ON-TARGETplus Rat Klf15 (85497) siRNA - SMARTpool

Protocol tips

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	1–2-Day-old Lewis neonatal rats were sacrificed by decapitation. Hearts were carefully removed, and left ventricles were cut into small pieces. Cardiac cells were then isolated by enzymatic dissociation using 0.05% collagenase I (Invitrogen, catalog no. 17100-017) and 0.05% pancreatin (Sigma, catalog no. p3292) dissolved in 1× Hanks' balanced salt solution (Sigma, catalog no. H4641). Cells were pre-plated for 1 h in DMEM (Invitrogen, catalog no. 11966) (supplemented with 10% horse serum, 5% newborn calf serum, 0.16% glucose and antibiotics) to separate myocytes from fibroblasts. After 1 h, cardiomyocytes were collected, counted, and plated in plates coated with 1% gelatin (Fluka, via Sigma). Overnight, cells were grown in DMEM supplemented with 10% horse serum, 5% newborn calf serum, 0.16% glucose, and antibiotics. Dharmacon ON-TARGETplus siRNA SMARTpools for non-targeting control (D-001810-10) and KLF15 (L-080131-01) were transfected (Lipofectamine 2000, Invitrogen) to a final concentration of 300 nm and incubated for 5 h. Medium was changed to medium containing 2% BSA, and after 72 h cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and stained with phalloidin 1:40 (Invitrogen, catalog no. F432) in PBS for actin. Images were taken with a Leica inverted microscope, and cell size (actin-positive area) was analyzed with Scion image.

Protocol tips

1–2-Day-old Lewis neonatal rats were sacrificed by decapitation. Hearts were carefully removed, and left ventricles were cut into small pieces. Cardiac cells were then isolated by enzymatic dissociation using 0.05% collagenase I (Invitrogen, catalog no. 17100-017) and 0.05% pancreatin (Sigma, catalog no. p3292) dissolved in 1× Hanks' balanced salt solution (Sigma, catalog no. H4641). Cells were pre-plated for 1 h in DMEM (Invitrogen, catalog no. 11966) (supplemented with 10% horse serum, 5% newborn calf serum, 0.16% glucose and antibiotics) to separate myocytes from fibroblasts. After 1 h, cardiomyocytes were collected, counted, and plated in plates coated with 1% gelatin (Fluka, via Sigma). Overnight, cells were grown in DMEM supplemented with 10% horse serum, 5% newborn calf serum, 0.16% glucose, and antibiotics. Dharmacon ON-TARGETplus siRNA SMARTpools for non-targeting control (D-001810-10) and KLF15 (L-080131-01) were transfected (Lipofectamine 2000, Invitrogen) to a final concentration of 300 nm and incubated for 5 h. Medium was changed to medium containing 2% BSA, and after 72 h cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and stained with phalloidin 1:40 (Invitrogen, catalog no. F432) in PBS for actin. Images were taken with a Leica inverted microscope, and cell size (actin-positive area) was analyzed with Scion image.

Publication protocol

1–2-Day-old Lewis neonatal rats were sacrificed by decapitation. Hearts were carefully removed, and left ventricles were cut into small pieces. Cardiac cells were then isolated by enzymatic dissociation using 0.05% collagenase I (Invitrogen, catalog no. 17100-017) and 0.05% pancreatin (Sigma, catalog no. p3292) dissolved in 1× Hanks' balanced salt solution (Sigma, catalog no. H4641). Cells were pre-plated for 1 h in DMEM (Invitrogen, catalog no. 11966) (supplemented with 10% horse serum, 5% newborn calf serum, 0.16% glucose and antibiotics) to separate myocytes from fibroblasts. After 1 h, cardiomyocytes were collected, counted, and plated in plates coated with 1% gelatin (Fluka, via Sigma). Overnight, cells were grown in DMEM supplemented with 10% horse serum, 5% newborn calf serum, 0.16% glucose, and antibiotics. Dharmacon ON-TARGETplus siRNA SMARTpools for non-targeting control (D-001810-10) and KLF15 (L-080131-01) were transfected (Lipofectamine 2000, Invitrogen) to a final concentration of 300 nm and incubated for 5 h. Medium was changed to medium containing 2% BSA, and after 72 h cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and stained with phalloidin 1:40 (Invitrogen, catalog no. F432) in PBS for actin. Images were taken with a Leica inverted microscope, and cell size (actin-positive area) was analyzed with Scion image.

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