Facscanto 2 cytometer

Flow cytometric analyses were performed using a FACS Canto II cytometer (BD Biosciences, San Jose, CA, USA) to assess the purity and CD137-mediated expression of NK cells. The following mAbs were used for staining human peripheral blood mononuclear cells (PBMCs): phycoerythrin (PE)-conjugated CD56, PE CD137L, PE NKp30, PE NKp44, PE NKp46, PE CD122, PE CD117, PE TRAIL, PE CD107a, PE CD158e, PE CCR5, PE CD25, PE CD226, PE NKG2D, PE FasL, PE NKG2A, PE NKG2C, fluorescein isothiocyanate (FITC)-conjugated CD3, FITC CD158a, FITC CD158b, allophycocyanin (APC)-conjugated CD137, APC anti-HER2, APC anti-epidermal growth factor receptor (EGFR), and 7-aminoactinomycin D (7-AAD); all mAbs were obtained from BD Biosciences. Stained cells were collected on a FACS Canto II cytometer (BD Biosciences), and data were analyzed using FlowJo version X software (FlowJo, LLC, Ashland, OR, USA).

Lymphomononuclear cells from the livers, spleens and peripheral blood were incubated with fluorochrome-labeled antibodies at 5×10⁵ / tube for 30 min at 4°C, and characterized using a FACS Canto II cytometer (BD Biosciences). For the intracellular cytokine staining, Caltag^TM, Fix&Perm^® reagents (Invitrogen, Carlsbad, CA, USA) were used following the manufacturer's instructions. The following antibodies were used: fluorescein isothiacyanate (FITC)-conjugated anti-CD4, FITC-conjugated anti-CXCR5, FITC-conjugated anti-CD19, Phycoerythrin (PE)-conjugated anti-PD-1, PE-conjugated anti-ICOS, PE-conjugated anti-PDL-1, PE-conjugated anti-ICOSL, PE-conjugated anti-IL-21R, allophycocyanin (APC)-conjugated anti-CD4 (eBioscience, USA), PE-conjugated anti-IL-21 (BD Biosciences, USA) and FITC-, APC- or PE-conjugated isotype antibodies (eBioscience, USA).
Lymphomononuclear cells from the livers and spleens were incubated with HBc-derived peptides HBc1-20 (1 mg / ml)at room temperature for 10 min. HBc-derived peptides HBc1-20 was purchased from Sangon Biotech (Sangon, Shanghai, China). The cells were then washed twice with PBS containing 1% BSA (BD Biosciences), and incubated with anti-CD4, anti-CXCR5 for 30 min at 4°C, and characterized using a FACS Canto II cytometer.

Aldehyde dehydrogenase (ALDH) enzyme activity was assayed with an AldefluorTM kit assay (Stemcell Technologies, Vancouver, BC, Canada) following the manufacturer’s instructions. Labelled cells were acquired using a FACS CANTO II cytometer (BD Biosciences) for analysis.
For CD133 analysis, cells were detached with trypsin, washed with PBS, resuspended in a blocking buffer solution (PBS supplemented with 2% bovine serum albumin (BSA)) and then stained with anti-human CD133-APC antibody or IgG control (Miltenyi Biotec, Auburn, CA, EEUU). After 15 min incubation at 4 °C, the dark cells were washed and acquired using a FACS CANTO II cytometer (BD Biosciences) for analysis.

Cells were pulse-labeled for 30 min with 10 μM BrdU (Sigma) before harvesting, fixed in 70% ethanol, incubated with 2 M HCl (20 min), washed, and incubated with FITC-conjugated anti-BrdU antibody (1 h/37 °C). To monitor DNA content, cells were incubated o/n with 50 μg/ml propidium iodide (PI; Sigma) and 10 μg/ml RNase A (Qiagen). Flow cytometry analyses were performed in a FACS Canto II cytometer (BD, San Jose, CA) and analyzed with FlowJo 9.4 (Tree Star, Ashland, OR). For cell cycle sorting, cells were stained with 5 μg/ml Hoestch 33342 (Invitrogen) for 30 min at 37 °C, resuspended in DMEM + 0.1% FBS and sorted in a BD Influx device (BD, San Jose, CA). For the analysis of cell viability, cells were incubated with 40 nM tetramethyl rhodamine ethylester (TMRE, Sigma) for 10 min at 37 °C, washed in PBS, stained with 1 μg/ml DAPI and analyzed in a FACS Canto II cytometer (BD). Data analysis was performed with FlowJo 9.4 (Tree Star).

Bone marrow (BM) cells were flushed from left femora with PBS. Red blood cells were lysed using lysis buffer (Thermo Fisher Scientific, A1049201). After being washed with PBS, cells were suspended in PBS with 2% FBS. Cells were stained with various fluorescein-labeled Abs and subjected to flow cytometric analysis using a Becton-Dickinson FACSCanto II Cytometer. FITC-anti-CD45, PE-anti-CD105, APC-anti-CD11b, and PECY5-anti-Gr1 antibodies were purchased from eBioscience. Results were analyzed by Flowjo7 data analysis software (Ashland, OR).

For flow cytometry, we harvested thymus, spleen and LN from mice (male 129P2_Ola × C57BL/6J F1 background at 8 weeks of age) and stained cells with a 100-fold dilution of phycoerythrin-Cy7-, fluorescein isothiocyanate (FITC)-, APC-Cy7-, Pacific-Blue-, APC- and phycoerythrin-conjugated monoclonal antibodies (mAbs). We purchased rat or hamster monoclonal antibodies to mouse CD4 (GK1.5), CD3 (145-2C11), B220 (RA3–6B2) and CD11b (M1/70) from Biolegend (USA) and CD8 (53-6.7) from eBioscience (USA) and CD11c (HL3) from BD Pharmingen (USA). We performed cell-surface staining according to standard techniques, and we analysed stained cells using a FACS Canto II cytometer and either CellQuest (BectonDickinson, USA) or FlowJo software (Tree Star, USA).