Calcein am

Cell viability was measured using a live staining with the fluorescent marker calcein-AM (PromoCell, Heidelberg, Germany). Three controls were included: a medium control with culture medium instead of treatment, a solvent control with 1% DMSO, and a positive control with heat-inactivated cells (95 °C, 20 min). Following treatment and recovery, with and without exogenous metabolic activation, 1.5 μL calcein-AM (10 μM in DMSO) was applied and incubated for 1 h at room temperature in the dark. Cells were analyzed using a flow cytometer (BD Accuri C6, Becton Dickinson, Franklin Lakes, NJ, USA). A total of three biological replicates were measured and evaluated by calculating the mean of the live staining calcein-AM. Only non-cytotoxic concentrations with a viability of at least 80% compared to the control were used for gene expression analyses.

Cell viability was determined by staining cells with calcein-AM (PromoCell, Heidelberg, Germany), which is converted to green fluorescent calcein by intracellular esterases in viable cells. Cells, grown and treated in 24-well plates, were dissociated from the culture plates with accutase and stained with 2.5 μg/mL calcein-AM at 37 °C for 1 h. Labeled cells were washed with phosphate-buffered saline without Ca²⁺ and Mg²⁺ and were measured by fluorescence activated cell sorting (FL2H). In the calcein assay, the total cell number does not contribute to the results; only the percentage of active cells in the remaining cell population is determined.

Cells (10⁵) were reversely transfected with 30 nM of siCon or siMcl-1 and seeded in 24-well plates. After 48 h, the cells were incubated with soluble TRAIL (50 ng/ml) or infected with AdV-TRAIL. Dox (1 μg/ml) was added, when TRAIL expression was intended. Cell viability was determined by calcein-acetoxymethyl (calcein-AM) staining at 24 h and 48 h after transduction with AdV-TRAIL. For measurement of cell viability, cells were harvested by trypsinization and stained with 2.5 μg/ml calcein-AM (PromoCell, Heidelberg, Germany) for 1 h at 37 °C. Subsequently, the labeled cells were washed with PBS and the percentage of calcein-AM-positive cells measured by flow cytometry (FL2H) with a FACS Calibur (BD Biosciences, Bedford, MA, USA).

The majority of the chemicals were purchased from Sigma (St. Louis, MO, USA), including hydroxyethylcellulose (#09368), Evans blue dye (#E2129), triphenyltetrazolium chloride (#T8877), HEPES buffer (#H3375), dimethyl-sulfoxide (DMSO #D5879 or #D2650), 2-Deoxy-d-glucose (#D8375), and laminin (#L2020). Additional chemicals used in experiments were MgSO₄ (Reanal, Budapest, Hungary, #20341), collagenase II (Biochrom GmbH, Berlin, Germany, #c2-22), fetal bovine serum (FBS, EuroClone, Pero MI, Italy, #ECS0180L), M199 (Lonza, Verviers, Belgium, #BE12-117F), bovine serum albumin (BSA, Santa Cruz Biotechnology, Santa Cruz CA, USA, #sc-2323), calcein AM (PromoCell GmbH, Heidelberg, Germany, #PK-CA707-80011-3), Dulbecco’s phosphate-buffered saline (DPBS, Gibco, Grand Island New York, NY, USA, #14080-055), heparin (Merck, Darmstadt, Germany, #375095), rosiglitazone (MedChemExpress, HY-14600), and pentobarbital (Produlab Pharma, Raamsdonksweer, The Netherlands, #17F015).

Quantification of apoptosis was performed by cell cycle analysis. Cells were harvested by trypsinization and lysed in hypotonic buffer. Isolated nuclei were stained for 1 h with 40 mg/mL propidium iodide (Sigma-Aldrich, St. Louis, MO, USA). Cells in G1, G2, and S-phase, as well as sub-G1 cells, were quantified by flow cytometry at FL3A using a FACS Calibur (BD Bioscience, Bedford, MA, USA). Due to the washing out of small DNA fragments, nuclei with less DNA than G1 (sub-G1) correspond to apoptotic cells.
Cytotoxicity was determined in cell culture supernatants by quantification of released lactate dehydrogenase (LDH) activity. Cells were stained by a cytotoxicity detection assay (Roche Diagnostics, Penzberg, Germany) and analyzed by an ELISA reader. Triton x100-treated cells (0.7%) were used as a positive control.
Cell viability was determined by staining cells with calcein-AM (PromoCell, Heidelberg, Germany; AM = acetoxymethylester), which is converted in viable cells through intracellular esterase activity to green-fluorescent calcein. Cells, grown and treated in 24-well plates, were harvested by trypsinization and stained for 1 h with 2.5 µg/mL calcein-AM at 37 °C. After labeling, cells were washed with PBS and measured by flow cytometry (FL2H).

Quantification of apoptosis was performed by cell cycle analysis. Harvested cells were lysed in hypotonic buffer, and isolated nuclei were stained for 1 h with 40 mg/mL propidium iodide (Sigma-Aldrich, St. Louis, MO, USA). Cells in G1, G2 and S-phase as well as sub-G1 cells were quantified by flow cytometry at FL3A with a FACS Calibur (BD Bioscience, Bedford, MA, USA). Due to the washing out of small DNA fragments, nuclei with less DNA than G1 (sub-G1) correspond to apoptotic cells.
Cell viability was determined by staining cells with calcein-AM (PromoCell, Heidelberg, Germany), which is converted through intracellular esterase activity in viable cells to green-fluorescent calcein. Cells, grown and treated in 24-well plates, were harvested and stained for 1 h with 0.5 µM calcein-AM at 37 °C. After labeling, cells were washed with PBS and measured by flow cytometry (FL2H).
Cell proliferation was determined by WST-1 assay (Roche Diagnostics) following the protocol of the supplier. The assay depends on the cleavage of the water-soluble tetrazolium (WST) salt by mitochondrial dehydrogenases in metabolically active cells.

Sterilized BCP granules were placed on confocal dishes after which OB cells were seeded at a density of 20,000 cells/well. Cells randomly received the following treatments: 1) only BCP granules (control), 2) BMP-2 loaded BCP granules (BMP-2), 3) BCP granules plus OB-EV_Dex (OB-EV_Dex), and 4) BMP-2 loaded BCP granules plus OB-EV_Dex (BMP-2+OB-EV_Dex). After various treatments for 1, 4, and 7 days, cell attachment and proliferation of OB cells were evaluated. Firstly, Calcein AM/DAPI staining was performed. In brief, cells were stained using 5 μM of fluorescence dye (Calcein AM, PromoCell GmbH, Heidelberg, Germany) for 40 min without light. DAPI staining dye (Invitrogen, Karlsruhe, Germany) was used at 10 μg/mL final concentration. Cells were stained at the indicated time-points and subsequently visualized via a fiuorescence microscope (Leica DM IRB, Wetzlar, Germany). In addition, a CCK-8 assay was also performed. OB cells were cultured on BCP granules in 48-well plates at an initial density of 2 × 10⁴ cells/well and subjected to four above-mentioned treatments for 1, 4, and 7 days prior to evaluation with the CCK-8 kit.