Cd8 qdot 605 3b5

Methods have been previously reported [20 (link)]. Briefly, cryopreserved PBMCs were thawed and stained with surface antibodies against CD3-APCefluor780 (UCHT1; eBiosciences, San Diego, CA); CD4-Qdot 655 (S3.5) and CD8-Qdot 605 (3B5) (both from Life Technologies, Eugene, OR); CXCR3-APC-CD183 (1C6), CCR4 BV421-CD194 (1G1), CCR5-PE-Cy7-CD195 (2D7), CCR7-Alexa Fluor700-CD197 (150503), CD38-FITC (HB7) and HLA-DR-PE (L243) (all from BD Biosciences, San Jose, CA). Cells were then incubated for 30 minutes and centrifuged at 1200 rpm for 10 minutes. PBMCs were fixed with 2% formalin before analysis. Stained cells were acquired using an LSR II flow cytometer (BD Immunocytometry Systems, San Jose, CA). Using 10-color flow cytometric analysis, we investigated T cell phenotypes classified by the markers listed above. Data were analyzed using FlowJo software (v9.8.5, TreeStar, Ashland, OR). Lymphocytes were analyzed based on forward scatter (FSC) and side scatter (SSC) profiles. A single cell population was selected with SSC-A vs. SSC-H and then with FSC-A vs. FSC-H before gating for other markers. Gates were set based on Fluorescence Minus One (FMO) control.

PBMCs and MMCs were stained with fluorescent antibodies against cell surface markers and analyzed using 10-color flow cytometry by previously reported methodology [3 (link)]. Briefly, PBMCs and MMCs were labeled with LIVE/DEAD fluorescent reactive dye (Invitrogen, Carlsbad, CA) and stained with surface antibodies against CD3-APCefluor780 (UCHT1; eBiosciences, San Diego, CA); CD4-Qdot 655 (S3.5) and CD8-Qdot 605 (3B5) (both from Life Technologies, Eugene, OR); CXCR3-APC-CD183 (1C6), CCR4 BV421-CD194 (1G1), CCR5-PE-Cy7-CD195 (2D7), CCR7-Alexa Fluor700-CD197 (150503), CD38-FITC (HB7) and HLA-DR-PE (L243) (all antibodies were purchased from BD Biosciences, San Jose, CA). Cells were then incubated for 30 minutes and centrifuged at 1200 rpm for 10 minutes. Both PBMCs and MMCs were fixed with 2% formalin before analysis. Using an LSR II flow cytometer (BD Immunocytometry Systems, San Jose, CA), CD3⁺ events were acquired from each sample. Data were analyzed using FlowJo software (v9.8.5, TreeStar, Ashland, OR). Lymphocytes analysis was based on forward and side scatter profiles after exclusion of dead cells (Fig 1). Gates were either set using negative sample or Fluorescence Minus One (FMO) control and were then applied to all samples from each participant.