Live dead cell staining kit

Nine days in vitro primary cortical neuronal cultures derived from embryonic day 17 to 18 (E17–18) P4htm^−/− and WT mice were used for cell survival studies. Pregnant mice were sacrificed via cervical dislocation, and cortexes from E17 to 18 embryos were removed of meninges, dissected, and trypsinized. After mechanical trituration, the cells were plated on poly-D-lysine-coated glass coverslips in 4-well plates at a density of 13,000 cells/cm². Neurons were cultured in MEM/B27 medium (Invitrogen) supplemented with sodium bicarbonate, sodium pyruvate, L-glutamine, penicillin, streptomycin, and 0.6% glucose. Cultures were incubated at 37 °C under 5% CO₂/95% air and 90% humidity without medium exchange up to 8 days, when they were subjected for glutamate treatment. For the in vitro cell death assay, primary neurons were exposed either to fresh medium containing 200 mM glutamate or to the medium without any additions (control) for 15 min, after which the cells were returned to the original medium and cultured for an additional 24 h.
Cell death was calculated using the Live-Dead Cell Staining kit (BioVision). The cells were visualized by fluorescence microscopy using an EVOS digital inverted microscope (Fisher Scientific) and quantified by Fiji-ImageJ software. Analysis was performed blinded to the experimental groups.

After repopulation, a Live-Dead cell staining Kit (BioVision, Milpitas, CA, USA) was used to distinguish live and dead cells and the cellular activity of the recellularized SIS was observed at 1, 2, 3 and 4 weeks. Live NPCs (stained green) were visualized using fluorescence microscopy, counted and recorded as the mean of 10 randomly-selected views from each specimen at 10 × 10 magnification, and were compared with the positive control group (NPCs cultured in standard DMEM-HG) to determine the cytocompatibility of SIS particles.

The live–dead cell staining kit (Biovision, USA) was performed according to manufacturer’s instructions. Briefly, cells were cultured on the specimens with a density of 5 × 10⁴ cells per well. Propidium iodide and calcium-AM were diluted to final concentrations of 5 and 2 μM in PBS, respectively. After 4 days, 100 μl of mixed solution was added to each specimen, and the cells were stained at 37 °C for 15 min.

To assess caspase 3/7 activity, Hif-p4h-1^−/− and wt MEFs and Hif-p4h-1^−/− MEFs transfected with empty vector or a vector encoding V5-tagged human recombinant HIF-P4H-1 were seeded in 96-well plates at a density of 20 000 cells/well in triplicates and treated with 100 ng/ml LPS for 24 h. Caspase 3/7 activity was assayed using the Caspase-Glo3/7 Assay (Promega) according to manufacturer’s instructions. To study LPS, staurosporine or cisplatin-induced cell death, the above MEFs were treated with 5 mg/ml LPS for 48 h, 2 μM staurosporine for 24 h or 50 µg/ml cisplatin for 24 h. The MEFs were plated in 6-well plates at a density of 200 000 cells/well, after the treatment washed with 0.15 M NaCl and 0.02 M phosphate, pH 7.4 (phosphate buffered saline, PBS), stained with a saturating concentration of 7-actinomycin D (7-AAD) and analysed by fluorescence-activated cell sorting (FACSCalibur, BD Biosciences). Alternatively, the cells were plated in 96-well plates at a density of 50 000 cells/well, after treatment stained with Live-Dead Cell Staining Kit (BioVision) and viability was determined by dynamic imaging using relative red (dead) and green (live) object count per well in IncuCyteTM zoom live-cell imaging system (Essen BioScience) according to the manufacturer’s instructions.

The damage to tumor cells induced by thermal ablation with MWNTs and NIR irradiation was evaluated in vitro. MCF-7 and MDA-231 cells (1.0 × 10⁴ cells/100 μl) were seeded onto 96-well plates for 24 hours and then incubated with various concentrations of MWNTs for 24 hours. The cells were exposed to irradiation with an 808-nm NIR laser at 5 W/cm² for 0-2 min. Cell survival efficiency was measured using the CCK8 assay as mentioned above at 16 hours post-irradiation.
To further verify the photothermal effect on cancer cells, the cells were stained with Live-Dead cell staining kit (Biovision, Mountain View, CA, US) at 16 hours after the photothermal treatment. Briefly, MCF-7 and MDA-231 cells (1.0 × 10⁴ cells/150 μl) were seeded onto confocal dishes (NEST Biotechnology Co. LTD, Jiangsu, China) and incubated overnight. Then 100 μg/ml MWNTs were added to the culture medium. After 24 h incubation, the cells were exposed to irradiation with an 808-nm NIR laser for 2 min at 5 W/cm² and stained with Live-Dead cell staining kit 16 hours later. Briefly, cells were stained with 0.5 ml staining solution containing 0.5 μl Live-Dye (1 mM) and 0.5 μl PI (2.5 mg/ml). After incubated for 15 min at 37 °C, signals were visualized by confocal microscopy (FV10i-W, Olympus, Tokyo, Japan).