El808 ultra microplate reader

The hTMSCs were sown on 24-well plates (Nunc; Roskilde, Denmark) with 2 × 10⁴ cells/well density and incubated for 24 h before being treated with the DNA-PEI NPs. The cells were washed with serum-free MEM-α to remove FBS and replaced in fresh MEM-α. The hTMSCs were treated with each of the DNA-PEI NPs for 4 h and then the medium was replaced by cell growth medium. After 24 h and 48 h of transfection with each N/P charge of DNA-PEI NPs, a cytotoxicity test was performed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a colorimetric assay used to measure the activity of cellular enzymes that decrease the water-soluble MTT reagents to its insoluble purple-colored formazan. In brief, 100 μL of the MTT solution (5 mg/mL in PBS) was added to each well of hTMSCs and the plates were incubated at 37°C in a 5% CO₂ atmosphere. After 4 h, the entire medium was removed, 500 μL of dimethyl sulfoxide (DMSO) was added, and the plates were incubated for 30 min under 100 rpm shaking to solve the formazan crystals. The optical density at 590 nm was measured using an enzyme-linked immunosorbent assay plate reader (EL808 Ultra Microplate Reader; Bio-Tek Instruments; Winooski, VT, USA). All experiments were accomplished in triplicate.

Concentrations of cytokines and chemokines were determined by ELISA using F8 Maxisorb Loose Nunc-Immuno Module (Thermo Scientific-Nunc A/S, Roskilde, Denmark). For measuring IL-4, IL-13, IL-17A, TGF-β1, IL-22, TNF-α the ELISA Ready-SET-Go! ^® Kit (eBioscience Inc., San Diego, USA) were applied and for IL-6, IL12/23 p40, IL-10 and INF-γ the Douset (R&D Systems, Minneapolis, USA) were used. The procedure was performed according to the manufacturer’s guidelines and the OD was measured at 450 nm by EL808 Ultra Microplate Reader (Bio-Tek Instruments Inc. Winooski, VT, USA).

To evaluate cell viability on various wettable surfaces (L-0.5, L-2.5, and L-4.5), we cut the corona discharge films into circles, inserted them into 24-well plates, and fixed them with a silicon mold. hADSCs (2 × 10⁴ cells) were seeded onto each surface and incubated at 37 ºC in an atmosphere of 5% carbon dioxide/95% air. After 1 and 5 days, cell viability on each gradient surface was measured using a cell counting kit (CCK-8; Dojindo, Tokyo, Japan). Briefly, 50 μL CCK-8 reagent was added to the hADSCs, the plates were incubated at 37 ºC for 4 h, and then the samples were gently pipetted. An aliquot from each well (100 μL) was transferred to a 96-well plate (BD Bioscience), and absorbance at 450 nm was measured with a microplate reader (EL808 Ultra Microplate Reader; Bio-Tek Instrument, Winooski, VT, USA). All experiments were performed in triplicate, and the results were presented as means ± standard deviation.

MDA-MB-231, SUM159, and 4T1 cells were plated in a 96-well plate in triplicate. After overnight incubation, the cells were transfected with STAT3 siRNA or Negative Control siRNA for 72 h. Subsequently, 20 µL of 5 mg/mL thiazolyl blue tetrazolium dye solution (Sigma-Aldrich, St. Louis, MO, USA) was added to each well of the plate and incubated at 37 °C for 4 h. One hundred µL of N, N-dimethylformamide solution (Sigma-Aldrich, St. Louis, MO, USA) was used to dissolve the formazan. Absorbance at 595 nm was read using an EL808 Ultra Microplate Reader (BioTek, Winooski, VT, USA).

A solution of MC-Cl, MC-Cl (+naïve BMP2), MC-Cl (+BMP2OpgY), and MC-BMP2 at RT was mixed with hPLSCs (1 × 10⁶ cells). Then hPLSCs-loaded hydrogels (100 uL) was separately plated onto the wells of 24-transwell membranes. For the control group, hPLSCs alone in equivalent numbers were seeded into the 24-transwell membranes. Cytotoxicity of hPLSCs was examined at 1, 4 and 7 days at 37 °C in a humidified incubator. The cytotoxicity was measured using a WST-1 Kit (Roche, Mannheim, Germany). WST-1 reagent (100 μL) was added to the wells and the plates were incubated at 37 °C for 4 h. The samples were then gently mixed, and an aliquot (100 μL) from each well was transferred to a 96-well plate (SPL Lifescience, Gyeonggi-do, Korea). The absorbance of the solution at 450 nm was measured using a microplate reader (EL808 Ultra Microplate Reader, Bio Tek Instruments, USA). The experiments of cytotoxicity were performed three times.

After 15 DALV, remaining medium from each shoot tip culture set up was measured and stored at -20 °C. Glucose uptake was assayed using the modified version of the original protocol [50 (link), 51 ]. A buffer containing 100 mM imidazole–HCl (pH 6.9), 5 mM MgCl₂, 2.25 mM NAD, 1 mM ATP as final concentrations were used for the measurement of soluble sugars using EL×808 ultramicroplate reader (BioTeK Inc, Germany) at 340 nm. Temperature of the plate reader was set at 30 °C. Further addition of auxiliary enzymes such as glucose 6-phosphate dehydrogenase and hexokinase, allowed the removal of endogenous hexose-phosphates and measurement of glucose respectively. A calibration curve with different concentrations of glucose was used to measure the glucose amounts in leftover MS medium. Glucose uptake was further determined by deducting the leftover glucose from the total supplemented glucose.