Pe anti cd8 antibody

To determine T helper and cytotoxic cells populations in HAM/TSP, ACs and normal groups; PerCP anti CD3 antibody (bio legend company cat no: 344813), Phicoerythrin (PE) anti CD4 antibody (bio legend company cat no: 317409) and PE anti CD8 antibody (bio legend company cat no: 301007) were used. Fresh peripheral blood samples were treated by lysis buffer for destroying the red blood cells and platelets. Samples were analyzed on a FACS caliber Becton Dichinson. All analyses were done in the lymphocyte gate.

J-Lat cells were exposed to NTP, then cultured for 24 h, as described previously. Prior to co-culture, J-Lat cells were washed with PBS and treated with 0.5% PFA or 10 µg/mL Mitomycin C (Sigma-Aldrich, St. Louis, MO, USA, Cat#M4287) for 2 h at 37 °C to inhibit proliferation of J-Lat cells, then washed 3× with PBS and supplemented with RPMI. Target J-Lat cells were then co-cultured with Cell Trace Far Red (CTFR) pre-labeled CD8+ T lymphocytes at effector-to-target (E:T) ratios of 1:1, 5:1, and 10:1. CD8+ T lymphocytes treated with a cocktail containing 2 µg/mL of an anti-CD3 antibody (Clone OKT3) (BioLegend, Cat#317302) and 2 µg/mL of an anti-CD28 antibody (Clone CD28.2) (BioLegend, Cat#302902) or 10 ng/mL PMA and 400 ng/mL Ionomycin served as positive controls for proliferation. Co-cultures were supplemented with 10 ng/mL IL-2 incubated for 1 and 7 days. Following incubation, cells were washed with FACS Buffer and stained with PE anti-CD8 antibody (BioLegend, Cat#344705), then stained with LIVE/DEAD™ Fixable Aqua Dead Cell Stain (ThermoFisher Scientific, Cat# L34957) to label dead cells. The percent of proliferated CD8+ T lymphocytes in co-cultures was enumerated using a BD LSRFortessa™ flow cytometer. Data were analyzed using FlowJo™ Software after doublet exclusion using forward scatter (FSC-A vs. FSC-H), and gating on live CD8+ T lymphocytes with a low intensity of CTFR.