GSM9207633: CITE_HTO; Mus musculus; OTHER
Cell suspensions were enriched for DCs by negative (sdLNs) or positive selection (thymus). Briefly, sdLN cell suspensions were stained with biotinylated antibodies against TER-119, TCR-β, CD3, NK1.1, CD19, and Ly6G (Thermo Fisher Scientific and Biolegend), and thymus cell suspensions were stained with biotinylated antibodies against CD11c. Samples were subsequently incubated with streptavidin microbeads (Miltenyi Biotec) and passed through LS columns attached to MidiMACS or QuadroMACS separators (Miltenyi Biotec) according to manufacturer protocol. Cells in the flow-through (sdLN) or eluted column-bound cells (thymus) were collected. After enrichment for DCs in the sdLNs and thymus tissue samples as described above, single cell suspensions were stained with fluorophore-conjugated antibodies as well as TotalSeqB hashtag antibodies specific against mouse CD45 and MHC-I (Biolegend) to maintain sample identity. Cell sorting was performed on FACSAria II (BD Biosciences) at the Cytometry & Cell Sorting Laboratory at NYUGSoM. 12,500 cDCs from each sample were sorted and pooled together in 50 μl PBS containing 10% FBS. cDCs = Live singlets lineage- (CD3- TCR-β- B220- CD19- NK1.1- NKp46- Siglec-F- Ly6G-) MHC-II+. The resulting cell mixture was prepared according to the 10X Genomics protocol at the Genome Technology Center at NYUGSoM.