Blood samples were drawn from patients following permission and processed at the University of Arizona Biorepository Laboratory. Peripheral blood mononuclear cells were cryopreserved for future analysis. A complete blood count was performed using an Ac-T 5diff CP machine (Beckman Coulter, Pasadena, CA). Cryopreserved PBMC (1–2x106/sample) were stained with LIVE/DEAD Fixable Dead Cell Stain-AQUA (Invitrogen) and T cell markers in various combinations.11 (link)
The following mAbs were used to differentiate T cell subsets: CD3—BV570 (BioLegend), CD4—APC (eBioscience), CD8β—ECD (Beckman Coulter), CD95—BV421 (BioLegend), CD28—PerCp/Cy5.5 (BioLegend), CCR7—FITC (BD Pharmogen), CD45RA—BV605 (BioLegend), CD57—BV570 (BioLegend), IFN-γ—APCe780 (eBioscience). Cells following various combination and incubations as described in our previous study,11 (link)
were analyzed on the BD LSR II instrument using DiVa acquisition (BDIS, Mountain View, CA) and the FlowJo analysis software (TreeStar Inc., Ashland, OR).
Twenty (20) of 37 patients in this study completed the entire cell surface analysis.
The following mAbs were used to differentiate T cell subsets: CD3—BV570 (BioLegend), CD4—APC (eBioscience), CD8β—ECD (Beckman Coulter), CD95—BV421 (BioLegend), CD28—PerCp/Cy5.5 (BioLegend), CCR7—FITC (BD Pharmogen), CD45RA—BV605 (BioLegend), CD57—BV570 (BioLegend), IFN-γ—APCe780 (eBioscience). Cells following various combination and incubations as described in our previous study,11 (link)
were analyzed on the BD LSR II instrument using DiVa acquisition (BDIS, Mountain View, CA) and the FlowJo analysis software (TreeStar Inc., Ashland, OR).
Twenty (20) of 37 patients in this study completed the entire cell surface analysis.
