Cell technology topic: Staurosporine induced mouse cardiomyocyte apoptosis model construction

Primary culture of cardiomyocytes and experimental protocols

The hearts of C57BL/6cr mice were harvested from 0 to 24 h after birth, and the cells were separated according to the modified Lader method. The pulsating heart was quickly placed in Hank's equilibrium solution without Ca ²⁺ and Mg ²⁺ . The heart was cut, placed in 100 g/L Trypsin solution at 4 ° C for 16-18 h, and the digestion was stopped with Trypsin inhibitor. Then, at 37 ° C, using collagenase II in a CO ₂ incubator, continue to incubate for 45-60 min on a shaker, and finally filter the cardiomyocytes with a 70 μm diameter nylon filter for 25 mmol/L HCO ^3- A cell suspension was prepared from M199 medium of 100 mL/L FSB, 1×10 5 u/L penicillin G and 50 g/L streptomycin, and the cardiomyocytes were purified by differential adherence separation. Add 10 μmol/L BrdU to the cell suspension, plant at a density of 6×10 ⁵ on a 96- or 24-well cell culture plate, and incubate at 37 ° C. The cultured cardiomyocytes were replaced with 10 μmol/L BrdU freshly for 24 h. M199 medium was cultured for 72 h and then used for experiment. Set negative, positive control and experimental groups, 20 wells per group; positive control added 4 μmol/L STS, and experimental group added 4 μmol/L STS and then added 250 μmol /L DIDS or 100 μmol/L NPPB, 100 μmol/L Quinine and 1 mmol/L BaCl _{2 were} incubated for 4 h, and fresh M199 medium was replaced and continued to incubate for 4 h.

Cell survival test

The cultured cardiomyocytes treated according to the experimental protocol were added with 10 μL of MTT per well of 100 μL of medium for 2 h, and then detected by a spectrophotometer. The results were expressed as cell viability, cell viability = A Test group / A control group × 100%. MTT method A value can reflect the mitochondrial dehydrogenase activity in cardiomyocytes, and can indirectly reflect the proliferation and survival of cardiomyocytes.

Apoptotic agarose gel electrophoresis

Collect 24 well-treated cells, discard the supernatant and add 50 μL of 200 mg/L RNase cell lysate (10 μmol/L Tris.HCl, 0.1 mol/L EDTA, 5 g/L SDS), incubate at 37 °C. Incubation was continued by adding 500 mg/L proteinase K for 1 h. After 1 h, add an equal volume of phenol, chloroform, isopropanol (25:24:1) and shake well. Centrifuge the supernatant and add an equal volume of chloroform for 1 time. Add 1/ of the transferred supernatant. 10 volumes of 3 mol/L sodium acetate and twice the volume of absolute ethanol were centrifuged overnight, washed with 700 mL/L of ethanol, and the TE solution was dissolved in 20 g/L agarose gel containing ethidium bromide. Electrophoresis, UV transmission instrument observation, record photos.

Detection of Caspase3 activity

Caspase3 activity was detected using the ApoONETM Homogeneous Caspase3 kit. The assay set up blank, negative control and experimental groups. Add 100 μL of mixed Caspase3 substrate ZDEVDR110 and buffer to the cells containing 100 μL of medium, and shake gently at room temperature. ~8 h, using a fluorescence detector, the excitation wave is 485 nm, the emission wavelength is 538 nm, and the fluorescence value is read.

Cardiomyocyte membrane integrity test

The cell culture supernatant detects the activity of lactate dehydrogenase (LDH). LDH can catalyze the formation of pyruvic acid from lactic acid. Pyruvate reacts with 2,4 dinitrophenylhydrazine to form dinitrophenylpyruvate, which is brown in alkaline solution. Red, the enzyme activity can be determined by colorimetry. 50 μL of the cell culture supernatant was taken, and the A value was measured at 490 nm after the reaction.