Cell proliferation assay kit

Cell viability was determined using a Cell Proliferation Assay Kit (ab211091) (Sigma Aldrich GmbH, Mannheim, Germany). For the test, U-937 cells were seeded in replicates (n = 2) on a 96-well microplate and treated with PMA in the presence or absence of bioactive compounds (10 μM) at 37 °C. Followed by incubation, the media were discarded, and serum-free medium was added along with MTT reagent and incubated for 3 h at 37 °C. To avoid interference by the MTT reagent, MTT solvent was added, and the 96-well microplate was kept on the shaker for 15 min. The absorbance was recorded at 590 nm, and the results are presented as percentage of the control in Section 2.2. Data are expressed as ±SEM of at least two measurements.

Cell viability was determined by CellTiter-Glo assay (Promega) according to the manufacturer’s instructions. Cell proliferation was determined by BrdU incorporation (Cell proliferation assay kit, Millipore) according to the manufacturer’s instructions.

Cell proliferation was evaluated by measuring 5-bromo-2′-deoxyuridine (BrdU) incorporation using a cell proliferation assay kit (Millipore; Burlington, MA, USA) following the manufacturer's instructions. Briefly, BrdU was added to each well of the 96-well plate and incubated for 24 h. The cells were washed with PBS for several times and then fixed with fixing solution for 30 min at RT, washed intensively, and then incubated with anti-BrdU monoclonal antibody for 1 h at RT. After washing, the cells were incubated with peroxidase-conjugated goat anti-mouse IgG for 30 min at RT, washed with PBS, and then incubated in TMB peroxidase substrate for 30 min at RT. The plate was read at 450 nm by ELISA plate reader (Multiskan EX; Thermo).

Human skin fibroblasts from A‐T patients and unrelated donors unaffected with A‐T were grown in complete Dulbecco's modified Eagle's medium (DMEM) at 37°C in 5% CO₂ and ambient (21%) or 3% O₂. Growth curves were constructed by seeding in triplicates 3.5 × 10⁴ cells/well in 12‐well plates and counting them after trypsinization every 24 h for 5 consecutive days. The number of population doublings (PDs) per passage was obtained by counting the cells after trypsinization according to the formula, PD = (N_f/N₀)log₂, where N₀ is the number of the initially seeded cells and N_f is the number of the cells at the following trypsinization. The number of CPD was calculated as the sum of PDs over passages. Colony formation efficiency was measured according to (Shiloh et al., 1982 (link)), and immunoblotting—according to (Jachimowicz et al., 2019 (link)). SA‐β‐gal activity was monitored using the Senescence Detection Kit (Biovision). SA‐β‐Gal–positive cells were scored in multiple fields. At least 100 cells were identified per condition.
EdU incorporation to cellular DNA was monitored using a Cell Proliferation Assay Kit (Millipore). Edu‐positive cells in randomly selected fields were counted using the ImageJ software (NIH). At least 100 cells were scored for each condition.

The effects of the peptides at concentrations of 2.5 μM, 20 μM, and 100 μM on the proliferation of the hTCEpi were assessed using a BrdU (5-bromo-2′-deoxyuridine) Cell Proliferation Assay Kit (EMD Millipore, MA, USA), according to the manufacturer’s instructions. In short, hTCEpi cells were seeded in a 96-well plate at a density of 3600 cells per well. After 24 h, BrdU was added to the culture media and the plates were incubated for an additional 6 h, after which the cells were fixed using the provided fixing solution. The incorporated BrdU was detected using the provided anti-BrdU monoclonal antibody. Absorbance was measured as OD at 450 nm using a microplate reader (FLUOstar Omega; BMG Labtech, Ortenberg, Germany). The medium only was used as the blank, and cells in the media were used as the control.