Cd45 fitc hi30

Freshly thawed decidual immune cells (1–2 10e⁶ cells) were incubated with Fc blocker (Human TruStain FcX, Biolegend) in PBS with 1% BSA for 10 minutes at 4C and then surface stained with CD45-FITC (HI30, Biolegend) for 30 minutes in the dark. Samples were then washed twice in PBS with 0.04% BSA and incubated with individual 3’ CellPlex oligos (10X Genomics) per manufacturer’s instructions for 5 minutes at room temperature. Pellets were washed three times in PBS with 1% BSA, resuspended in 300 uL FACS buffer and sorted on BD FACS Aria Fusion into RPMI (supplemented with 30% FBS) following addition of SYTOX Blue stain for live versus dead discrimination. Sorted live cells were counted in triplicates on a TC20 Automated Cell Counter (BioRad), washed and resuspended in PBS with 0.04% BSA in a final concentration of 1500 cells/uL. Single cell suspensions were then immediately loaded on the 10X Genomics Chromium Controller with a loading target of 20,000 cells. Libraries were generated using the V3 chemistry (for gene expression) and Single Cell 3 Feature Barcode Library Kit per manufacturer’s instructions (10X Genomics, Pleasanton CA). Libraries were sequenced on Illumina NovaSeq with a sequencing target of 30,000 gene expression reads and 5,000 multiplexed CellPlex reads per cell.