Viability cytotoxicity assay kit

A Live/Dead cell assay was performed using a fluorescent Viability/Cytotoxicity assay kit (Biotium, Fremont, CA, USA) to visualize the live and dead cells on the surface of the spheroids. At day 15, the treatments were completely removed from each well and replaced with 100 µL of 2 µM calcein AM/4 µM EthD-III staining solution. The plate was then incubated in the dark at room temperature for 30 min and subsequently imaged on an EVOS-FL Cell Imaging fluorescence microscopy at 4X magnification (Thermo Fisher Scientific, Waltham, MA, USA). Green fluorescent protein (GFP, imaging calcein AM) is representative of viable cells, whereas red fluorescent protein (RFP, imaging EthD-III) indicates necrotic/dead cells. Fluorescent intensity was analyzed by NIH ImageJ software (Version 1.53c).

Cell death was assessed by EthD-III/calcein AM staining (Viability/Cytotoxicity Assay Kit, Biotium, USA). EthD-III/calcein AM staining was also used for the cell death assay. The AM stain produced a bright green fluorescence in live cells. EthD-III entered dead cells, thereby producing red fluorescence in dead cells. Cells were simultaneously stained with 2 mM calcein AM and 4 mM EthD-III for 45 min at room temperature. Samples were analyzed using flow cytometry (BD FACSVerse, BD Biosciences, USA). The percentage of dead cells was determined by counting the ratio of red-positive cells to blue-positive cells.

The cell behavior after seeding and culturing on different polymeric materials was assessed by evaluating density, viability, and proliferation. APCs were labeled with the fluorescent marker 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil) (Cell-TrackerTM CM-DiI, Molecular Probes, Leiden, Netherlands) and seeded onto the scaffold as described above. After 5 days of incubation, cellularized scaffolds were washed with PBS, fixed with PFA 4%, then counterstained with DAPI, mounted with antifade mounting medium.
The viability of the APCs seeded onto PCL- and PLGA-based scaffold was assessed using the viability/cytotoxicity assay kit (Biotium Inc, US). Five days after seeding, the culture medium was removed, the scaffold washed with PBS and incubated for 30 min with a solution of Calcein [1:2000], EthD-III [1:500] and Hoechst [1:100] in serum-free medium at 37°C, 5% CO². Viable cells were identified using fluorescence microscopy. The ability of cells to proliferate once seeded onto scaffolds was evaluated by Click-iT® EdU Assay (Life Technologies, UK). Fluorescent images of the scaffolds were obtained using a Zeiss Fluorescent Microscope (Zeiss Axio observer Z1, Zeiss) and number of cells quantified using Image-Pro Plus software. All the functional assays in this study were performed on three cell lines, in technical triplicates.

Positively and negatively charged self‐assembling peptides were mixed 30 min prior to experiment except where otherwise stated. The viability of the cell lines was measured by using CellTiter‐Glo 2.0 Reagent (Promega G9248). The luminescent signal was measured in accordance with the manufacturer's instructions. Measurements were carried out with BioTek Neo2SM microplate reader and relative viability was calculated. Live‐Dead assay was carried out using Viability/Cytotoxicity Assay kit (BIOTIUM 30002). After peptide treatment, cells were treated with calcein and ethidium homodimer in accordance with the manufacturer's instructions. Fluorescent images were taken by using Keyence bz‐x710 microscope.

Prior to injection, AcGFP1- and MAHS-transgenic ASCs were washed three times with DPBS (+ , +), routinely passaged and resuspended at a density of 5000 cells/µL in viability/cytotoxicity assay kit (Biotium #30002, Fremont, CA) solutions diluted to final concentrations of Calcein-AM [2 µM] and EthD-III [4 µM] in PBS. The resuspended solutions were immediately injected onto glass coverslips surrounded by PDMS barriers at an injection rate of 1,000 µL/min. Samples were imaged on an inverted microscope as described above. A 36-position tile scan of ~ 0.53 cm² area was obtained for each sample with 1 µm steps to focus each image. Six cell platings were used as biological replicates for each experimental group.
The custom MATLAB script was used to calculate percentage of Calcein-AM-expressing cells as described above. Analysis of the extent to which the relationship between needle gauge and cell viability varied with genotype was performed through 2-way analysis of variance (2-way ANOVA) and Tukey’s honestly significant difference (Tukey’s HSD) test as described above. The dataset utilized for generating all graphical representations and conducting statistical analyses is available in the supplementary materials.

AcGFP1- and MAHS-expressing ASCs were routinely passaged, resuspended in 100% bovine calf serum (BCS, Fisher Scientific #SH3007303HI, Hampton, NH) and deposited directly into liquid nitrogen (flash freezing) or in a cell freezing container for controlled freezing (− 1 °C/min) down to − 80 °C. After 7 days, cryovials were removed from liquid nitrogen storage, centrifuged, and resuspended in viability/cytotoxicity assay kit (#30002; Biotium, Fremont, CA) solutions diluted to final concentrations of Calcein-AM [2 µM] and EthD-III [4 µM] in PBS. Samples were transferred into 24 well plates and incubated for 30 min at room temperature. Cells were imaged as described above. Three separate cell suspensions were used as biological replicates.
A custom MATLAB script was written to quantify cell viability. For all analysis, quantification of each experimental group was obtained from averaged cell counts of 36 positions per sample. Pairwise comparisons at each tested cryopreservation period were performed using two sample t-tests. All statistical tests were performed in MATLAB. Statistical tests and data visualization were performed in MATLAB as described above. The dataset utilized for generating all graphical representations and conducting statistical analyses is available in the supplementary materials.

Viability of cardiac PCs and CAECs exposed to 1 μg/ml (5.8 nM) S protein or PBS vehicle was evaluated using the Viability/Cytotoxicity Assay Kit (Biotium #30002), according to manufacturer’s guidelines. Cytoplasmic Calcein-AM identified live cells, while nuclear Ethidium Homodimer III (EthD-III) the dead cells. Cells treated with 0.1% w/v saponin for 10 min served as positive control for EthD-III staining. Per each patient, experiments were performed in duplicates.