3 replies to this topic

[zodiac1505]

I have been trying to do T helper differentiation and to stain for intracellular cytokines. I think the differentiation works fine since I can see loads of cytokines by ELISA but I can never see anything if I stain the cells and do FACS. I used the staining kit from BD and antibodies from R&D and eBioscience. I usually restimulate the cells with PMA+ ionomycin. I have tried both golgiplug and brefeldin A. Does anyone have any idea what could be wrong?  

[Merlita]

Have you used any permeabilization solution? Intracellular staining need it to make a 'hole' for antibody go to the intracell cytokine.

[Merlita]

zodiac1505, on 02 September 2010 - 11:23 PM, said:

I have been trying to do T helper differentiation and to stain for intracellular cytokines. I think the differentiation works fine since I can see loads of cytokines by ELISA but I can never see anything if I stain the cells and do FACS. I used the staining kit from BD and antibodies from R&D and eBioscience. I usually restimulate the cells with PMA+ ionomycin. I have tried both golgiplug and brefeldin A. Does anyone have any idea what could be wrong?  

Have you used any permeabilization solution? Intracellular staining need it to make a 'hole' for antibody go to the intracell cytokine.

[zodiac1505]

The BD staining kit already has permeabilisation and fixation solutions.So I guess they are enough