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Found 3 matching solutions for this experiment
| Upstream tips |
Protocol tips |
Downstream tips |
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After treatments, MAEC and HL-1 cells were washed three times with cold PBS and lysed in RIPA buffer (Beyotime, Shanghai, China) containing the Protease Inhibitor Cocktail and protease inhibitor PMSF for 30 min. Lysates were centrifuged at 12000rpm for 5 min at 4 °C. The protein samples were boiled in SDS-loading buffer for 10 min and were separated by 10% SDS-PAGE. Immunoblotting was performed with primary antibodies against parkin (1: 500), LC3 (1: 1000), P62 (1: 1000), ERRα (1: 1000), eNOS (1: 200), p-eNOS (1: 200), TOM20 (1: 1000), cleaved-caspase-3 (1: 1000), and GAPDH (1: 1000) followed by the incubation with HRP-labeled secondary antibody. Protein bands were visualized using ECL Plus detection reagents with Amersham ECL Detection Systems (Amersham, Buckinghamshire, UK). Densitometric analysis was performed using Image Lab Software (Bio-Rad, USA). |
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| Protocol tips |
| After treatments, MAEC and HL-1 cells were washed three times with cold PBS and lysed in RIPA buffer (Beyotime, Shanghai, China) containing the Protease Inhibitor Cocktail and protease inhibitor PMSF for 30 min. Lysates were centrifuged at 12000rpm for 5 min at 4 °C. The protein samples were boiled in SDS-loading buffer for 10 min and were separated by 10% SDS-PAGE. Immunoblotting was performed with primary antibodies against parkin (1: 500), LC3 (1: 1000), P62 (1: 1000), ERRα (1: 1000), eNOS (1: 200), p-eNOS (1: 200), TOM20 (1: 1000), cleaved-caspase-3 (1: 1000), and GAPDH (1: 1000) followed by the incubation with HRP-labeled secondary antibody. Protein bands were visualized using ECL Plus detection reagents with Amersham ECL Detection Systems (Amersham, Buckinghamshire, UK). Densitometric analysis was performed using Image Lab Software (Bio-Rad, USA). |
| Upstream tips |
Protocol tips |
Downstream tips |
|
After treatments, MAEC and HL-1 cells were washed three times with cold PBS and lysed in RIPA buffer (Beyotime, Shanghai, China) containing the Protease Inhibitor Cocktail and protease inhibitor PMSF for 30 min. Lysates were centrifuged at 12000rpm for 5 min at 4 °C. The protein samples were boiled in SDS-loading buffer for 10 min and were separated by 10% SDS-PAGE. Immunoblotting was performed with primary antibodies against parkin (1: 500), LC3 (1: 1000), P62 (1: 1000), ERRα (1: 1000), eNOS (1: 200), p-eNOS (1: 200), TOM20 (1: 1000), cleaved-caspase-3 (1: 1000), and GAPDH (1: 1000) followed by the incubation with HRP-labeled secondary antibody. Protein bands were visualized using ECL Plus detection reagents with Amersham ECL Detection Systems (Amersham, Buckinghamshire, UK). Densitometric analysis was performed using Image Lab Software (Bio-Rad, USA). |
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| Protocol tips |
| After treatments, MAEC and HL-1 cells were washed three times with cold PBS and lysed in RIPA buffer (Beyotime, Shanghai, China) containing the Protease Inhibitor Cocktail and protease inhibitor PMSF for 30 min. Lysates were centrifuged at 12000rpm for 5 min at 4 °C. The protein samples were boiled in SDS-loading buffer for 10 min and were separated by 10% SDS-PAGE. Immunoblotting was performed with primary antibodies against parkin (1: 500), LC3 (1: 1000), P62 (1: 1000), ERRα (1: 1000), eNOS (1: 200), p-eNOS (1: 200), TOM20 (1: 1000), cleaved-caspase-3 (1: 1000), and GAPDH (1: 1000) followed by the incubation with HRP-labeled secondary antibody. Protein bands were visualized using ECL Plus detection reagents with Amersham ECL Detection Systems (Amersham, Buckinghamshire, UK). Densitometric analysis was performed using Image Lab Software (Bio-Rad, USA). |
| Upstream tips |
Protocol tips |
Downstream tips |
| This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry |
Incubate cells with CYTO-ID dye (2μl of CYTO-ID in 1ml of 1X assay buffer) for 30 min |
Incase of Low CYTO-ID Green dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time.
Incase of High CYTO-ID Green staining, it could be that the cell culture medium was depleted of nutrients and so change media 4-8 hours before the experiment.
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| Upstream tips |
| This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry |
| Protocol tips |
| Incubate cells with CYTO-ID dye (2μl of CYTO-ID in 1ml of 1X assay buffer) for 30 min |
| Downstream tips |
Incase of Low CYTO-ID Green dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time.
Incase of High CYTO-ID Green staining, it could be that the cell culture medium was depleted of nutrients and so change media 4-8 hours before the experiment.
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