Antisense LNA GapmeR targeting (Exiqon™/Qiagen, Germany) MIR99AHG were diluted in Opti-Mem medium (Life Technologies™) and mixed in a 1:1 ratio with Lipofectamine RNAiMAX 3000 (Life Technologies™). As a negative control, non-targeting scrambled LNA GapmeR (Exiqon™/ Qiagen, Germany) was transfected to the macrophages. For mouse and human macrophage cultures, two GapmeR MIR99AHG designs (5’-CAGTTGCGTGGAGTAA-3’ and 5’-CTAGCTTTGAAGTCGT-3’) were used to transfect murine macrophages and for the in vivo experiment in mice one GapmeR MIR99AHG design was used (5’-CAGTTGCGTGGAGTAA-3’). Cultured cells were transfected with 25 nM of GapmeR for 48 hours and the medium was replaced with DMEM or RPMI medium supplemented with 10% FCS for downstream experiments. In vitro dose response experiments were performed to optimize ASO transfections, investigating the effect of different concentrations of scrambled GapmeR negative control on BMDM cell viability. BMDMs treated with scrambled GapmeR negative control for 24, 48 or 72 hours at either 10nM or 50 nM did not cause any cell toxicity in cell culture plates seeded with 200’000 macrophages as confirmed by Cell Titre Blue assay (Figure S4A-C ).
Lipofectamine rnaimax 3000
Manufactured by Thermo Fisher Scientific
Sourced in Germany
Lipofectamine RNAiMAX 3000 is a transfection reagent designed for the efficient delivery of small interfering RNA (siRNA) and other nucleic acids into eukaryotic cells. It facilitates the introduction of genetic material into cells, enabling the study of gene function through RNA interference (RNAi) techniques.
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2 protocols using lipofectamine rnaimax 3000
1
LNA GapmeR Silencing of miR-99a Host Gene
2
LNA GapmeR Silencing of miR-99a Host Gene
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Antisense LNA GapmeR targeting (Exiqon™/Qiagen, Germany) MIR99AHG were diluted in Opti-Mem medium (Life Technologies™) and mixed in a 1:1 ratio with Lipofectamine RNAiMAX 3000 (Life Technologies™). As a negative control, non-targeting scrambled LNA GapmeR (Exiqon™/ Qiagen, Germany) was transfected to the macrophages. For mouse and human macrophage cultures, two GapmeR MIR99AHG designs (5’-CAGTTGCGTGGAGTAA-3’ and 5’-CTAGCTTTGAAGTCGT-3’) were used to transfect murine macrophages and for the in vivo experiment in mice one GapmeR MIR99AHG design was used (5’-CAGTTGCGTGGAGTAA-3’). Cultured cells were transfected with 25 nM of GapmeR for 48 hours and the medium was replaced with DMEM or RPMI medium supplemented with 10% FCS for downstream experiments. In vitro dose response experiments were performed to optimize ASO transfections, investigating the effect of different concentrations of scrambled GapmeR negative control on BMDM cell viability. BMDMs treated with scrambled GapmeR negative control for 24, 48 or 72 hours at either 10nM or 50 nM did not cause any cell toxicity in cell culture plates seeded with 200’000 macrophages as confirmed by Cell Titre Blue assay (Figure S4A-C ).
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