Facscalibur platform

To assess T cell entry into the spinal cord after CCI, flow cytometry was used to measure CD3-positivity levels in mononuclear cells extracted from the dorsal horn, as CD3 is a well-known T cell marker (34 (link)). On day 3 of CCI, after an overdose intraperitoneal injection of urethane (2 g/kg), the rats' lumbar spinal cord segments were harvested. The dorsal horn tissues ipsilateral to the site of CCI or sham surgery were isolated, placed in 0.01-M PBS, and homogenized into single-cell suspensions with a cell strainer. Homogenates were washed with 0.01-M PBS, suspended in a 30%/70% discontinuous-gradient Percoll solution (Sigma-Aldrich), and centrifuged at 390 × g for 30 min.
Mononuclear cells were collected, washed with 0.01-M PBS, and resuspended in a fluorescence-activated cell sorting buffer solution for 30 min at 4°C. The cells were labeled with fluorescein isothiocyanate–conjugated mouse anti-CD3 antibodies (1:100 dilution; eBioscience, San Diego, CA, USA) for 20 min at room temperature, and ≥10,000-cell samples were analyzed with the FACSCalibur platform running with CellQuest software (Becton Dickinson; Franklin Lakes, NJ, USA) to determine the percentage of mononuclear cells that were CD3-positive.

Cells (5 × 10⁵) were seeded into 6-well plates containing complete 1640 culture solution, without FCS, for 48 h. Apoptosis rate was determined using an apoptosis detection kit (Annexin V PE/7-AAD, BD Biosciences, Franklin Lakes, NJ, United States), and analyzed by flow cytometry on a FACSCalibur platform (Becton Dickinson, San Jose, CA, Unied States).

Approximately 1 × 10⁸ cells were harvested from the HL5 culture, washed in phosphate-buffered saline (PBS) and pelleted by centrifugation. Their nuclei were prepared using a nuclei extraction kit NE-PER (Pierce), resuspended in 2 mL PBS containing 1 mM EDTA, 200 μg/mL RNase A, and 50 μg/mL propidium iodide, and then analyzed on a FACS Calibur platform (Becton Dickinson) using an excitation wavelength of 488 nm. To ensure single-nucleus measurement, the gate was set using the FLS-A and FL2-W parameters of the doublet discrimination module.

The phenotype of primary hDPSCs (n = 4) at passage 3 (P3) was characterized using flow cytometry. Briefly, 2 × 10⁵ cells for each marker were resuspended in 50 µL of the PBS buffer and incubated with selected human-specific monoclonal antibodies, directly labeled with PE: CD73 (clone AD2), CD90 (clone 5E10), CD105 (clone 266), CD45 (clone HI30), CD31 (clone WM59), CD54 (ICAM-1) (clone HA58), CD106 (VCAM-1) (clone 51-10C9), HLA ABC (clone G46-2.6), HLA DR (clone G46-6) (all from BD Pharmingen, San Jose, CA, USA), CD44 (IgG2a, clone 156-3C11), (Thermo Fisher Scientific, Rockford, MI, USA) and Stro-1 (clone STRO-1) (Invitrogen, Thermo Fisher Scientific, Rockford, MI, USA). All samples were incubated for 30 min in the dark at 4 °C. Mouse isotype-matched IgG₁ and IgG₂ were applied as negative controls (BD Pharmingen, San Jose, CA, USA). The cells were analyzed using flow cytometry with a FACS Calibur platform (Becton Dickinson, San Jose, CA, USA). The expression levels of the selected antigens were evaluated using the CellQuest software (Becton Dickinson, San Jose, CA, USA).

Apoptosis was assessed by Annexin V-fluorescein isothiocyanate/propidium iodide (annexin V-FITC/PI) binding kit (Becton–Dickinson) using flow cytometry. To this aim, the cells were seeded at 4 × 10⁴ cells/cm² in 6-well plates and incubated for 24 h at 37 °C in a humidified atmosphere of 5% CO₂. The cells, were then maintained in standard conditions (medium supplemented with 10% FBS) for 24 h, after which the cells were exposed to 1% LD with or without 10% PRP for 30 min (Fig. 1). After the treatments, the mediums were collected and the cells were detached with 0.05% of DPBS/EDTA. The chondrocytes were washed twice with DPBS without Ca²⁺ and Mg²⁺. Procedures for labelling of of Annexin V-FITC/PI were carried out according to the manufacturers’ instructions. Flow cytometry data acquisition was performed on a FACSCalibur platform (Becton–Dickinson) equipped with 488 and 633 nm lasers and running CellQuest Software (Becton–Dickinson).Fig. 1

Cell viability. The effects of LD alone or with 10% PRP on cell viability were assayed in chondrocytes maintained for 24 h in medium (starved-cells), in medium containing 10% FBS (FBS-cell) or 10% PRP (PRP-cells). Date are expressed as mean ± SD. The experiments were repeated thrice, in triplicates. *P < 0.005 vs cell untreated of each experimental group (PBS)

The cells (5 × 10⁵) were seeded into 6-well plates containing complete RPMI 1640 culture medium without fetal bovine serum for 48 h. The rate of apoptosis was then detected with an apoptosis detection kit (Annexin V PE/7-AAD BD Biosciences, Franklin Lakes, NJ, USA) by flow cytometry analysis using a FACSCalibur platform (Becton Dickinson, San Jose, CA, USA).