Apoptosis necrosis kit

To induce necrosis, McCoy B cells were suspended at 1 × 10⁶ cell/ml in culture medium and heated at 55 °C for 30 min. To confirm cell death and display of phosphatidylserine (PS), an apoptosis/necrosis kit (Abcam) was used as per the manufacturer’s protocol. Heat-treated and control (live) cells were incubated with CytoCalcein Violet (EX/EM: 405/450 nm), membrane-impermeable DNA Nuclear Green DCS1 dye (Em/Ex: 490/520 nm) and the PS sensor dye Apopxin Deep Red (Em/Ex: 630/660 nm), and imaged using a Nikon-Ti-2 epifluorescence microscope. Cells with permeabilized membranes (DCS1 positive) and cells displaying PS (Apopxin-positive) were quantified using NIS Elements General Analysis software.

The impact of both street and standard MXP samples on cell death type induced in 5637, SH-SY5Y, and MRC-5 cells was studied by using an apoptosis/necrosis kit (Abcam, Cambridge, UK) following the manufacturer protocol. Briefly, the cells were seeded as in the cytotoxicity evaluation described in Section 2.9, but instead of WST-1, the apoptosis/necrosis kit was added, i.e., the cells were washed with 100 µL of assay buffer and, then, 200 µL of assay buffer with 2 µL of apopxin green indicator (100×; labeling apoptotic cells) and 1 µL of 7-AAD (200×; for visualization of necrotic cells as well as late apoptotic cells) were added to the cells per one well for 30 min. After that, the cells were washed with PBS and fixed with 2% formaldehyde solution in PBS and subjected to fluorescence microscopy to capture apoptotic and necrotic cells, the samples were prepared in three replicates, from each 6–10 region of interest were monitored and the results were evaluated by Image J. Untreated cells served as a control.

Freshly isolated acinar cells were incubated for 30 min with supplemented Medium-199 in 48-well plates (Greiner Bio-One, Hungary), then AZA containing (0.1–1-10–100-1000 μg/mL) media was added, and cells were incubated for another 60 min at 37 °C. Cerulein (100–1000 nM) was applied as positive control. We determined the intracellular ATP content, which is proportional to the number of viable cells, using the CellTitre-Glo 3D (Promega) luminometric assay on a CLARIOStar plate reader. The total protein amount was determined using by Bradford assay in Spectrophotometer (Thermo Scientific™ NanoDrop™ One). Blank-corrected data were normalized to the total protein amount.
In addition, living, apoptotic, and necrotic cells were stained in 8-well chamber slides (Sarstedt, Germany) with Apoptosis-Necrosis Kit (Abcam) and were visualized under a Zeiss LSM880 confocal microscope using a 40× oil immersion objective (Zeiss, NA: 1.4). The total number of cells was calculated in FIJI (NIH, USA) using the built-in Cell Counter plugin and the ratio of the live, apoptotic and necrotic cells were given as the % of the total cell number.