HiPerFect Transfection Reagent

Protocol tips

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	Dilute siRNA in culture medium without serum. Incubate the samples for 5–10 min at room temperature

Protocol tips
Dilute siRNA in culture medium without serum. Incubate the samples for 5–10 min at room temperature

Publication protocol

AR42J cells were used between passages 7 and 13. For each group, including the controls, cells were suspended in 2 ml DMEM, which contained FBS and antibiotics, and added to 24-well plates (triplicates, density of 8 × 104 cells/well). Cells were incubated under standard cell culture conditions. After reaching 70% subconfluence, the medium was replaced by 500 μl fresh DMEM. For the calpain-2 knockdown, 0.002 nmol calpain-2 siRNA was diluted in 100 μl DMEM and mixed with 3 μl and 1.5 μl HPF, respectively. While the solution incubated at room temperature for 10 min, the transfection complexes were formed and subsequently were added drop-wise onto the cells, resulting in a final siRNA concentration of 3.33 nM. Control cells were incubated with control siRNA (scrRNA) at the same concentration and dilution, but without gene silencing effect on calpain-2 or with DMEM only. Thereafter, the cells were incubated under standard cell culture conditions. In order to induce cell differentiation, 100 nM dexamethasone were added to all samples 6 h after transfection. After further 48 h, a part of the siRNA-treated cells and untreated control cells were harvested for Western blot analysis to investigate the calpain-2 knockdown at the protein level. Additionally, cells were used for apoptosis experiments. siRNA-treated cells and control cells were exposed to H2O2 (final concentration: 250 μM). The medium was removed and replaced by DMEM 1 h after H2O2 addition. Cells without H2O2 exposure served as control. Then, the cells were incubated for further 5 h.

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