Pe conjugated anti human cd235a

To assess epithelial cell marker expression on normal tissue, single cells were blocked in phosphate‐buffered saline (PBS) containing 2% FCS, 10% DNase, rat immunoglobulin (Jackson Immunolabs), and antibodies to CD16 and CD32 Fcγ II and III receptors (WEHI Monoclonal Antibody Facility) for 10 min at 4°C. Cells were then incubated with the following antibodies for 25 min at 4°C: PE‐conjugated anti‐human CD31 (BD Pharmingen; clone WM59; 1/40), PE‐conjugated anti‐human CD45 (BD Pharmingen; clone H130; 1/120), PE‐conjugated anti‐human CD235a (BD Pharmingen; clone GA‐R2; 1/120), FITC‐conjugated anti‐human CD236 (EpCAM; Stem Cell Technologies; clone VU‐1D9; 1/40), and APC‐Cy7‐conjugated anti‐human CD49f (integrin a6; clone GoH3; 1/120). Cells were then washed with PBS/2% FCS and resuspended in 7‐AAD (0.2 mg/ml) for live‐cell discrimination. Cells were sorted on a FACSAria flow cytometer (Becton Dickinson). For normal tissue, lineage‐negative (depleted for CD45, CD31, CD235a lineage‐positive cells), epithelial cells (EpCAM⁺CD49f^– + EpCAM⁺CD49f⁺ + EpCAMCD49f⁺) were sorted.

Cells were blocked with rat immunoglobulin (Jackson ImmunoLabs) and antibody to Fc receptor–binding inhibitor (eBioscience) before incubation with the following primary antibodies: phycoerythrin (PE)–conjugated anti-human CD31 (BD Pharmingen), PE-conjugated anti-human CD45 (BD Pharmingen), PE-conjugated anti-human CD235a (BD Pharmingen), BV650-conjugated anti-human epithelial cell adhesion molecule (EpCAM) CD326 (BioLegend), and biotin-conjugated anti-human ITGA6 (eBioscience). Where required, cells were incubated with allophycocyanin-Cy7–conjugated streptavidin (BD Pharmingen). Cells were either stained with 4′,6-diamidino-2-phenylindole (DAPI) for viability or fixed with 1% paraformaldehyde and stained with the Zombie Aqua Fixable Viability Kit (BioLegend). Viable cells were sorted on a FACSAria flow cytometer (Becton Dickinson). Data were analyzed using FlowJo software (Tree Star).