1. Activated cell populations can be prepared from in vivo-stimulated tissues or from in vitro-stimulated cultures (e.g., antigen-specific activation or mitogen-induced). For cytokine and chemokine detection, it is critical to include a protein transport inhibitor such as brefeldin A (BioLegend Cat. No.420601) or monensin (BioLegend Cat. No.420701) in the last 4-6 hours of cell culture activation. The cells can be suspended and distributed to 12 x 75 mm plastic tubes or microwell plates for immunofluorescent staining. For details on stimulation methods, please see our stimulation guide for cytokines\/chemokines: (www.biolegend.com\/media_assets\/support_protocol\/BioLegend_StimulationGuide_101711.pdf)2. Different cytokines\/chemokines have different production peaks. In order to obtain optimal staining signals, the stimulation conditions for each stimulant need to be optimized.3. Some antibodies recognizing native cell surface markers may not bind to fixed\/denatured antigens. For this reason, it is recommended that staining of cell surface antigens be done with live, unfixed cells PRIOR to fixation\/permeabilization and staining of intracellular targets. Altering the procedure such that cells are fixed prior to staining of cell surface antigens requires that paraformaldehyde-denatured antigen reactive antibody clones be empirically identified. You can also take a look at our Fixation Webpage (www.biolegend.com\/fixation) to get an idea of how epitopes stain after fixation with 4% PFA. Please note that the ability to stain post-fixation depends on the fluor, antigen expression, and several other factors.