calman prussin (calman_prussin at nih.gov)
> Thu, 8 Feb 1996 19:55:28 GMT
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> 
> To: immuno at net.bio.net
> 
> From: calman prussin <calman_prussin at nih.gov>
> 
> Subject: Re: intracellular cytokine labelling
> 
> Date: Thu, 8 Feb 1996 19:55:28 GMT
> 
> 
> I have extensive experience with cytokine flow in the human system
> with some in the mouse.
> First of all, what mAbs are you using? Choice of mAbs is critical.
> Recently, Pharmingen has
> begun directly labelling anti-cytokine mAbs in the mouse system. Try
> PE as a fluorochrome for
> best S/N.
> 
> As far as stimulus, try PMA/ionomycin. For cytokine flow it always
> works better than mitogen.
> Once you have that working, you can change to a less potent stimulus.
> Check out the Nov '95
> JEM for a paper by O'Garra and Oppenshaw-it shows some of the best
> mouse staining in print.
> 
> Best of luck,
> 
> Calman Prussin
> NIAID/ NIH
>     ------------------------------------------------------------------------
> 
> 
>       Next message: Mr N.C. Eastmond: "Re: Fluoridation vs. Breast
>      feeding"
>       Previous message: Jan Robert Becker - PhD -: "Re: ban Dihydrogen
>      monoxide"
>       Maybe in reply to: Cees de Heer: "intracellular cytokine
>      labelling"
> 





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