Dead cell reagent

The cells were seeded and cultured as described in Section 4.5. The Muse™ Annexin V with Dead Cell Assay Kit was used for the determination and differentiation of the four following distinct subpopulations of the cells localized in the one sample, i.e., live cells, early and late apoptotic as well as death cells. Briefly, following trypsinization and centrifugation, 1 × 10⁵ cells were placed in 100 μL of cell culture medium including FBS, and mixed with 100 μL of Muse™ Annexin V with Dead Cell Reagent (Merck Life Science Sp.z.o.o., Millipore, Poznan, Poland). This reagent includes fluorescently labeled Annexin V which connects to the phosphatidylserine being expressed outside of the cell membrane of apoptotic cells. The second fluorescent component of the Annexin V with Dead Cell Reagent, i.e., 7-aminoactinomycin D (7-AAD) labels the death and late apoptotic cells. After 20 min of incubation in the dark at room temperature, the measurement of apoptosis index was determined by the device recognized as miniaturized fluorescence detector and microcapillary cytometer, i.e., the Muse™ Cell Analyzer (Millipore, Germany).

Flow-cytometry analyses were performed using the Muse Cell Analyzer system and Muse 1.5 Analysis software (Merck, Darmstadt, Germany). For cell cycle analysis, the cells collected were first fixed in 70% ethanol for 12 hours, diluted to 3 × 10⁵ cells/ml with Muse Cell Cycle Reagent (Merck) and incubated in the dark for 30 minutes prior to analysis. The number of events for analysis was set at 5000.
The cell cycle analyses were repeated but following a slightly different protocol whereby the media for cells that have been treated with BLE and GA at IC₂₀ and IC₅₀ for 48 hours, were replaced with fresh MEM, followed by a further 48-hours incubation (37 °C and 5% CO₂)^{26 (link)}. Flow cytometry analyses were then performed as above.
The results for cell cycle analysis were expressed as DNA content profiles. For apoptosis analysis, Caco-2 cells were collected and resuspended in complete MEM at a concentration of 6 × 10⁵ cells/ml. Two hundred microlitres of cell suspension was mixed with 200 μl of Muse Annexin V and Dead Cell Reagent (Merck) and incubated in the dark for 20 minutes prior to analysis. The number of events for analysis was set at 5000. The results for apoptosis analysis were expressed as apoptotic profiles.