Apoptosis assay kit

Cell proliferation was examined using Cell Counting Kit 8 (Invitrogen, Carlsbad, CA). That is, 100 μL of cell suspension (2,000 cells • well-1) were seeded into a 96-well plate. Next, after 0, 24, 48, and 72 hours (h) of culture, we added 10 μL of Cell Counting Kit 8 reagent and incubated for 1 h prior to making detections. Cell viabilities were examined by measuring optical density at 450 nm with a specrophotometric plate reader (BioTek, VT, USA). Each experiment was repeated in triplicate.
For apoptosis assays, HO8910 or A2780 cells were harvested after transfection and double stained using fluoresce in isothiocyanate (FITC)-conjugated Annexin V and propodium iodide (PI). Next, the percentage of early apoptotic cells was analyzed on a flow cytometer (Becon Dickinson FACSCalibur, NY, USA). Apoptosis was examined by One Step TUNEL (TdT-mediated dUTP Nick-End Labeling) Apoptosis Assay Kit (Beyotime, Shanghai, China) in accordance with manufacturer protocols. HO8910 or A2780 cells were fixed with 4 % paraform for about 30 minutes (min), then stained with Hoechst 33342 (Beyotime, Shanghai, China) for 20 min and photographed under light fluorescence microscopy (Leica, Wetzlar, Germany). We performed three independent replicated experiments for these analyses.

The compound Radix Sophorae Flavescentis was provided by Shanxi Zhendong Pharmaceutical Co., Ltd., Changzhi, China; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Jingke Biotechnology Co., Ltd., Beijing, China; the apoptosis assay kit was from the Beyotime Institute of Biotechnology, Haimen, China; terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) reagent was purchased from Roche Diagnostics, Mannheim, Germany; the reverse transcription kit was obtained from Nanjing Vazyme Biotech Co., Ltd. (Nanjing, China); SYBR-Green universal qPCR Master Mix was from Roche Diagnostics; the real-time PCR system was purchased from Bio-Rad (Hercules, CA, USA); and the inverted fluorescence microscope was obtained from Leica Microsystems (Mannheim, Germany).

TUNEL staining was conducted as described in a previous study [34 (link)]. In brief, liver tissues of septic mice were fixed, embedded, and sectioned into 4 μm thick slices. The Apoptosis Assay Kit (Beyotime) was used to detect apoptotic cells in liver tissue sections according to the manufacturer's instruction. The liver slices were deparaffinized, rehydrated with graded ethanol dilutions, incubated with 20 μg/ml Proteinase K (Beyotime) at 37°C for 30 min, and washed with PBS three times. Then, the slices were incubated with 50 μl TUNEL working solution at 37°C for 60 min, followed by incubation with DAPI (Beyotime) for 15 min protected from light. The slices were washed with PBS three times, and the fluorescence signals (488 nm excitation wavelength) were observed by fluorescence microscopy. For statistical analysis, three fields under ×200 magnification were observed and TUNEL-positive cells were counted, as previously described [35 (link)].

Western blotting and immunofluorescence were performed using the following antibodies: phospho‐PERK (p-PERK), phospho‐eIF2α (p-eIF2α), cleaved‐Caspase-3 (c-Casp3), β‐actin (Cell Signalling Technologies; Danvers, MA); cleaved‐PARP (c-PARP), CHOP, Bax, Bcl‐2, Grp78, PERK, eIF2α, ATF-4, Beclin-1, LC3B-I/II (Abcam, Cambridge, UK). 2‐DG (purity ≥98%) and HCQ (purity >99%) were obtained from Sigma‐Aldrich Chemical (Agent of Shanghai, China). The Apoptosis Assay Kit and FITC Annexin V Apoptosis Detection Kit with PI (propidium iodide) were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). The Cell Counting Kit‐8 was purchased for cell viability detection (CCK‐8, Dojindo Laboratories, Minato‐ku, Tokyo, Japan). All of the other chemicals and reagents were of the highest commercial grade availability.

GSCs were obtained according to previous studies [18 (link)] by growing GL261 in serum-free DMEM/F12 medium (HyClone, USA) supplemented with 20 ng/mL murine bFGF (PeproTech, USA) and 20 ng/mL EGF (PeproTech, USA) at 37°C with 5% CO₂. The culture medium was refreshed by 50% every 2-3 days. The cells were passaged when they reached 90% confluence. Spherical cells at the third passage (P3) were examined for the expression of CD133 by immunofluorescence staining. After three days, the expression of GFAP in the spheres was detected by an immunofluorescence assay.
We extracted GSC antigens from apoptotic lysates. GSCs of the third passage (P3) were digested with 0.05% trypsin (HyClone, USA) for 5 min and processed overnight with hydrogen peroxide (100 μM),which is known to induce apoptosis. In order to evaluate apoptosis, an apoptosis assay kit manufactured by Beyotime in China was utilized. Then, GSCs were stained with FITC-Annexin V/PI (KeyGen Biotech, China) for 15 min, and apoptotic antigen labelled with FITC-Annexin V was enriched by fluorescence-activated cell sorting (FACS; BD FACSAria III, USA). The purity of these apoptotic GSCs was verified by flow cytometry test. After sorting, the enriched apoptotic GSCs were added to imDCs at a 3 : 1 (stimulator : responder) ratio in RPMI-1640 medium and incubated for 24 h at 37°C with 5% CO₂.