connexin 43 siRNA (r)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Transfect in the presence of 8 μM transfection reagent. Incubate cells at 37C in a CO2 incubator for 5–24 hours.	Study expression 24–72 hours post-transfection

Protocol tips
Transfect in the presence of 8 μM transfection reagent. Incubate cells at 37C in a CO2 incubator for 5–24 hours.
Downstream tips
Study expression 24–72 hours post-transfection

Publication protocol

To determine the effect of reduced Cx43 expression on communication between Müller cells and pericytes, rMC-1 was transfected with Cx43 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or scramble siRNA (Qiagen, Valencia, CA, USA) as a negative control in the presence of 8 μM transfection reagent (Lipofectin; Invitrogen, Carlsbad, CA, USA). Transfected cells were washed and grown as rMC-1 monoculture and rMC-1/pericytes for 3 days. To confirm the effects of Cx43 downregulation and avoid “off target” effects, three Cx43 siRNAs targeted to different locations of the Cx43 transcript were independently transfected in rMC-1. Connexin 43 siRNA-1 (Santa Cruz Biotechnology), Cx43 siRNA-2 (Ambion, Austin, TX, USA), and Cx43 siRNA-3 (Invitrogen, Grand Island, NY, USA). Note, Cx43 siRNA-1 is referred to as Cx43 siRNA. The efficacy of each of the Cx43 siRNAs in reducing Cx43 expression was tested at different concentrations. In this study, we used 40-nM concentration of Cx43 siRNA to achieve approximately 30% reduction in Cx43 level to mimic the HG-induced Cx43 downregulation.

To determine whether Cx43 upregulation rescues cells from HG-induced apoptosis, rMC-1 were grown for 7 days in HG medium and transfected with plasmid pEGFPN1 containing full-length Cx43 cDNA or empty vector as control using transfection reagent (Lipofectamine 2000; Invitrogen) at a ratio of 1 μL of transfection reagent (Invitrogen) for every 1 μg plasmid DNA. In parallel, cells were grown in normal (N) or HG medium for 7 days to serve as control. Transfected cells were subsequently grown in the presence of G418 (Invitrogen) at 500 μg/mL until stable colonies formed.

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