Transcription factor perm wash buffer

Frequency and phenotype of peripheral blood and mucosal CD161⁺ CD4⁺ T cells were determined as previously described [17 (link)]. Briefly, thawed samples were washed, stained with LIVE/DEAD Fixable Aqua Dead Cell dye (ThermoFisher, Waltham, MA, USA), blocked for Fc receptors using normal mouse serum (ThermoFisher), and surface-stained with an antibody cocktail. Samples were surface-stained at room temperature for 30 min. Surface staining was performed at 37 °C for panels, including CCR5 antibodies. Cells were then washed and fixed in 2% paraformaldehyde. Cells were fixed in Cytofix/Cytoperm or in Transcription Factor Fixation/Permeabilization buffer (both from BD Biosciences, San Jose, CA, USA) as appropriate for transcription factor analysis. Intracellular staining was performed using the relevant mAbs in Perm/Wash or Transcription Factor Perm/Wash buffer as appropriate (both from BD Biosciences). The LEGENDScreen was performed as previously described [18 (link)]. Samples were acquired on a five-laser, 16-parameter BD LSRII SORP; an 18-parameter LSR Fortessa; or a four-laser, custom-built LSR Fortessa (all from BD Biosciences). Data were analyzed with FlowJo v.9.9.4 or higher (BD Biosciences). See Supplemental Experimental Procedures for specific antibodies used throughout the study.