GCGR Stealth siRNA ((Invitrogen, Carlsbad, CA, USA) or negative control siRNA (Invitrogen) was mixed with Lipofectamine RNAiMAX (Invitrogen) in an OptiMEM serum-free medium (Invitrogen) for 5 min at room temperature and then added to cells at a final concentration of 10 pmol/L. Forty-eight hours post transfection, cells were harvested for western blotting and cell proliferation assays.
Optimem serum
Manufactured by Thermo Fisher Scientific
Sourced in United States
OptiMEM serum is a specialized cell culture medium formulated to support the growth and maintenance of a variety of cell types. It is designed to optimize cell performance and viability in various experimental and research applications.
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Lab products found in correlation
4 protocols using optimem serum
1
GCGR Stealth siRNA Knockdown Assay
2
Macrophage Transfection with siRNA
3
In-vitro Validation of DNA Vaccine
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In-vitro expression of DNA vaccine candidate was checked by transfection of the same in Vero cell line. For transfection experiments, Vero cells were seeded at density of 3 × 105 cells/ml in 6 well plates and kept in CO2 incubator to attain 80–90% confluency. After 24Hrs, once the cells reached the desired confluency, transfection was carried out in OptiMEM serum free medium with Lipofectamine 2000 reagent (Thermo Fisher). Two different concentrations (4 µg and 8 µg) of DNA construct was used for transfection experiments. We keep the amount of transfection reagent constant while plasmid DNA concentration varied to achieve 1:1 and 1:2 ratios of volume to mass. After transfection, media was replenished with fresh DMEM media (Biowest) containing FBS. After 72Hrs, plates were fixed with 1:1 acetone and methanol. Anti-S1 rabbit polyclonal antibody (NB100-56048) from Novus Biologicals was added to each well and incubated for 1Hr followed by incubation with FITC labelled anti- rabbit antibody (Merck). Cells were washed three times with PBS and stained with DAPI counter stain. Fluorescence images were captured using an inverted microscope (Zeiss AX10) at 20X magnification.
4
Overexpressing uc.412 in Macrophages
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MCs were transfected with uc.412 plasmid (oeuc.412; VectorBuilder) or empty vector control (oeVec) with Lipofectamine 2000® (Invitrogen; Thermo Fisher Scientific, Inc.). Briefly, 2.5 µg plasmid was diluted into 125 µl Opti-MEM serum-free medium (Gibco; Thermo Fisher Scientific, Inc.), and 6 µl Lipofectamine 2000 was diluted with 125 µl Opti-MEM serum-free medium and incubated room temperature for 5 min. The two solutions were incubated at room temperature for 20 min and then added to the cells seeded in 6-well plates at a density of 2×105 cells/well. Culture medium was replaced after 8 h. After 24 h, puromycin was used to select cells that were puromycin resistant following transfection. After a further 24 h, these cells were used for further experiment. The transfection efficacy was detected by RT-qPCR.
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