Gill Webster commented on the use of monensin in cytokine assays, which I highly recommend as well. As usual, I thought I'd add my 2 cents worth. Monensin is not simply a protein transport inhibitor. The reason its use is preferable with CD4 prestaining is that it inhibits the acidification of endosomes (it is a Na/H transporter and equilibrates the proton gradient). By blocking acidification, proteolysis of the antibody conjugate is inhibited; for fluorescein conjugates, inhibiting acidification keeps the fluorescein fully fluorescent. Monensin does block transport of the proteins out of the Golgi, but so does Brefeldin A. However, BfA does not have the desired characteristic of inhibiting endosomal destruction of the internalized fluorochrome. Note that studies done by PharMingen (I believe) showed that monensin is not as good for cells when doing 24 hour stimulations. In this case, CD4 detection is better done by including it with the intracellular reagents. Indeed, even for the short stimulations you can also just use the anti-CD4 antibody with your anti-cytokine antibodies. Since the CD4 is internalized, it is still cell-associated and will be available for intracellular staining). mr (PS, if you need a literature reference for this technique, see Mitra et al., Intl Immunol., 11:1801)
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