Celltiter glo 3d cell viability kit

CellTiter-Glo 3D Cell Viability kit (Promega) was used to quantify ATP content as an indicator of metabolically active cells. Cardiac spheres were dissociated using 0.25% Trypsin–EDTA and replated into a 96-well plate (Corning) at 5 × 10⁴ per well in 100 µL maturation medium. The kit was thawed at 4 °C a day before and the Cell Titer-Glo 3D reagent was added to the maturation medium (1:1) in each well containing cells followed by shaking for 10 min at RT. Some wells considered as blank reagent were filled with maturation medium and Cell Titer-Glo 3D reagent. Measurement was performed at Top Count NXT Microplate Luminescence Counter (PerkinElmer) with integration time of 1 s per well after 20 min incubation at RT.

Cell viability of 3D cultures was detected by fluorescence Live/Dead assay after cellular aggregate formation. Calcein AM solution (Cat. no C1359, Sigma-Aldrich) was used to stain live cells, while cell membrane-impermeable Ethidium homodimer I solution (Cat. no E1903, Sigma-Aldrich) for nuclei of dead cells. Spheroids were incubated for 1 h at 37 °C, then washed in 1X PBS, and imaged in a fluorescence microscope (Eclipse Ti Nikon Corporation, Tokyo, Japan).
Cell viability of 3D cultures was also determined by an adenosine-5′-triphosphate (ATP) assay with the Promega CellTiter-Glo^® 3D Cell Viability kit (Promega Italia S.r.l., Milan, Italy). Relative Luminescence units (RLUs) were recorded with a LuMate^® luminometer (Awareness Technology Inc., Palm City, FL, USA) and the resulting data were analyzed through LuMate Manager software (version 2.0).

An adenosine triphosphate (ATP) assay was performed using the CellTiter-Glo 3D Cell Viability kit (Promega, Madison, WI). Each retinal organoid was placed in an individual well of a white Costar 96-well plate (Corning) with 100 µL of media. To each sample, 100 µL of reagent was applied, and the samples were incubated for 30 minutes at room temperature on an elliptical shaker under dark conditions. Luminescence readings were taken using a FLUOstar Omega Plate Reader (BMG Labtech, Ortenberg, Germany). ATP levels for each retinal organoid were determined from an ATP dilution series after being normalized to media-only controls. Data are presented as percentage ATP levels relative to the average ATP levels of four untreated retinal organoids. All treatment groups were n = 4. The normal distribution and variance of data were confirmed (Brown–Forsythe and Bartlett's tests), and a one-way analysis of variance (ANOVA) with Dunnett's multiple comparisons was performed on the dataset. For correlation of retinal organoid size with ATP levels, retinal organoid sizes were determined by drawing around a standardized image and using the area measurement function within ImageJ (National Institutes of Health, Bethesda, MD). Area measurements were plotted against ATP levels, and a Pearson r-test was performed.

Viable cell content in encapsulated islets was determined by intracellular ATP quantification (RLU, relative light unit) using the CellTiter-Glo^® 3D Cell Viability kit (Ref 69682, Promega, Charbonnieres-les-Bains, France) following the manufacturer’s recommendations. The ATP content standard curve for MPIs was obtained from pseudo-islets immediately after encapsulation in alginate patches at several densities. A linear correlation was observed between the luminescent signal of the CellTiter-Glo^® 3D Cell Assay and the fluorescent Cyquant DNA Assay (Ref C7026, Waltham, MA USA) regardless of the culture conditions. Cell death within the encapsulated islets was evaluated by quantifying the lactate dehydrogenase activity (Ref 11644793001, LDH, absorbance unit (AU), Roche, Meylan, France) in culture supernatants according to the manufacturer’s recommendations. ATP luminescence and LDH absorbance were evaluated on a FLUOstar OPTIMA luminometer (BMG Labtech, Champigny-sur-Marne, France).