Hi everyone,
We are performing some intracellular staining for flow with Ag-stimulated (5 days) human PBMCs. We use cytofix/cytoperm for intracellular staining of IFN-gamma (BV711) in CD4 T cells. We then observe a very high nonspecific binding. Of note, we don’t see this binding with the same clone conjugated to other fluorochromes. Any idea of how to manage this issue? Thanks a lot!
hi
1- check your cell viability before staining ( dead cells deleted by centrifugation )
2- PE / PE-CY5 ( 250 kD) or APC (105 kD) conjugated has poor stability , you can brief centrifuge conjugated antibodies before used for staining .
good luck
Hi, I tend to agree viability is a potential issue for 5 day stimulated cells.Monensin or brefeldin A over that time frame could exacerbate the toxic effects. Perhaps add the monensin of the last 6-24hrs (if you have not tried that already :)
And....titration is always good practice. Maybe try using less antibody.
Hi, are you staining a combination of more BV conjugated mAbs?
If so, your problem could be solved by using the BV staining buffer (it should be provided with the antibodies).
Regards, Julia
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