True nuclear tf buffer set

For cell sorting experiments, splenic CD4⁺ T cells from LCMV Cl13-infected mice were isolated using the EasySep mouse CD4 T cell isolation kit (STEMCELL; Cat#19852). Enriched CD4⁺ T cells were then stained using LCMV-specific GP_66-77 PE tetramer from National Institutes of Health along with CD4 and CD44 antibodies in FACS buffer. The staining was performed in the dark for 1 hr at room temperature, followed by three washes with FACS buffer. Gp66-specific CD4⁺ T cells were sort-purified on a FACS-Aria sorter. For FACS analyses, single cell suspensions obtained from spleenocytes from LCMV Cl13-infected mice were stained with GP_66-77 PE tetramer in conjunction with other surface antibodies in FACS buffer, followed by three washes in FACS buffer. Samples were then acquired on either an Aurora Cytek (Cytek Biosciences) or LSR-II (BD Biosciences). In some experiments, after surface staining, cells were fixed with buffer from the True-Nuclear TF Buffer Set (BioLegend) for 1 hr. Then cells were then washed with permeabilization buffer and stained with antibodies against TFs in permeabilization buffer. Analyses were performed using Flowjo software version 10.8.1.

Standard flow cytometric methods were used for staining of cell-surface proteins. For intracellular staining, cells were stained with surface markers, fixed, and permeabilized with BD Cytofix/Cytoperm Kit (BD Biosciences) or True Nuclear TF Buffer Set (BioLegend). Flow cytometric tests were performed on FACSCelesta (BD Biosciences). Data were analyzed with FACSDiva (BD Biosciences) and FlowJo software (BD Biosciences). Gating strategy and antibodies used for staining are provided in Supplementary Material.

AM epithelial cells were harvested and suspended in cell staining buffer (0.5% BSA in PBS with 2 mM EDTA) followed by incubation with primary antibody for LGR5 at 4 °C for 30 min. After washing with PBS, the cells were incubated with appropriate fluorochrome-conjugated secondary antibody in the dark at 4 °C for 30 min. True-Nuclear™ TF Buffer Set (BioLegend) was utilized for OCT4 (Abcam, ab18976) after the membrane staining of LGR5. Isotype-matched IgG control antibodies were used as negative controls. ALDH activity was identified by a nonimmunological method (ALDEFLUOR Kit, STEMCELL) and the inhibitor of ALDH enzyme (ALDEFLUOR DEAB Reagent, STEMCELL) was used as negative controls. Samples were analyzed by BD LSRII flow cytometer. Data were processed and analyzed by FlowJo software.