Homo sapiens
Protocols: ADRCs were isolated from human lipoaspirates using an automated processing Celution® 800/IV device (Lorem Cytori Therapeutics) and used immediately for further enrichment of CD31-postitive ADRCs. ADRCs were subjected to red cell lysis before enrichment of CD31-postitive ADRC subpopulations was accomplished by magnetic cell sorting (MACS) using MS separation columns and CD31 microbead kits (Miltenyi Biotech). To enable single-cell RNA sequencing (scRNA-seq), at least 1,000,000 CD31+ ADRCs from each of four subjects (one male and three females) were methanol-fixed and stored at -80°C. Briefly, the cells were resuspended in 500 µl PBS with 1% BSA and 1 U/µl RNAsin PLUS RNase Inhibitor (Promega) and incubated on ice for 5 min to ensure the integrity of the RNA in the cells. The cells were passed through a 40 µm Flowmi® Cell strainer (VWR) to create a single-cell suspension. The cells were centrifuged at 400g for 3 min at 4°C, and washed in 1 ml RNase-free PBS and subsequently centrifuged at 400g for 3 min at 4°C, before the cell pellet was resuspended in 200 µl RNase-free PBS and 800 µl ice-cold methanol gently added dropwise to the cells. The cells were fixed for 30 min at -20°C and subsequently stored at -80°C until use. Before sequencing, fixed cells were thawed and rehydrated. Briefly, the cells were centrifuged at 1,000g for 10 min at 4°C and the supernatant was removed. The cell pellet was resuspended (1,000 cells/µl) in a rehydration buffer (3x saline sodium citrate, 0.04% BSA, 1 U/µl RNAsin Plus, 40 mM Dithiothreitol) and incubated at 4°C for 30 min. Immediately following rehydration, 8,000 cells were loaded onto the 10x Genomics Chromium controller (10X Genomics). Libraries were prepared according to the instructions of the manufacturer using the 10x Genomics Single-Cell 3' v3, Chromium Single Cell B Chip Kit, 48 runs (10X Genomics).
isolate
not applicable
individual
Pt 2
organism
Homo sapiens
organism
Homo sapiens
immunophenotype
CD31-positive
sex
female
scientific_name
Homo sapiens
common name
human
organism part
subcutaneous adipose tissue
cell_type
adipose-derived regenerative cell
age
69
developmental stage
adult
ENA-LAST-UPDATE
2023-04-20