Cellevent caspase 3 7 green readyprobes reagent

hB cells (1 × 10⁶ cells) and hMSCs (0.1 × 10⁶ cells) were added in 2 mL onto 35-mm culture dishes (BD Biosciences) and cultured for 24 h. hB cell apoptosis was determined in three ways. First, hB cells were stained with anti-CD19 antibody conjugated with APC and then with FITC-annexin and propidium iodide for 15 min (FITC-Annexin V Apoptosis Detection Kit, BD Biosciences). hB cells were analyzed using a flow cytometer (FACSCalibur) and the data were processed using Cell Quest Pro software (BD Biosciences) 38 (link). Second, hB cells were stained with anti-CD19 antibody conjugated with APC and then labeled with FITC-ApoStat (Intracellular Caspase Detection ApoStat kit, Bio-Techne, Minneapolis, MN, USA) for 1 h. hB cells were analyzed using a flow cytometer (FACSCalibur) and the data were processed using Cell Quest Pro software 39 (link). Third, CellEvent Caspase-3/7 Green ReadyProbes Reagent (Thermo Fisher Scientific, Carlsbad, CA, USA) was directly added to the co-culture of hB cells and hMSCs 40 (link). Time-lapse imaging was performed with a Biostation IM-Q microscope (Nikon) and images were acquired in two channels (phase contrast and green filter) every 10 min for 24 h 4 (link). Images were analyzed by using Imaris software 9.3.0. Green-fluorescent cells were considered apoptotic.

Cells used in the cell adhesion assay were stained with the CellEvent Caspase‐3/7 Green Ready Probes Reagent to visualize apoptotic cells and with the cell membrane‐impermeable propidium iodide from the Ready Probes Cell Viability Imaging Kit, blue/red (both from Thermo Fisher Scientific) to visualize dead cells with porous membrane according to the manufacturer’s instructions. Images of the cells were obtained using a Olympus IX71 microscope and DP70 camera (Olympus, Center Valley, Philadelphia, PA, USA).

The following reagents were acquired from Sigma Aldrich (reagent, Cat-no.): N-acetylcysteine, A7250; propidium iodide (PI), P4170; NaCl, S7653; KCl, P9333; MgCl₂, M1028; CaCl₂, 21114; D-glucose, 158968; HEPES, H3375; NaOH, S5881; l-glutamine, G7513; sodium pyruvate, 25030; Oligomycin, 75351; FCCP, C2920; antimycin A, A8674; rotenone, R8875; DMEM, D5030; Tris-HCl, T3253; KH₂PO₄, P5655; BSA, A3803; ATP-releasing reagent, FLSAR; ATP determination assay, A22066. The Lactate Dehydrogenase Activity Assay Kit, 88953 and CellEvent^® Caspase-3/7 Green Ready Probes^® Reagent, C10423 were both obtained from Thermo/Fisher. Collagenase CLSPA, LS005275 (Worthington Biochemical Corporation). MitoQ was a kind gift from Prof. M. Murphy (University of Cambridge, UK).

The apoptosis assays were employed to estimate the effect of double labelling of MSC with Luc and VT680, as well as to quantify the cell death induced in MSC after the treatment with TNFa and IFNγ (20 ng/ml, each) for 48 h. To estimate the effect of double labelling, 10⁵ MSC were incubated with 5 µl APC Annexin V (Biolegend, 640920) and 5 μg/ml Propidium Iodide (Sigma-Aldrich, P4170) in 100 μl, for 15 min in the dark. To determine apoptosis of MSC in pro-inflammatory conditions, 10⁵ cells (collected as both floating and attached cells) were stained with CellEvent™ Caspase-3/7 Green ReadyProbes™ Reagent (ThermoFisher Scientific, R37111), according to the manufacturer’s instructions. Cells stained with Annexin V and PI or CellEvent™ Caspase-3/7 were analysed by flow-cytometry. At least 100,000 events were recorded for each sample, using a CytoFLEX Flow Cytometer (Beckman Coulter, U.S.A.) and the acquired data were analysed using CytExpert version 2.1 software.
Furthermore, apoptosis induced by hypoxia, in the presence and absence of the pro-inflammatory cytokines was also evidenced by time-lapse fluorescence microscopy using a PAULA Smart Cell Imager (Leica Microsystems, Germany). After 24 h of treatment, CellEvent™ Caspase-3/7 reagent was added onto the cells and the cells were imaged for the next 24 h at a time interval of 20 min.

In order to confirm the V-AgNPs mediated apoptosis, we analyzed caspase 3/7 activation in 2D cultured A549 cells using fluorescence microscopy. For 2D cultured cells, 1 × 10⁵ cells were seeded per well in a 6-well plate. After treating them with LD25-2D and LD50-2D doses of V-AgNPs for 24 h, 2 drops of CellEvent™ Caspase-3/7 Green ReadyProbes™ Reagent (ThermoFisher Scientific, Denmark) per ml of cell culture medium was added to plate. Cells were incubated with this reagent for 30 min at 37 °C in the incubator with 5% CO₂ supply. ReadyProbe™ Reagent that has a DNA binding dye covalently linked to DEVD peptide. Active form of caspase 3/7 breaks this covalent bond leading to attachment of dye to DNA which yields bright green fluorescence. After 30 min, imaging was done using fluorescence microscope (Leica DM 4000B) using 10X magnification. DAPI was used as counterstain for cell nuclei. Untreated cells served as control for this experiment.

Detection of caspase-3/7 was performed using a fluorogenic, no-wash indicator of activated caspase-3/7 (CellEvent Caspase-3/7 Green Readyprobes Reagent, Thermo Fisher). HUVECs were seeded on a 35 mm glass bottom dish. Then, the cells were incubated at a glucose level of either 5.6 or 0.56 mmol/L with or without 3-HB. After 24 h, cells were loaded with the reagent. Nuclei were visualized by Hoechst 33342 (NucBlue Live Cell Stain, Thermo Fisher, Waltham, MA, USA). Positive cells were obtained with a fluorescence microscope (Eclipse Ti, Nikon, Tokyo, Japan) and analysed with CellInsight CX5 (Thermo Fisher).
A pan caspase inhibitor zVAD-fmk (Selleck, Osaka, Japan) was added at the concentration of 10 μmol/L at the start of the low-glucose culture. After 24 h, cell viability was assessed with the CCK-8 assay and the LDH release assay.

Cells were plated into 96-well plates (1 × 10³ cells/well) in 200 µl of growth medium; drugs were added the next day. Confluency was measured every 2 h, using the IncuCyteS3 Live-Cell Imaging Analysis System (Essen Bioscience). For apoptosis studies, cells were treated with inhibitors added to media containing the CellEvent Caspase 3/7 Green ReadyProbes reagent (Thermo Fisher Scientific)^{26 (link)}.