I'm trying to stimulate human monocytes from fresh PBMC isolations with 2ug/mL imiquimod and 2ug/mL ssRNA40. I do this in 10% FCS complete RPMI for 18-20 hours before staining and analysing CD14+ or CD16+ monocytes by FACS. The LPS positive control produces lots of TNFa, IL-6 etc but the imiquimod and ssRNA40 do not show anything. This is puzzling to me because there is lots of literature, for TLR8/ssRNA at least, which demonstrates robust production of inflammatory cytokines.
Does anyone have any tips on how I can improve the assay? Am I doing anything wrong?
Many thanks!
Control cells stimulated with LPS secreted cytokines shows there is nothing wrong with culture or method. May be concentration of imiquimod or ssRNA40 could be a issue (try different concentration). Also, something wrong with the fixation and permeabilization during ICS? I never did ICS from monocytes but for T cells we used to add brefeldin A to trap the cytokines inside the cell during incubation period.
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