Anti btla pe clone mih26

PBMCs, naive CD4⁺ T cells or Treg subpopulations were stained for 20 min at 4°C in staining buffer (2% FCS in PBS) with the following conjugated mAbs: anti-BTLA-PE (clone MIH26, Biolegend); anti-CD45RA-PE Violet-615 (clone 562, Miltenyi); anti-CXCR5-fluorescein (FITC) (clone REA103, Miltenyi); anti-HVEM-PE Cyanine-7 (PE-Cy7) (clone 122, Biolegend); anti-CD25-APC (clone REA 570, Miltenyi); anti-CD3-Alexa Fluor 700 (clone UCHT-1, BD Pharmingen); anti-CD4-APC Violet-770 (clone REA 623, Miltenyi); anti-CCR6-PE-Cy7 (clone 11A9, BD); anti-CXCR3-APC (clone REA232, Miltenyi). Dead cells were excluded using 4’,6-diamidino-2-phenylindole (DAPI) and single cells were discriminated from aggregates or doublets using SS-W versus SS-H and FS-W versus FS-H plots. Cell acquisition was performed using 10-color Flow Cytometer Gallios-Navios (Beckman Coulter). At least 1x10⁶ cells were analyzed using FlowJo 7.6.5 software (TreeStar) with the strategy depicted in Figure 1 by using Fluorescence Minus One (FMO) controls to define gates.

Leukemic cells, B lymphocytes and NK cells from patients and HD were identified by flow cytometry. For this purpose, the following antibodies were employed: anti-CD19-APC and anti-CD3-PE (Immunostep, Salamanca, Spain), anti-CD3-FITC and anti-CD56-APC (both from Cytognos, Salamanca, Spain). Leukemic cells were identified as CD19+. NK cells were defined as CD56+CD3−. BTLA and HVEM expression was evaluated using anti-BTLA-PE (clone MIH26, Biolegend, San Diego, CA, USA) and anti-HVEM-PE (clone 122, Biolegend). Cells were analyzed in a Cytoflex S flow cytometer and CytExpert 2.3 software (Beckman Coulter, Brea, CA, USA).