GSM9362945: Donor 6 ADT; Homo sapiens; OTHER
200 million splenocytes were resuspended in PBS containing 2% FBS and 2 mM EDTA, followed by incubation with Human TruStain FcX for 10 minutes. Cells were then labeled with a biotinylated lineage antibody cocktail (anti-CD3 [Cat# 100243], anti-CD19 [Cat# 302204], and anti-CD335 [Cat# 331906], all from BioLegend) for 15 minutes at 4˚ C. Following magnetic separation using the EasySep™ Release Human Biotin Positive Selection Kit (STEMCELL Technologies, Cat# 17663), the unlabeled fraction was collected and subjected to LIVE/DEAD Fixable Aqua Dead Cell Staining Dye (Invitrogen, Cat# L34966) for 10 minutes. The cells were then stained with PE -anti-Lin (CD3/CD19/NKp46), PE-anti-CD89, FITC-anti-CD11c, APC-anti-HLA-DR, and FITC- anti-CD66b, purified anti-CADM1 antibodies for 30 minutes at 4˚ C. The target population (Lin [CD3/CD19/CD56]⁻CD89⁻CD66b⁻CD88⁻HLA-DR⁺) was isolated using a MA900 Multi- Application Cell Sorter (SONY). Purified cells were blocked using antibody staining buffer, which contained human TruStain FcX (BioLegend), and then stained with FITC-anti-chicken, PE-anti- SIGLEC6, and APC-anti-AXL for 30 min at 4°C. After washing three times, cells were then incubated with Biolegend TotalSeq_B_Universal_Cocktail_v1 (Cat# 399904) and custom antibodies purchased from the Biolegend TOTALseq catalogue for 30 min at 4°C. Stained cells were washed three times and encapsulated for CITE-seq. Single-cell suspensions were loaded onto the 10x Chromium platform, followed by 5′ gene expression library, immune repertoire library, and cell surface protein library.