Brdu incorporation assay

Bromodeoxyuridine (BrdU) incorporation assay (Abcam, USA) performed to detected DNA synthesis ability of AML cells according to the manufacturer’s instructions. In brief, cells were seeded into 6‐well plates and treated with the stimulus for forty-eight hours. BrdU solution (10 μM) was incubated for 2 h and washed with PBS. Cells were fixed with paraformaldehyde for 30 min, followed with Triton X-100 for 15 min. After, cells were treated with BrdU detection antibody (1:1000, ThermoFisher, China) at 4 °C for overnight. Cells were washed with ice-cold PBS for three times. Secondary antibodies which were incubated for 2 h. DAPI was treated for staining of cell nuclear. At last, cells were examined by fluorescence microscopy (Olympus, Japan) with the Image-Pro Plus software (Olympus). Each experiment was performed three times.

The proliferative activity of subconfluent sl-pHCs was determined with regard to distribution in the cell cycle and population doubling time (PDT). The cells were stained with Muse® Cell Cycle Kit and Muse® Count and Viability Kit, respectively. The analysis was performed on Muse Cell Analyzer – the reagents and analyser derived from Luminex/Merck (Poznan, Poland). The number of cells was determined routinely, and obtained data were used to calculate PDT with an algorithm published by Heuer et al. [11 (link)] and Cell Calculator [12 ]. The proliferation of sl-pHCs was also evaluated based on the BrdU incorporation assay (Abcam, Symbios, Straszyn, Polska). For BrdU analysis, cells were seeded at density 2.5 × 10³on a 96-well plate. All tests were performed accordingly to well-described methods [8 (link), 13 (link)] and in compliance with protocols provided by the manufacturers of the assays. Furthermore, colony-forming unit (CFU-E) occurrence efficiency was assessed after ten days of culture using the earlier protocol for stromal [8 (link)] and cancer cells [14 (link)].

Detection of BrdU incorporation into HTR-8/SVneo cells to evaluate cell proliferation based on BrdU incorporation assay (Abcam). The cells were placed on 96-well plates (5 × 10³ cells per well) and incubated for 24 h, then 10 M BrdU was added for another 15 h. The cells were then immobilized for 30 min and incubated with 100 μL of BrdU for 60 min. After incubating with 100 μL IgG for 30 min, 100 μL TMB substrate was added and incubated in darkness at room temperature for 30 min. Cell proliferation was evaluated at 450 nm using a microplate reader.