Realtime glo annexin 5 apoptosis and necrosis assay

Apoptosis and necrosis were detected using the RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay (Promega, Walldorf, Germany). EA.hy926 cells were plated in a black 96-well assay plate (Corning Incorporated Costar®, Merck, Poznan, Poland) at a concentration of 8500 cells per well. The test was carried out after 48 h of culture in complete cell culture medium, according to the manufacturer’s instruction. Measurements were performed using the Infinite M200 pro plate reader (Tecan Group Ltd, Männedorf, Switzerland) at 0, 1, 3, 7, 9, 11, 13, and 24 h. Naringenin dilutions were prepared from a stock solution in DMSO, therefore, an additional control of 0.1% DMSO was introduced.

The cells were seeded in a sterile, white 96-well tissue culture plate at a concentration of 10,000 or 30,000 cells/100 µL volume/well, depending on the cell line. All cell lines were incubated for 24 h at 37 °C and with 5% CO₂. After 24 h, the supernatant was discarded and the cells with GP-2250 were stimulated at the concentration of 0.0–2000 µmol/L with a total of 100 µL/well. Analysis was performed with RealTime-Glo Annexin V Apoptosis and Necrosis Assay (Promega, Madison, WI, USA) according to the manufacturer’s protocol. This assay measures the exposure of phosphatidylserine on the outer leaflet of the cell membrane during the apoptotic process, and the loss of membrane integrity during secondary necrosis. Annexin V binding is detected with a simple luminescence signal, and necrosis is detected with a fluorescent DNA binding dye. The luminescence signal and the fluorescence signal were measured on the GloMax Discover Detection System GM 3000 + 3030 (Promega) after 2 h, 6 h and 24 h.

iCell-Cardiomyocytes² (Fujifilm Cellular Dynamics Inc., Madison, WI, United States) were seeded into 96 well-plates (Greiner Bio One, ref. 655946) according to the manufacturer instructions, at a density of 50 k cells/well. Seven days after seeding, the hiPSC-CMs were used to assess cytotoxic effects of doxorubicin, captopril and sunitinib. To assess effects on viability and cytotoxicity, two assay combination kits were used: 1) RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay [Promega, JA1011 (Kupcho et al., 2019 (link))] and 2) ApoLive-Glo Multiplex Assay [Promega, G6410 (Flörkemeier et al., 2022 (link))]. The assays were performed according to manufacturer instructions. The experiments were repeated at 24, 48, and 72 h in order to assess any time-dependent change in live cells or activation of cell-death pathways.

The RealTime-Glo^™ Annexin V Apoptosis and Necrosis Assay (Promega, Madison, WI, USA) is a live-cell (non-lytic) real-time (kinetic) assay that measures the exposure of phosphatidylserine (PS) on the outer leaflet of the cell membrane during the apoptotic process. Measurement of PS exposure is a reliable and well-validated mean of assessing apoptosis [40 (link)]. Briefly, A549 cells were plated at a density of 10,000 cells/well in a white opaque tissue culture 96-well plate (Fisher Scientific, Hampton, NH, USA). The next day, cells were treated with 4X concentration of PFD and PFD–D-Lip, followed by addition of 100 μL of 2X assay buffer and incubated at 37 °C/5% CO₂. Luminescence signal was monitored at regular time points of 0, 3, 6, 12 h using a Tecan microplate reader [41 (link)].

To assess the time-dependent dose-response of paclitaxel exposure on apoptosis induction in mature brain organoids, the RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay was used (Promega, Germany). Apoptosis induction is measured by Annexin V binding to trans-localized phosphatidylserine, whereas necrosis is detected by a membrane-impermeable reagent binding to DNA. The sensitivity of the assay was previously tested by the company in 3D cultures (Data available on the company webpage). Furthermore, Annexin-V based assays have been previously used to assess cytotoxicity in 3D cultures (Xiao et al., 2020 (link); Parseh et al., 2022 (link)). Three organoids per batch (day 58 in culture) were incubated with medium, vehicle or paclitaxel (1, 10, 100, 1,000, 10,000 nM) for 14 h, transferred to a white round bottom 96-well ultra-low attachment plate (MS-9096WZ; PHCbi, Netherlands) in NDM and the assay reagents were added according to the manufacturer’s instructions. Luminescence and fluorescence measurements were done 17, 18, 20, 22, 24, 26, 28, 32, 40 and 48 h after the start of the exposure with a SpectraMax iD3 plate reader (molecular devices, San Jose, CA) and normalized to the first measurement (17 h) as well as to the vehicle control. Effects of more than 20% compared to the VC were interpreted as relevant.

Approximately 4 × 10⁴ RPE, HCT116 TP53^+/+ or HCT116 TP53^−/− cells were seeded per well on a 96-well plate, and treated with 1 µM 4NQO, 1 µM A-1155463, or both compounds. Control cells were treated with 0.01% of DMSO. Activation of apoptosis was assessed using RealTime-Glo Annexin V Apoptosis and Necrosis Assay (Promega). Luminescence was measured using Victor X3 plate reader.