Facscanto 2

The total analysis of lymphocytes and subpopulations was determined in accordance with what was observed by H. T. Maecker Maecker et al. (2000). The analysis of lymphocyte subpopulations was carried out using an 8-color BD FACS Canto™ II (Becton Dickinson, San Jose, CA) flow cytometer. The Treg lymphocyte subpopulation was identified by monoclonal antibodies labeled CD45 V500, CD3 V450, CD4 PerCP-Cy5.5, CD25 PE, CCR4 PE-Cy7, CD 27 Alexa 647, (BD Biosciences, San Jose, CA) and analyzed for medium of BD FACS Canto™ II (Becton Dickinson, San Jose, CA) using FACSDiva™ software.
Once the peripheral venous blood sample is drawn, it is then prepared for the flow cytometry analysis. To prepare the sample, 50 μl of blood and 50 μl of mixed antibodies are combined at room temperature, away from direct light sources. After about 30 minutes of incubation, time necessary for the binding between membrane antigens and antibodies, the erythrocyte component is eliminated using BD FACS™ Lysing Solution and subsequent two washes with PBS liquid and centrifugation at 1500 rpm for 5 minutes. The sample is then re suspended in PBS liquid for the flow cytometric analysis.

In this part of the study, 5*10⁴/100 μl rMSCs were seeded on each scaffold. After 1, 4, and 7 days of co-culture, the viability of the cells cultured in 3D TPMS porous scaffolds were assessed using the Cell Counting Kit (CCK8; Invitrogen, United States). Briefly, at various intervals, the culture wells in each group were replenished with fresh medium containing CCK-8 solution, as recommended in the manufacturer’s procedure. After one hour incubation at 37°C, the solution absorbance was measured at 450 nm.
After 24 h of culture, the cells of each group were trypsinized and pooled for cell cycle analysis. The cells were stained with propidium iodide (PI, Cell Cycle and Apoptosis Analysis Kit, Beyotime, China), and the PI-elicited fluorescence of individual cells was measured using flow cytometry (FACS Canto Ⅱ, BD). After 7 days of co-culture, the Calcein-AM/PI Double Stain Kit (Yeasen, China) was used for live/dead assay using flow cytometry (FACS Canto Ⅱ, BD).

A total of 4 × 10⁵ stimulated OVA-specific CD8⁺ T cells were added to 1 × 10⁵ B16F10-mCherry-OVA and treated with fresh condition medium (1:2 dilution). After 48 h, cells were stained using anti-Annexin V, 7-AAD (BioLegend, 640930) and anti-CD8 for 20 min at 4 °C, and were immunophenotyped by flow cytometry using an LSRFortessa or a FACSCanto II (Becton Dickinson). For CGRP, 4 × 10⁵ stimulated OVA-specific CD8⁺ T cells were added to 1 × 10⁵ B16F10- mCherry-OVA and treated with CGRP (100 nM). After 24 h, the cells were stained using anti-Annexin V, 7-AAD (BioLegend, 640930) and anti-CD8 for 20 min at 4 °C, and were immunophenotyped by flow cytometry using an LSRFortessa or a FACSCanto II (Becton Dickinson). Cytokine expression levels were analysed after in vitro stimulation (PMA–ionomycin; see ‘Intracellular cytokine staining’).

To fulfill the criteria of the International Society for Cellular Therapy, MSCs were analyzed for the expression of CD34, CD45, CD73, CD90, CD105, and CD44 (fluorochrome-conjugated monoclonal antibodies from BD Bioscience) by flow cytometry (FACSCanto II Becton Dickinson, BD Bioscience, USA). Cellular apoptosis was evaluated by Annexin V and 7AAD staining, detected by FACSCanto II Becton Dickinson (BD Bioscience, USA). Unstained MSCs were used as negative controls to assess background fluorescence. Cell analysis was performed using FlowJo software (TreeStar Inc., USA).

For analysis of lentivirus transduction rate of NK-92 cells, the GFP expression in Ctrl-NK-92 and CAR-NK-92 was analyzed by a FACS machine (FACSCanto II, Becton-Dickinson, USA). For analysis of EGFR and PD-L1 surface expression, 1 × 10⁶ cancer cells were incubated with influorescence-labeled antibody in 200 μl phosphate-buffered saline (PBS) with 2% bovine serum albumin (BSA) for 30 min at room temperature in dark, washed, and then analyzed by the FACS machine (FACSCanto II, Becton-Dickinson, USA). The PE-labeled mouse anti-human EGFR antibody (555997) and isotype control (555743) were purchased from BD Bioscience. The PE/Cy7-labeled mouse anti-human PD-L1 (329718) and isotype control (400325) were purchased from BioLegend.

HepG2 cells were treated with various concentrations of SC-III3 and were harvested by trypsinization, fixed with 70% ethanol and stored at -20°C for at least 1 day. Following fixation, cells were subjected to a PBS wash and then stained with DNA staining solution comprising 2.5 mg/mL propidium iodide (PI) and 50 mg/mL RNase A in PBS. Samples were incubated at 37°C for 30 min away from light and then analysed on a FACSCanto II flow cytometer (FACSCanto II, Becton Dickinson, USA).

The Vδ2 T cells and PMN-MDSC frequency and phenotype were evaluated utilizing the following monoclonal antibodies: anti-Vδ1 (Life technology), anti-NKG2A, anti-NKG2D (Beckman Coulter), anti-NKG2C (R&D system), anti-Vδ2, anti-CD3, anti-CD15, anti-CD33, anti-HLA-DR, cocktail of antibodies anti-CD3, -CD56, -CD19, anti-CD14, anti-CD11b (BD Biosciences). In brief, the cells were washed twice in PBS, 1% BSA, and 0.1% sodium azide and were stained with the mAbs for 15 min at 4°C. The cells were then washed and fixed with 1% paraformaldehyde and analyzed using a FACS Canto II (Becton Dickinson). For intracellular staining, membrane staining was performed as above described. After fixation cells were incubated with anti-IFNγ (BD Biosciences) for 30 min at room temperature. CD107a detection was accomplished by antibody staining during cell stimulation. After washing cells were analyzed using a FACS Canto II (Becton Dickinson). Apoptosis induction of Daudi cells were accomplished by evaluating Annexin V ligation to Daudi (Annexin V-FITC Apoptosis Detection Kit, eBiosciences) following the manufacturer’s instruction. Then cells were stained with anti-CD19, anti-Vδ2, anti-CD3, anti-CD15.