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Neonatal rats at postnatal day 0 to day 2 were sacrificed by decapitation and subsequent heart removal. Ventricular myocytes were then isolated using the Cellutron Neomyocytes isolation system (Cellutron Life Technology, Baltimore, MD) following the manufacturer's instructions [22]. Cells collected were suspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 µM BrdU, and 100 U/ml penicillin/streptomycin (Invitrogen, Carlsbad, CA). To selectively enrich cardiomyocytes, the cells were preplated in 100 mm non-coated dishes for 1 hour. The resulting suspended cells were counted with a hemocytometer and then plated evenly on 1% gelatin-coated plates in appropriate cell densities. The plated cells were then cultured in a 5% CO2 incubator at 37°C for at least 24 hours before the medium was changed to meet the needs of the follow-up experiments. The siRNA's specific for Rab7 (siRab7: Cat. # SI03021725) and the siRNA targeting luciferase serving as a control siRNA (siLuc: 5′-AACGTACGCGGAATACTTCGA-3′) were all purchased from Qiagen (Valencia, CA). LipofectamineTM 2000 transfection reagent (Invitrogen) was used for siRNA transfection following the manufacturer's protocol. Transfection of cultured NRVMs with siRNA was generally started at 48–72 hours after NRVMs were plated . Six hours after the transfection, the siRNA-containing medium was replaced with the fresh medium containing 2% FBS. . For the Rab7 knockdown experiments, Rab7 siRNA were introduced along the second round of sip62 or siLuc transfection |
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| Protocol tips |
| Neonatal rats at postnatal day 0 to day 2 were sacrificed by decapitation and subsequent heart removal. Ventricular myocytes were then isolated using the Cellutron Neomyocytes isolation system (Cellutron Life Technology, Baltimore, MD) following the manufacturer's instructions [22]. Cells collected were suspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 µM BrdU, and 100 U/ml penicillin/streptomycin (Invitrogen, Carlsbad, CA). To selectively enrich cardiomyocytes, the cells were preplated in 100 mm non-coated dishes for 1 hour. The resulting suspended cells were counted with a hemocytometer and then plated evenly on 1% gelatin-coated plates in appropriate cell densities. The plated cells were then cultured in a 5% CO2 incubator at 37°C for at least 24 hours before the medium was changed to meet the needs of the follow-up experiments. The siRNA's specific for Rab7 (siRab7: Cat. # SI03021725) and the siRNA targeting luciferase serving as a control siRNA (siLuc: 5′-AACGTACGCGGAATACTTCGA-3′) were all purchased from Qiagen (Valencia, CA). LipofectamineTM 2000 transfection reagent (Invitrogen) was used for siRNA transfection following the manufacturer's protocol. Transfection of cultured NRVMs with siRNA was generally started at 48–72 hours after NRVMs were plated . Six hours after the transfection, the siRNA-containing medium was replaced with the fresh medium containing 2% FBS. . For the Rab7 knockdown experiments, Rab7 siRNA were introduced along the second round of sip62 or siLuc transfection |
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