Infinite m microplate reader

The metabolic activity and proliferation of the differently isolated DPSC fractions were measured using the resazurin/Alamar blue^® assay (Bio-Rad, Munich, Germany). Briefly, cells were seeded at 0.5 × 10⁴ cells/cm² and at the indicated points in time (1, 3, 7, 14 and 21 days, respectively), culture medium was replaced by medium containing 10% (w/v) Alamar blue^® reagent. After incubation for 3 h at 37 °C and 5% CO₂, triplicate samples of the supernatant were analyzed by measuring fluorescence according to manufacturer’s instructions (570 nm excitation and 630 nm emission wavelength) in an infinite-M microplate reader (Tecan, Männedorf, Switzerland). The relative amount of Alamar blue^® reduction in the samples was calculated using a 100% reduced Alamar blue^® control as a reference. Blanks and negative controls were routinely included in each run.

The In-Cell Western assay is a proven method for the rapid quantification of proteins in cells [28 (link), 29 (link)]. BV-2 microglia were seeded into a 96-well plate at 5 × 10⁴ cells/ml, and cells treated at 70% confluence. At the end of each experiment, cells were fixed with 8% formaldehyde solution (100 µL) for 15 min, followed by washing with PBS. The cells were then incubated with primary antibodies overnight at 4 °C. The following antibodies were used: rabbit anti-iNOS (Abcam), rabbit anti-phospho-p65 (Cell Signalling Technology), rabbit anti-phospho-IκBα (Santa Cruz Biotechnology), rabbit anti-NLRP3 (Abcam) and rabbit anti-phospho-p38 (Cell Signalling Technology) antibodies. Thereafter, cells were washed with PBS and incubated with anti-rabbit HRP secondary antibody for 2 h at room temperature. Then, 100 µl of HRP substrate was added to each well and absorbance measured at 450 nm with a Tecan Infinite M microplate reader. Readings were normalised with Janus Green normalisation stain (Abcam).

The in-cell western (cytoblot) analysis is a proven method for the rapid quantification of proteins in cells [18 (link), 19 (link)]. PBMCs were seeded into a black 96-well plate at 5 × 10⁴ cells/mL. At 70% confluence, cells were stimulated with spike glycoprotein S1 (100 ng/ml) for different periods. At the end of each experiment, cells were fixed with 8% paraformaldehyde solution (100 μL) for 15 min, followed by washing with PBS. The cells were then incubated with primary antibodies overnight at 4 °C. The following antibodies were used: rabbit anti-phospho-p65 (Cell Signalling Technology), rabbit anti-phospho-IκBα (Santa Cruz Biotechnology), rabbit anti-total IκBα (Santa Cruz Biotechnology), rabbit anti-phospho-p38 (Cell Signalling Technology) and rabbit anti-NLRP3 (Abcam) antibodies. Thereafter, cells were washed with PBS and incubated with anti-rabbit HRP secondary antibody for 2 h at room temperature. Then, 100 μL HRP substrate was added to each well and absorbance measured at 450 nm with a Tecan Infinite M microplate reader. Readings were normalised with Janus Green normalisation stain (Abcam).