Dxflex flow cytometry system

Impact of TP at 10^-8 M concentration on the characteristic features of BM-MSCs were determined by investigating cell surface markers and by evaluating BM-MSCs’ differentiation capacities. For immunophenotyping, 2x10⁵ BM-MSCs were seeded into 6-well tissue culture plates as triplicates, incubated in the presence of 10^-8 M TP for 24 hours followed by collecting cells with trypsinization and labeling with antibodies abovementioned. Cells were immediately read with Beckman Coulter DxFLEX flow cytometry system and analysis was performed on CytExpert software. For evaluation of BM-MSCs’ differentiation capacities, 1x10⁴ BM-MSCs were seeded into 96-well cell culture plates and once they reached 80% confluency, differentiation was initiated by commercial chondrogenesis (Thermo Fisher Scientific, #A1007101), osteogenesis (Thermo Fisher Scientific #A1007201), and adipogenesis (Thermo Fisher Scientific, #A1007001) kits, either supplemented with 10^-8 M TP or not. Differentiation media were replaced twice a week. On the 21^st day of differentiation, cells were fixed with 10% neutral-buffered formalin solution. Chondrogenesis, osteogenesis, and adipogenesis were evaluated by Alcian Blue, Alizarin Red, and Oil Red-O staining, respectively.

K562 cell line (ATCC #CCL-243) was maintained in RPMI medium (Gibco, #11875093) supplemented with 10% FBS (Pan Biotech, #P30-3306) and 1% penicillin/streptomycin antibiotic solution (Sigma Aldrich, #P4333). For evaluating cytotoxic effects of BM-MSCs on the K562 cell line, BM-MSCs were seeded into 35-mm cell culture dishes (1x10⁴ cells/dish) and cultured overnight to allow attachment. Then, the medium was discarded, and BM-MSCs were either treated with 10^-8 M TP for 24 hours followed by co-culturing with K562 cells (1x10⁵ cells/dish) for an additional 24 hours; otherwise, K562 cells were added on BM-MSCs in the presence of 10^-8 M TP and incubated for 24 hours. The effects of TP on the K562 cells were determined by incubating them with medium supplemented with 10^-8 M TP for 24 hours, whereas the effects of BM-MSCs on K562 cells were evaluated by co-culturing cells for 24 hours. K562 cells’ viability was determined by labeling them with 4′,6-diamidino-2-phenylindole (DAPI, Biolegend, #422801) by incubating the cells for 15 minutes under dark conditions at room temperature. The cells were read with Beckman Coulter DxFLEX flow cytometry system, and analysis was performed with CytExpert software.

Healthy human bone marrow-derived mesenchymal stem cells (BM-MSCs, passage 3) were purchased (Gibco, #A15652) and cultured in low-glucose DMEM (Thermo Fisher Scientific, #11054020) supplemented with 10% Fetal Bovine Serum (FBS - Pan Biotech, #P30-3306), 1% L-glutamine (2 mM, Sigma Aldrich, #G7513) and penicillin/streptomycin antibiotic solution (100 U/0.1 mg/ml, Sigma Aldrich, #P4333) at 37 °C degrees containing 5% CO₂ in humid environment. For the evaluation of cell surface markers, cells were detached with trypsin-EDTA (Thermo Fisher Scientific, #25300054), collected by centrifuging at 300 X G for 5 minutes, and then suspended in Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% sodium azide. Next, the cells were labeled with anti-CD73 FITC (Clone:AD2, Biolegend, #344016), anti-CD90 PE (Clone: 5E10, Biolegend, #328110), anti-CD105 PerCP/Cy5.5 (Clone:43A3, Biolegend, #323216), anti-CD34 PE/Cy7 (Clone:581, Biolegend, #343515), anti-CD11b Alexa Fluor 700 (Clone:ICRF44, Biolegend, #301355), anti-HLA-DR Pacific Blue (Clone:L243, Biolegend, #307633), and anti-CD45 (Clone: J33, Beckman Coulter, #A96416) antibodies by incubating under dark conditions for 15 minutes at room temperature. The cells were immediately acquired on Beckman Coulter DxFLEX flow cytometry system. Analyses were performed on CytExpert software.