In vitro expression was performed in HeLa cells (ATCC, catalog #CCL-2). Cells were seeded in Minimal essential medium (MEM) with 10% FBS, 1X Glutamax, and 1X sodium pyruvate at 37 °C at 15,000 cells in a total volume of 100 µL per well and grown for 24 h. Extracted mRNA was transfected with Lipofectamine L2000 (Life Technologies, catalog #11668019) at 1333 ng of mRNA per well and incubated for 18–20 h at 37 °C. To detect intracellular protein expression, cells were first fixed and permeabilized (Cytoxfix/Cytoperm; BD Biosciences catalog #554714) and then stained with a proprietary primary antibody (1/2000 dilution of 1 mg/mL stock). Data were acquired on the BD LSRFortessa™ Flow Cytometer (BD Biosciences) using BD FACSDiva™ version 8.0 software (BD Biosciences) and analyzed using FlowJo software version 10.4 (BD Biosciences). Mean fluorescence intensity was normalized using a neat mRNA positive control and non-translating negative control. The gating strategy can be found in Supplementary Fig. 8 .
Lipofectamine l2000
Manufactured by Thermo Fisher Scientific
Lipofectamine L2000 is a transfection reagent used for the introduction of nucleic acids, such as DNA or RNA, into mammalian cells. It facilitates the uptake of these molecules by the cells, enabling various research applications involving gene expression and genetic manipulation.
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Lab products found in correlation
4 protocols using lipofectamine l2000
1
Protein Expression in HeLa Cells
2
BJ Fibroblast mRNA Transfection
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BJ fibroblasts (ATCC; catalog #CRL-2522) were cultured in complete media (Eagle’s minimum essential medium [EMEM] with L-glutamine; 10% FBS). Cells were seeded in 96-well plates (20,000 cells/well) 24 h prior to transfection; extracted mRNA was transfected with Lipofectamine L2000 (Life Technologies, catalog #11668019) at 250 ng/well and incubated for 48 h at 37 °C before collection of the supernatant for analysis. An hEPO mRNA standard was included as an assay control.
3
BJ Fibroblast mRNA Transfection Protocol
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BJ fibroblasts (ATCC; catalog #CRL-2522) were cultured in complete media (Eagle's minimum essential medium [EMEM] with L-glutamine; 10% FBS). Cells were seeded in 96-well plates (20,000 cells/well) 24 hours prior to transfection; extracted mRNA was transfected with Lipofectamine L2000 (Life Technologies, catalog #11668019) at 250 ng/well and incubated for 48 hours at 37 °C before collection of the supernatant for analysis. A hEPO mRNA standard was included as an assay control.
4
In Vitro mRNA Expression in HeLa Cells
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In vitro expression was performed in HeLa cells (ATCC, catalog #CCL-2). Cells were seeded in Minimal essential medium (MEM) with 10% FBS, 1X Glutamax, and 1X sodium pyruvate at 37 °C at 15,000 cells in a total volume of 100 µL per well and grown for 24 hours. Extracted mRNA was transfected with lipofectamine L2000 (Life Technologies, catalog #11668019) at 1333 ng of mRNA per well and incubated for 18-20 hours at 37 °C. To detect intracellular protein expression, cells were first fixed and permeabilized (Cytoxfix/Cytoperm; BD Biosciences catalog #554714) and then stained with a proprietary primary antibody (1/2000 dilution of 1 mg/mL stock). Data were acquired on the BD LSRFortessa™ Flow Cytometer (BD Biosciences) using BD FACSDiva™ version 8.0 software (BD Biosciences) and analyzed using FlowJo software version 10.4 (BD Biosciences). Mean fluorescence intensity was normalized using a neat mRNA positive control and non-translating negative control. The gating strategy can be found in Supplementary Figure 8.
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