αcd4 fitc

Anti-γδTCR-PE, αTCRβ-APC-eFluor780, αCD11c-FITC, αCD45.1-BV421, αSlamf4-PE, αSlamf4-FITC and αGranzymeB-FITC (eBioscience, San Diego, CA); αCD4-FITC and αTCRβ-FITC (BD Biosciences, San Jose, CA). αCD3-PB, αCD3-BV421, αCD8α-APC, αCD8α-PE-Cy7, αCD8β-PE, αCD8β-PerCP/Cy5.5, αCD4-BV650, αSlamf2-PE, αCD45.2-AlexaFluor 647, αCD45.2 FITC, αCD11b-PECy7, αCD11c-APC, αI-A/I-E-PB, αCCR9-PE, and αα4β7-APC were purchased from Biolegend (San Diego, CA). Data were collected using a BD LSRII and analyzed with FlowJo software (Tree Star, Ashland, OR).

Multicolor flow cytometric analysis was performed on host cells from peritoneal fluid samples. Staining for surface markers was performed using α-CD3 APCCy7, α-CD4 FITC, α-CD8PEy7, CD11b PerCP, α-CD14 APC, CD20 PE, α-HLA-DR BV421 and α-CD45 BV500 antibodies (BD Biosciences, CA, USA). Cells (1.0 × 10⁶) were stained for 25 min at 4°C in the dark with a cocktail of antibodies followed by fixation (eBiosciences) and wash. Appropriate fluorescence-minus-one (FMO) and/or isotype controls were used. Stained cells were acquired on a 3-laser instrument (Canto II, BD Bioscience), and data were acquired with FACSDiva 6.1.3 (BD Bioscience). The relative percentages and the absolute count of each subpopulation were determined using FlowJo 10.1 software (TreeStar San Diego CA, USA).