Apoptosis necrosis detection kit

A431 cell death was analyzed at 0, 24, and 48 h after mechanical vibration using an Apoptosis/Necrosis Detection Kit (ab176749; Abcam, Cambridge, UK) according to the manufacturer's instructions. This kit enables discrimination between apoptotic and necrotic cells. The cells were visualized with an Eclipse Ti‐S inverted microscope (Nikon, Tokyo, Japan) and images were captured using NIS‐Elements D version 4.30 software followed by processing and quantification of live/dead cells with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

RAW264.7 macrophages (5 × 10⁵ cells/well) were seeded and stimulated with LPS or with LPS in the presence of LDL for 24 h. They were then treated with DS-Ce6 (equiv. 5 μM Ce6) for 1 h and the medium was replaced with fresh cell culture medium. The cells were irradiated using a 670 nm laser (50 mW, irradiated area 3.8 cm²) for 10 s and further incubated for 1 min and 2 h. For comparison, non-irradiated cells were prepared. Apoptosis induction was detected using a commercial Apoptosis/Necrosis Detection Kit (Abcam). The fluorescence signal was examined using a model LSM 780 Meta NLO confocal microscope (Carl Zeiss). The rate of apoptosis was quantified as the percentage of annexin V-positive cells per at least 40 cells (n = 5) [53 (link)].

Both MSCs LYS and MSCs CM were tested in vitro for their anti-proliferative activity on MSTO-211H, NCI-H2452 and NCI-H2052 cells in 96 multi-well plates (Sarstedt, Nümbrecht, Germany), as previously described [23 (link),24 (link)]. Briefly, 1:2 serial dilutions MSCs LYS and MSCs CM were performed in 100 µL of culture medium/well, and then, 10³ tumor cells were added to each well. Cell growth was evaluated after 7 days of culture by measuring the optical density at 550 nm in an MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-H-tetrazoliumbromide) assay [25 (link)]. Cell death was assessed by Hoechst 33342 and propidium iodide dual staining and by using the Apoptosis/Necrosis Detection Kit (Abcam, Cambridge, UK). Caspase-3 activity was measured by the Caspase-3 Assay Kit (Abcam) following the supplier’s protocols. The distribution of the cells in the cell cycle (determined by PI staining and flow cytometry analysis) was determined as described elsewhere [26 (link)].

An Apoptosis/Necrosis detection kit (Abcam) was used to simultaneously monitor apoptotic, necrotic and healthy cells through staining with Apopxin Green Solution (green Ex/Em = 490/525 nm) to detect phosphatidylserine (PS) as marker of initial/intermediate stages of apoptosis, with 7-AAD (/-aminoactinomycin D) (red Ex/Em = 546/647 nm) to detect loss of plasma integrity, characteristic of late apoptosis and necrosis. Briefly, 8 × 10⁵ HepG2/SB3 and HepG2/Ctrl cells were seeded onto coverslip and grown until semi-confluence. After overnight treatment with 100 μM H₂O₂, cells were washed twice and incubated with staining probes and incubated at room temperature for 1 h. After washing, the slides were mounted with ELVANOL (Sigma-Aldrich, St. Louis, MO, USA) and observed under a fluorescence microscope (Axiovert 200M, Carl Zeiss MicroImaging GmbH, Gottingen, Germany).

Apoptosis was detected using the Apoptosis/Necrosis Detection Kit (Abcam) according to the manufacturer's instructions. Apoptotic cells were stained with Apopxin Green Indicator, and viable cells were stained with CytoCalcein Violet 450. Images were acquired using a microscope (Olympus IX51, Olympus) and analyzed using ImageJ.

To measure apoptosis, AT2s isolated from newborn mouse lungs were exposed to normoxia or hyperoxia and treated with saline or T3 (0.8 ng/mL) for 72 hours and then stained with Apopxin green and 7-aminoactinomycin D (7-AAD) using an Apoptosis/Necrosis Detection Kit (Abcam, catalog ab176749). An LSRII flow cytometer (BD Biosciences) was used to quantify Apopxin green (Ex/Em = 490/525 nm) and 7-AAD (Ex/Em = 550/650 nm) fluorescence, and FlowJo software 10.8 (BD Biosciences) was used to analyze the results as described by the manufacturer (58 (link)).
AT2 proliferation was measured in cells exposed to normoxia or hyperoxia and treated with saline or T3 (0.8 ng/mL) for 72 hours using a CyQuant cell proliferation assay kit (Invitrogen, catalog MP 07026) and a fluorescence spectrophotometer (SpectraMax i3X) according to manufacturer’s instructions (59 (link)).