Cells were infected with d0-GFP or Ld0-GFP at MOIs of 0.1, 1, and 10 PFU/cell or with mock (10% DMEM). After 24 h of infection, the cells were harvested and stained with annexin V, Pacific Blue flow cytometry kit (Invitrogen, CA, USA) and PI. Apoptotic cell death was determined by FACS analysis using the BD FACSDiva Software on a FACSAria II cell sorter (Becton Dickinson, NJ, USA). ELISA analysis was used to determine the expression of ICD determinants in the supernatants of treated cells. Cells were infected with d0-GFP or Ld0-GFP at MOIs of 0.1, 1, and 10 PFU/cell and mock (10% DMEM). After 24 h of infection, the supernatants were harvested. The released ATP was measured by an ATPlite Luminescence kit (PerkinElmer, MA, USA), and the HMGB1 was measured by an HMGB1 ELISA kit (Tecan, Switzerland).
Atplite luminescence kit
Manufactured by PerkinElmer
Sourced in United States
The ATPlite Luminescence kit is a laboratory assay product used to quantify the levels of adenosine triphosphate (ATP) in biological samples. It provides a sensitive and reliable method for measuring ATP concentrations, which is a fundamental indicator of cellular energy status and viability.
Automatically generated - may contain errors
4 protocols using atplite luminescence kit
1
Virus-Induced Apoptosis and ICD
2
Cell Viability Assay in 96-well Plates
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Cells were plated in poly-HEMA coated or uncoated 96-well plates at a density of 11,000 cells per well. After 24 h, ATP was measured using the ATPlite Luminescence kit (PerkinElmer no. 6016943) per the manufacturer’s protocol.
3
Cell Viability Assay by ATP Measurement
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Cells were seeded in 96-well plates at 10 × 103 cells/well and treated as indicated. Cells were then rinsed twice with RPMI followed by 50 μl of RPMI and 25 μl of lysis solution for 5 min. Fifty-microliter aliquot was subjected to ATP measurement by the ATPlite luminescence kit (PerkinElmer).
4
HepG2 Compound Exposure Assay
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Two different compound exposure scenarios were performed: a single exposure in 2D and a 4-day repeated exposure in 3D. A schematic overview of the tested exposure scenarios for each HepG2 model has been depicted in Figure 1 . For the single exposure scenario in 2D, medium was replaced by freshly diluted compound in medium 24 h post seeding. DDRs were monitored after 24, 48 and 72 h by live cell confocal imaging (Figure 1A and B ). For the 4-day repeated exposure scenario in 3D, each day medium was replaced by freshly diluted compound in medium for four consecutive days (Figure 1C and D ). The imaging was started 24 h after the last exposure. For both scenarios, five compounds in eight concentrations were tested. DMSO, PBS or ultrapure H2O were used as solvent controls. The ATP-lite luminescence kit (Perkin Elmer) was used according to supplier’s protocol to measure cell viability. Measurements were performed 72 h or 24 h post single or repeated exposure, respectively. Absolute IC50 values have been calculated over the ATP-lite data by determining the intersect of the fitted concentration–response curve with the 50% viability baseline via GraphPad prism 8.1.1.
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