GSM9635515: NTCP_Positive_A; Homo sapiens; RNA-Seq
Isolation of NK cells from peripheral blood  Peripheral NK cells were isolated from two representative F3/F4 advanced liver fibrosis MASH patients (A and B) selected based on age and sex and were sorted on the basis of NTCP expression as described below.                                                                                                                                                                                                                                                                                     Peripheral blood (20 mL) was obtained by venipuncture and collected into 5 mL sodium citrate tubes. Samples were diluted 1:1 with phosphate-buffered saline (PBS), gently mixed, and layered over an equal volume of Ficoll-Paque (GE17-1440-02; Merck) in a separate tube. Tubes were centrifuged at 515 × g for 20 minutes at room temperature without acceleration or braking. The lymphocyte-containing interface was collected under sterile conditions, transferred to a new tube, and washed with PBS or cell wash buffer by centrifugation at 515 × g for 10 minutes to remove residual contaminants.NK cells were isolated from the lymphocyte fraction using a Human NK Cell Isolation Kit (Catalog #17955; STEMCELL) via negative selection, following the manufacturer's instructions. Briefly, non-NK cells included T cells, B cells, stem cells, dendritic cells, monocytes, granulocytes, and erythroid cells were magnetically labeled with a biotin-conjugated antibody cocktail (NK Cell RapidSpheres™) and removed, leaving untouched NK cells for downstream experiments. Following removal of dead cells via the Dead Cell Removal Kit (Miltenyi Biotec).                                                                                                           Fluorescence-activated cell sorting: NK cells [3X106/100 ul of PBS] were stained with 500 nM preS1(2-48)-myr-FITC with a cocktail of NTCP antibodies targeted for different extracellular epitopes, such as anti-NTCP (BS-1958R, Thermo), anti-NTCP (D-5) Alexa Fluor 488 (sc-518115, Santa Cruz Biotechnology), for 45 minutes at 4°C (these antibodies showed a fluorescent signal without NK permeabilization). NK cells were then sorted based on NTCP expression (NKNTCP- and NKNTCP+) via a FACSAria III Cell Sorter (BD FACSAria) for fluorescence-activated cell sorting (FACS). The cells that did not present NTCP-positive staining represented the NTCP-negative population. This was confirmed by comparable staining patterns following the use of isotype IgG labeled with the relevant fluorochrome [isotype negative control]. Approximately 10,000 cells from each NK cell subpopulation (NKNTCP+ and NKNTCP-) were loaded into the Chromium Controller system. The RNA libraries were subsequently prepared via the Chromium Single Cell 3' Reagent Kit v.3 (10X Genomics) according to the manufacturer's instructions.