Intracellular cytokine detection

From: Fumeaux Thierry (fumeauxt@usa.net)
Date: Tue Aug 28 2001 - 03:22:59 EST 

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Dr Thierry Fumeaux
Chef de Clinique adjoint
Soins Intensifs de Medecine
Hopital Cantonal Univesitaire
Rue Micheli-du-Crest 24
CH - 1211 GENEVE
022 / 372 33 11 
022 / 372 56 86

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------------------------------------------------------- Ce message a volontairement ete envoye sans accent pour permettre une compatibilite avec toutes les plateformes ------------------------------------------------------- Dr Thierry Fumeaux Chef de Clinique adjoint Soins Intensifs de Medecine Hopital Cantonal Univesitaire Rue Micheli-du-Crest 24 CH - 1211 GENEVE 022 / 372 33 11 022 / 372 56 86

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Hello ! I am trying to detect and quantitatively measure intracellular cytokines (TNF and IL-10) in total leukocytes of patients and healthy volunteers. I set the method with LPS stimulated-PBMC and with a permabilisation method. However, I still have problems : - Isotype control does not seem to be OK, because its fixation on cells is highly variable among patients : I use the same IgG type and the same fluorochrome for cytokine and isotype - shoud I use another method, like Fc bloker or working with 2 % BSA ? - Surface markers (labelled with FITC) are not very useful after fixation and permeabilization of cells and forward and side scatters are not sufficient to detect monocytes among neutrophils : does anyone have a solution ? should I use 2 % formaldehyde instead of 4 % ? What lysing solution performs best ? - Finally, after taking fresh blood, should I incubate the cells a few hours with momensin or another bloker, in order to rise sensitivity ? All suggestions and help are wellcome ! Thierry Fumeaux LABO SIM - local 7198 7e etage CMU Departement Genetique et Microbiologie 1, Rue Michel Servet 1211 GENEVE 4

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