Apc efluor 780 conjugated cd3 clone ucht1

The isolation and cryopreservation of the brain-infiltrating lymphocytes (BILs) have been previously described (21 (link)). In brief, fresh brain tissue was finely minced in dissociation solution (HBSS with 20 mM HEPES pH7.0, 5 mM glucose, and 50 U/ml penicillin/streptomycin), then digested overnight at room temperature in dissociation solution containing 0.5 mg/ml Type IV collagenase (Worthington Biochemical Corp., Lakewod, NJ, USA) and 5% filtered human serum (Mediatech Inc., Manassas, VA, USA). BILs were obtained by fractionation on a 30%: 70% Percoll^® (SigmaAldrich, St. Louis, MO, USA) step gradient in RPMI containing 20 mM HEPES. BILs were stained with the following antibodies: APC-efluor^® 780-conjugated CD3 (clone UCHT1; eBioscience Inc., San Diego, CA, USA), PE/Cy7-conjugated CD4 (clone SK3; eBioscience Inc.), PerCP/Cy5.5-conjugated CD8 (clone RPA-T8; eBioscience Inc.), APC-conjugated TCR αβ (clone IP26; eBioscience Inc.), FITC-conjugated TCR γδ (clone B1.1; eBioscience Inc), and PE-conjugated CD103 (clone B-Ly7; eBioscience Inc). Data were acquired on an analytical LSRII flow cytometer (Becton Dickinson, San Jose, CA, USA), and were analyzed with FlowJo software (TreeStar Inc., Ashland, OR, USA); histograms were exported into CorelDrawX6 (Corel Corporation, Ottawa, ON, Canada). Statistical analysis and graphing utilized R-project programs (www.r-project.org).

Phenotypic data were acquired on an analytical LSRII flow cytometer (Becton Dickinson, San Jose, CA). The following antibodies were used: APC-efluor® 780-conjugated CD3 (clone UCHT1; eBioscience Inc., San Diego, CA), PE/Cy7-conjugated CD4 (clone SK3; eBioscience Inc.), PerCP/Cy5.5-conjugated CD8 (clone RPA-T8; eBioscience Inc.), APC-conjugated T cell receptor (TCR) αβ (clone IP26; eBioscience Inc.), FITC-conjugated TCR γδ (clone B1.1; eBioscience Inc.), PE-conjugated CD69 (clone FN50; eBioscience Inc.), PE-conjugated TCR Vδ2 (clone B6; Biolegend), and FITC-conjugated TCR Vδ1 (Clone TS8.2; GeneTex Inc., Irvine, CA). Data were analyzed with FlowJo software (TreeStar Inc., Ashland, OR); plots were exported to CorelDRAW X6 (Corel Corporation, Ottawa, Canada). Statistical analyses and graphing utilized R-project programs (www.r-project.org).