Ki67 apc

Surface and intracellular staining were performed using routine protocols as described before [52 (link)]. The following antibodies were used: Ki67-APC (Biolegend, San Diego, CA, USA), CD44-APC and CD326/EpCAM-APCVio779 from Miltenyi Biotec (Bergisch Gladbach, Germany). Apoptosis was evaluated by Annexin V/Iodure Propidium staining using the Annexin V-FITC Apoptosis Detection Kit (Invitrogen™ eBioscience™).
ALDH activity was measured by flow cytometry with ALDEFLUOR (StemCell Technologies, Vancouver, BC, Canada) as recommended by the manufacturer. Briefly, 10 × 10⁵ cells were suspended in ALDEFLUOR assay buffer containing an ALDH substrate (BODIPY-aminoacetaldehyde-diethyl acetate, BAAA-DA) and incubated at 37  °C for 45 min. A negative control for each sample was generated by incubation with the ALDH inhibitor diethylaminobenzaldehyde. Cells were washed with the ALDEFLUOR buffer and analyzed by flow cytometry in a flow cytometer (BD Biosciences, San Jose, CA, USA).

Liver tissues were cut into pieces and digested at 37 °C for 45 min with DMEM containing hyaluronidase (1.5 mg/mL, Sigma–Aldrich, USA), collagenase type 1A (1.5 mg/mL, Sigma–Aldrich, USA), and deoxyribonuclease I (20 U/mL, Sigma–Aldrich, USA). The samples were passed through a 70-μm nylon cell strainer to produce a single cell suspension and resuspended in a cold flow cytometry buffer (1% bovine serum albumin and 0.1% NaN₃ in PBS). Fc receptors were blocked with a rat anti-mouse CD16/CD32 antibody (BD Pharmingen, USA). Cells were stained with the following fluorochrome-conjugated anti-mouse antibodies: CD45-BV421, CD11b-BV510, F4/80-FITC, and Ki67-APC (all from BioLegend, USA). The number of cells was measured using 7-amino-actinomycin D (eBioscience, USA). Flow cytometry was performed using a Gallios flow cytometer (Beckman, USA), and data were analyzed using Kaluza software version 1.3.

The following antibodies were purchased from Biolegend: CD3 BV510, CD49b (DX5) APC-Cy7, NKp46 BV421, CD11b PE, Ly49D PE, Streptavidin BV650, CD27 biotin, KLRG1 BV605, KLRG1 PerCP/Cy5.5, CD69 PerCP/Cy5.5, and Ki67 APC. The following antibodies were purchased from eBioscience: CD107a PerCP-eFluor710, Ly49I PE, CD94 FITC, IFNγ APC, and Ly49H PE-Cyanine7. The following antibodies were purchased from BD Biosciences: NKG2A/C/E BV605. The following antibodies were purchased from Miltenyl Biotec: NKG2D (CD314) biotin, Ly49D biotin, and Ly49D PerCP-Vio700. To determine viability Invitrogen Fixable live/dead Aqua stain was used.

Surface markers of MSCs were assayed by flow cytometry analysis (Attune™ NxT Acoustic Focusing Cytometer, A24863, Thermo Fisher Scientific), and the same voltage parameters for FSC and SSC were used in all the assays; FSC was 90, and SSC was 255. Fluorescently labelled antibodies were CD147‐FITC (Biolegend, Cat# 306204), CD90‐PerCP/Cyanine5.5 (Biolegend, Cat# 328117), CD44‐PE/CY7 (Biolegend, Cat# 103029), CD34‐FITC (BD Biosciences, Cat# 560942), CD73‐APC (Biolegend, Cat# 344005), CD105‐PE (Biolegend, Cat# 800503), CD14‐FITC (Biolegend, Cat# 301803), CD45‐APC (BD Biosciences, Cat# 555485), CD29‐PE (Biolegend, Cat# 303003), PCNA‐PE (Biolegend, Cat# 307908) and Ki‐67‐APC (Biolegend, Cat# 350513).