Flow cytometry staining buffer

Cells were dissociated with accutase and checked for viability. Suspensions were then washed twice with Flow Cytometry Staining buffer (R&D Systems). Cells were stained in suspension with conjugated antibodies for TrKA(PE), TrKB(FITC) and TrKC(PE), for 1 h at room temperature. Suspensions were washed twice with Staining buffer and then processed. Cytometry was done using a CytoFLEX S (Beckman Coulter) and data was analysed with FlowJo (BD Life Sciences).

After dissociation by trypsin, cells were suspended in flow cytometry staining buffer (R&D Systems, Minneapolis, MN, USA) at a final cell concentration of 1 × 10⁶ cells/mL. After 30 min of incubation with mouse anti-human CD14 R-PE, CD34 FITC, CD44 FITC, CD45 APC, CD73 APC, CD90 R-PE, CD105 PE-Cy 7, and HLA-DR FITC (all purchased from eBioscience TM, Thermo Fisher Scientific, Waltham, MA, USA), cells were washed twice with 2 mL of flow cytometry staining buffer. The labeled cells were suspended in 500 µL of flow cytometry staining buffer, and analyzed on Attune NxT flow cytometer (Thermo Fisher Scientific).

Apoptosis was evaluated using APC Annexin V (BioLegend), according to the manufacturer's instructions. Cells were analysed by flow cytometry (FACS) and the apoptotic fraction was defined as annexin V positive. HEL cells were treated with increasing concentration of curcumin for 24 and 48 hours. After incubation, cells were harvested, resuspended in Flow Cytometry Staining Buffer (#FC001, R&D Systems) and labelled with 1 μg/μL propidium iodide (PI). Live cells (PI negative fraction) were counted by FACS.

Immuno‐phenotype analysis was performed before and after hMSC expansion on the FC40/DMEM interface. hMSC immune‐phenotype was determined by multi‐parameter flow cytometry by using a previously developed protocol.3 Briefly, cells were harvested from the substrate as described previously, counted and the cell suspension concentrated to achieve 1 × 10⁶cells mL⁻¹. 200 μL of cell suspension was then loaded onto a V‐bottom 96 well plate and centrifuged at 220 g for 5 min. The supernatant was then aspirated, the cell pellet resuspended in flow cytometry staining buffer (R&D Systems, UK) and centrifuged again. The cells were stained with mouse anti‐human monoclonal antibodies (BD Biosciences, UK) for 30 min in the dark at room temperature. The fluorescently‐labelled antibodies were selected based on a panel recommended by the International Society for Cell Therapy (ISCT)22 and these were: CD73 (PE‐Cy7), CD90 (APC), CD105 (PE), CD34 (PE‐Cy5) and HLA‐DR (FITC). After incubation, the samples were washed twice with staining buffer as described before. The stained samples were then analysed on the Guava EasyCyte 8HT flow cytometer (Merck Millipore, UK) equipped with 488 nm and 640 nm excitation lasers. A minimum of 10 000 gated events were recorded for each sample. Post‐acquisition analysis and compensation were performed using the FlowJo software (Treestar Inc, USA).

Propidium Iodide (P1304MP, ThermoFisher Scientific, USA) staining was employed to determine cell cycles. In detail, the collected cells were aliquoted to 1 × 10⁶ cells/100 μL into FACS tubes, followed by twice washing using 1xPBS. Then, cells were centrifuged for 5 min at a speed of 300×g. Furthermore, cells were resuspended using 100 μL of Flow Cytometry Staining Buffer (Cat: # FC001, R&D systems). 10 μL of PI staining solution was added to a control tube of otherwise unstained cells to adjust flow cytometer settings for PI. Cells were mixed gently and incubated for 5 min in the dark. PI fluorescence of samples was determined (using the FL-2 or FL-3 channel) with a FACScan™ instrument. Each detection was repeated at least 3 times. For cell death ratio detection, Annexin V-FITC/PI double-labeled FACS was carried out following the manufacturer’s protocol (Cat: 40302ES20, Yeasen, Shanghai). Flowjo software was used for gate setting FACS original data, and the ggcyto R package (https://bioconductor.org/packages/release/bioc/html/ ggcyto.html) was used for visualization.

Adherent cells from p3 were dissociated and resuspended in flow cytometry staining buffer (R&D Systems, Minneapolis, MN, USA) at a final cell concentration of 1 × 10⁶ cells/mL. Human cells were incubated with the following fluorescent monoclonal mouse anti-human antibodies: CD29 APC (Thermo Fisher Scientific, San Diego, CA, USA); CD44 FITC (Thermo Fisher Scientific); CD73 APC (eBioscience^TM, Thermo Fisher Scientific); CD90 BV510 (BD Biosciences, San Jose, CA, USA); CD105 PE-Cyanine7 (eBioscienceTM); CD14 PE (eBioscienceTM); CD34 APC-eFluor 780 (eBioscienceTM); CD45 Pacific Orange (eBioscienceTM). Canine cells were incubated with the following fluorescent monoclonal mouse antibodies: CD29 APC (Thermo Fisher Scientific); CD44 FITC (Thermo Fisher Scientific); CD73 APC (eBioscienceTM); CD90 APC (eBioscienceTM); CD105 PE-Cyanine7 (eBioscienceTM); CD14 PE (Thermo Fisher Scientific); CD34 PE (eBioscienceTM); CD45 FITC (eBioscienceTM). Cells were washed twice with 2 mL of flow cytometry staining buffer and resuspended in 500 μL of flow cytometry staining buffer. Fluorescence was evaluated by flow cytometry in Attune NxT flow cytometer (Thermo Fisher Scientific). Data were analyzed using Attune NxT software (Thermo Fisher Scientific).