To minimize batch to batch variability across sites during sample processing, we used a lyophilized antibody cocktail consisting of eight premixed antibodies that was fabricated and lyophilized for the flow cytometry staining panel used in this study (BioLegend, San Diego, CA). Because the final panel consisted of three antibodies containing brilliant violet technology, one of these antibodies was added in the appropriate concentration after the lyophilized cocktail was resuspended with appropriate stabilization buffers (BD Biosciences, Franklin Lake, NJ). We included a live/dead stain to allow gating on live cells in the analysis. The composition of the lyophilized cocktail was as follows: CD4 (clone OKT4) FITC, CD3 (clone SK7) Alexa Fluor700, CD45RA (clone HI100) APC/Fire760, CD127 (clone A019D6) PE, CD25 (clone BC96) PE/Cyaine7, CD194/CCR4 (cloneL291H4) APC, CD183/CXCR3 (clone G026H7) Brilliant Violet 421, CD45RO (clone UCHL1) Brilliant Violet 711, CD196 (CCR6) (clone G03E3) PE/Dazzle 594.
We also designed a separate lyophilized cocktail that we included with each sample to serve as the fluorescence minus one (FMO) control for CCR4, CCR6 and CXCR3. This cocktail included all antibodies listed above except these three chemokine receptors. The appropriate concentration of each antibody (minus one) was added to each FMO control using separate liquid reagents for CCR4, CCR6 and CXCR3.
We also designed a separate lyophilized cocktail that we included with each sample to serve as the fluorescence minus one (FMO) control for CCR4, CCR6 and CXCR3. This cocktail included all antibodies listed above except these three chemokine receptors. The appropriate concentration of each antibody (minus one) was added to each FMO control using separate liquid reagents for CCR4, CCR6 and CXCR3.
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