Easysep human pe positive selection kit 2

CR, CAR, CR + CAR, and NTD T cells (1e6) were co-cultured with SKOV3 cells for 24 h in triplicate. Following this, the co-cultured cells were labeled using a CD45-PE antibody (BioLegend) for 15 min at room temperature and sorted using an EasySep™ Human PE Positive Selection Kit II (Stemcell). Briefly, CD45-PE-stained cells were mixed with a PE selection cocktail and RapidSpheres™. CD45-PE-positive cells were labeled with magnetic beads and sorted using a magnet. The sorting efficiency was assessed using an FASC greater than 95%. Total RNA was extracted from the sorted cells using the RNAeasy Mini Kit (QIAGEN), and RNA was detected with integrity and deep sequenced by Novogene, Beijing. The functional analyses of DEGs were performed using DAVID and KEGG for Ingenuity canonical pathway enrichment. The heatmaps and volcano plots of differentially expressed genes in each group and gene set enrichment analysis (GSEA) results were depicted using R (version 3.6.3).

CD44+, CD44-, CD44v6+, and CD44v6- cells were obtained by magnetic activated cell sorting (MACS). MACS sorting of the CD44 and CD44v6 epitopes was conducted using a PE-conjugated anti-human antibody and EasySep™ Human PE Positive Selection Kit II (STEMCELL Technologies, Vancouver, Canada), according to the standard protocol (https://cdn.stemcell.com/media/files/pis/DX22359-PIS_1_0_0.pdf). CD44v6+ LCSCs were cultured as described previously [30 (link)].

Further enrichment of trophoblasts was established by magnetic bead retraction. For this, the EasySep human PE positive selection kit II was used (Stem Cell Technologies, #17684). All incubations were performed at room temperature in the dark: 100 µL of FcR blocker and 20 µL of PE-labeled ITGA6 (CD49f) antibody were added to the cells for 15 min. Further steps included addition of 30 µL of Selection Cocktail and 30 µL of RapidSpheres, both followed by 15 min of incubation. The step of bead retraction was repeated three times by replacing the flow-through each time again on the column. After the final step, cells were washed in PBS/2% FBS/1 nM EDTA and resuspended in TS-basal medium. This medium has been described previously (14 (link)) and contains DMEM/F-12 with 0.05 mM 2-mercapto-ethanol, 0.2% FBS, 1% penicillin/streptomycin (p/s), 0.3% bovine serum albumin (BSA), 0.5% KnockOut Serum Replacement (KSR), and 1% Insulin-Transferrin-Selenium (ITS)-X. A summary of reagents and growth factors is displayed in Supplementary Table 1.