Apoptosis induced by staurosporine

Summary

Staurosporine is a potent inhibitor of protein kinase C and most other kinases, including tyrosine protein kinases. Directly inhibits topoisomerase II activity by blocking the transfer of phosphodiester bonds from DNA to activated tyrosine sites. Can be used to induce apoptosis in CHO cells.

Operation method

Construction of staurosporine-induced apoptosis model in mouse cardiomyocytes

Principle

Staurosporine is a potent inhibitor of protein kinase C and most other kinases, including tyrosine protein kinases. Directly inhibits topoisomerase II activity by blocking the transfer of phosphodiester bonds from DNA to activated tyrosine sites. Can be used to induce apoptosis in CHO cells.

Materials and Instruments

Pregnant C57BL 6cr mice
staurosporine distilled water dehydrogenase ZVAD-fmk LDH kit Caspase3 kit collagenase II quinone barium chloride cell counting kit STS TrypsinEDTA DIDS NPPB
Microscope Test tubes Tubes Pipettes Pipette guns Tube boxes Ice boxes Refrigerators Incubators Centrifuges Slides Coverslips

Move

I. Culture of primary cardiomyocytes and experimental protocols

1. Postnatal 0~24 h C57BL/6cr mouse hearts were removed and cells were isolated according to the modified Lader method. Obtained booted hearts were quickly placed in Hank's equilibrium solution without ^Ca2+,^Mg2+.

2. The hearts were sheared and placed in 100 g/L Trypsin solution at 4°C for digestion for 16 to 18 h. Digestion was aborted with Trypsin inhibitor.

3. The digestion was then continued by incubation with collagenase II in a CO2 incubator at 37°C for 45~60 min on a shaker.

4. Finally, cardiomyocytes were obtained by filtration with a 70 μm diameter nylon filter, and cell suspension was made with M199 medium containing 25 mmol/L ^HCO3-, 100 mL/L FSB, ^{1×105 u/L} penicillin G, and 50 g/L streptomycin, and the cardiomyocytes were purified according to the differential applanation and separation method.

5. 10 μmol/L BrdU was added to the cell suspension, which was grown at a density of ^6×105 in 96- or 24-well cell culture plates and incubated in a 37°C incubator.

6. Replace the cultured cardiomyocytes with fresh M199 medium containing 10 μmol/L BrdU after 24 h and incubate for 72 h for experimental use.

7. Set up negative and positive control and experimental groups, each with 20 wells; add 4 μmol/L STS to the positive control and 4 μmol/L STS to the experimental group, and then add 250 μmol/L DIDS or 100 μmol/L NPPB, 100 μmol/L Quinine and 1 mmol/L BaCl2 to the experimental group, respectively, and incubate for 4 h. Replace the fresh M199 medium and continue incubation. M199 medium was replaced with fresh M199 medium and incubated for another 4 h for the detection of various indexes.

Cell survival test

1. Cultured cardiomyocytes treated according to the experimental protocol were incubated for 2 h with 10 μL of MTT per 100 μL of medium, and then detected with a spectrophotometer, and the results of the experiments were expressed as the cell survival rate, which was expressed as cell survival rate = A test group/A control group×100%. and survival.

Apoptosis agarose gel electrophoresis assay

1. Collect the treated cells from 24-well plates, discard the supernatant and add 50 μL of RNase cell lysate (10 μmol/L Tris-HCl, 0.1 mol/L EDTA, 5 g/L SDS) containing 200 mg/L, incubate at 37℃ for 1 h and then add 500 mg/L Proteinase K. 2. After 1 h, add an equal volume of proteinase K to the cell lysate, and then add 500 mg/L Proteinase K to the cell lysate.

2. After 1 h, add equal volume of phenol, chloroform, isopropanol (25:24:1) and shake well, centrifuge the supernatant and then add equal volume of chloroform extraction once, then add 1/10 volume of 3 mol/L sodium acetate and two times the volume of anhydrous ethanol in the transferred supernatant overnight and centrifuged, 700 mL/L ethanol washed, the DNA was solubilized by TE solution, and the DNA was extracted in a 20 g/L agarose gel containing ethidium bromide, and then electrocuted in an agarose gel containing ethidium bromide. The DNA was electrophoresed in 20 g/L agarose gel containing ethidium bromide, and observed under UV transilluminator, recorded and photographed.

Detection of caspase 3 activity

Caspase3 activity was detected using ApoONETM Homogeneous Caspase3 kit. Blank, negative control and experimental groups were set up. 100 μL of medium containing 100 μL of experimental cells was added with 100 μL of mixed caspase3 substrate ZDEVDR110 and buffer, and the cells were incubated for 6-8 h at room temperature with slight shaking, and the fluorescence value was read by a fluorescence detector at the excitation wavelength of 485 nm, and the emanation wavelength of 538 nm.

Lactate dehydrogenase (LDH) activity of cell culture supernatant was detected. LDH can catalyze the formation of pyruvate from lactate, which reacts with 2,4 dinitrophenylhydrazine to form dinitrophenylhydrazone pyruvate, which is brownish-red in alkaline solution, and the enzyme activity can be found out by colorimetry. The enzyme activity can be determined by colorimetry. 50 μL of cell culture supernatant was taken, and the A value was measured at 490 nm after the reaction.

Caveat

1. Strict sterilization. Use three sets of instruments to take materials.

2. Strictly carry out aseptic operation to prevent bacterial, fungal and mycoplasma contamination and avoid chemical substance contamination.

3. Flame sterilize the mouth of the bottle and the pipette before aspirating the liquid; always use Hank'S liquid to cool the pipette after being flame sterilized. Prevent scalding and scalded cells. When aspirating liquids, avoid bottle mouth and pipette contact collision.

4. Before the centrifuge tube is put into the table, the mouth and wall of the tube should be sterilized.

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