title: B13
organism: Bos taurus
description: Protocols: Blood Sampling, Processing and Cell Separation Peripheral blood from the jugular vein of bTB infected and control animals was collected in 9 ml vacutainer tubes containing EDTA anticoagulant. For cell separation, whole blood was diluted 1:1 with phosphate buffered saline (PBS) in 50-mL conical falcon tubes. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coat fractions using Histopaque-1077 (Sigma-Aldrich Ireland Limited, Vale Road, Arklow, Co Wicklow, Ireland) and the Leucosep system (Greiner Bio-One Ltd. Brunel Way, Stroudwater Business Park, Stonehouse, Great Britain) with gradient centrifugation at 1034 x g for 25 min. PBMCs were collected and washed in PBS to remove platelets. Contaminating red blood cells were removed using cell lysis buffer (Life Technologies).Preparation of Cell Suspensions, γδ T Cell Labelling and SortingPBMCs were washed and re-suspended in PBS containing 2% bovine serum albumin (BSA). Cell suspensions were labelled with primary antibodies (Abs, 1-2 μg/mL of each), incubated at 4 °C for 10-20 min, washed and re-suspended in 2% BSA PBS. Cells were then incubated in the dark for 30-45 min on ice with secondary Abs (1:500, 1:700, 1:1000) conjugated with different fluorochromes, washed twice and re-suspended in 2% BSA PBS. The cell samples were analyzed using the FACSAria Fusion Sorter (BD Biosciences) and one hundred thousand events were acquired for analysis. Compensation was used to eliminate residual spectral overlaps between individual fluorochromes. Side and forward scatter area/forward scatter width parameters or characteristics were used for the identification of viable single lymphocytes and exclusion of doublets. Propidium iodide (Invitrogen/Life Technologies Corp., Paisley, UK) was used for the exclusion of dead cells. Sorted γδ T cells and subsets were identified as having viability greater than 95% and purity greater than 99%. The γδ T cell samples were centrifuged and cell pellets were snap frozen on dry ice first and then stored at -80°C. Primary and secondary antibodies used for cell profiling/phenotyping by flow cytometry and isolation by fluorescence activated cell sorting (FACS) were bovine mb GB21A IgG2b, mb BAG25A IgM, mb CACTB32A IgG1 (Monoclonal Antibody Center, Pullman, WA, USA); goat anti-mouse IgG2b AF 488, anti-mouse IgM AF 647 and anti-mouse IgG1 PE/Cy 5.5 (Life Technologies Corporation, Invitrogen, USA). Total RNA was extracted from the cell pellets. RNA quantity, integrity and purity were assessed using a NanoDrop spectrophotometer and the Agilent 2100 Bioanalyzer (Novogene, Genomic Centre Cambridge, CB4 0FW, UK). All RNA samples used for transcriptomic analysis (n= 6 per group) had RIN values > 8. TruSeq (Illumina, Novogene, Genomic Centre Cambridge, CB4 0FW, UK.)