Cytotox one homogenous membrane integrity assay

Lactate dehydrogenase (LDH) was quantified from basal media using CytoTox-ONE Homogenous Membrane Integrity Assay (Promega). For each bacterial strain, two biological experiments in technical duplicate were performed.

Cells were infected as described above and loss of cellular membrane integrity was measured by lactate dehydrogenase (LDH) release in the supernatant. LDH release was quantified using the CytoTox-One homogenous membrane integrity assay (Promega, Austria) as previously described [37 (link)]. A ratio was calculated by normalizing infected samples to their corresponding not infected control (set to 1).

Lactate dehydrogenase (LDH) was measured using CytoTox‐ONE Homogenous Membrane Integrity Assay (Promega Corp., G7890) according to the manufacturer's instructions. Briefly, 4.0 × 10⁴ cells were plated into each well of a 96‐well plates for overnight incubation and subjected to the experimental treatments indicated. At the designated time‐points, samples were equilibrated to room temperature and 100 μL freshly prepared resazurin containing solution was added to individual wells holding 100 μL cell culture media. Samples were incubated at room temperature for 10 minutes and fluorescent measurements were made using a fluorescence microplate reader (excitation 560 nm, emission 590 nm; Molecular Devices, Inc., SpectraMax M2).

The hiPSC-CMs were seeded onto 24-well plates or four-chamber slides and separately treated with PBS, EC-Exo (1 μg/mL), EC-Exo^NC (1 μg/mL), EC-Exo^{anti−miR−100−5p} (1 μg/mL), mimic NC (100 nM), and miR-100-5p mimic (100 nM). Mimic NC and miR-100-5p mimic were transfected using the Lipofectamine RNAi_MAX Kit (Thermo Fisher Scientific, 13,778,075, USA). Then, cardiomyocytes were cultured under normal or oxygen–glucose deprivation conditions (glucose-free DMEM without serum, 1% O₂/5% CO₂/94% N₂) for 48 h. Apoptosis was evaluated using an In Situ Cell Death Detection Kit (Roche Applied Science, 12,156,792,910, Germany). Lactate dehydrogenase (LDH) leakage in the culture medium was determined using a CytoTox-One homogenous membrane integrity assay (Promega, G7891, USA). The ATP content was measured in homogenized hiPSC-CMs using an ATP Bioluminescence Assay Kit (Sigma-Aldrich, 11,699,709,001, USA). The Ca²⁺ transients were evaluated by incubating the hiPSC-CMs with a Ca²⁺ indicator, Fura-2 AM (Beyotime, S1052, China), and electrically stimulating the cells. Then, the ratio of fluorescence emitted at 340 nm and 380 nm was recorded using a Ca²⁺-recording system (IonOptix, USA) [21 (link)].

Cell viability was determined by measuring LDH released using a CytoTox-ONE Homogenous Membrane Integrity Assay (Promega Corporation, Madison, WI, USA). The reaction mixture was added to the cells, and fluorescence was quantified at the excitation and emission wavelengths of 560 and 590 nm, respectively, using a Synergy HT microplate reader (BioTek, Shoreline, WA, USA).

Human polymorphonuclear leukocytes (hPMNs) were isolated using a Ficoll-Paque method and cytotoxicity assays were performed as described by Reyes-Robles et al. (58 (link)). Briefly, PMNs were seeded at 2 × 10⁵ cells per well in RPMI medium without phenol red (Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gemini Bio-Products). Either 1.25% or 2.5% supernatant was added (percentage is the percentage of total reaction volume that was culture supernatant). To measure cell viability, the metabolic dye CellTiter (Promega) was added at a final concentration of 10% per well and incubated for 2 h at 37°C and 5% CO₂. Absorbance at 492 nm was measured using the PerkinElmer EnVision plate reader. For extracellular infections, bacteria were subcultured for 3 h in 5 mL of TSB, washed twice with PBS, and normalized to an OD₆₀₀ of 1. hPMNs were added to a flat-bottom tissue culture treated plate at 2 × 10⁵ and incubated at room temperature for 30 min. Bacteria were added at a multiplicity of infection (MOI) of 100. After a 2-h infection at 37°C and 5% CO₂, hPMN viability was determined by lactate dehydrogenase (LDH) release (CytoTox-ONE homogenous membrane integrity assay; Promega), measured using the PerkinElmer EnVision plate reader.