The mitochondrial targeting sequence (MTS) of succinate dehydrogenase complex subunit C was fused with green fluorescence protein (GFP) gene and red fluorescence protein (dsRED), resulting in MTS-GFP and MTS-dsRED, respectively. Lentiviral expression vectors were constructed by cloning MTS-dsRED and MTS-GFP genes into a lentiviral vector plasmid with EF1α promoter. For lentivirus production, HEK293T cells were seeded on 10-cm dishes at 70%–80% confluency. The cells were co-transfected with 4 μg lentiviral expression vector plasmid along with 4 μg pLP1, 4 μg pLP2, and 3 μg pLP/VCVG (Packaging plasmids, Invitrogen). Transfection mixtures were prepared in 1 mL of Opti-MEM containing the DNA and 30 μL of 293 Tran (Origene, USA) according to the manufacturer's protocol. After transfection, lentiviral supernatant samples were collected after 24, 48, and 72 h. The collected lentiviral supernatants were concentrated via ultracentrifugation, and the medium was exchanged with α-MEM medium. To label mitochondria with dsRED and GFP, UC-MSCs were seeded into 150-mm plates at 70%–80% density and then incubated with lentiviral supernatants and 4 μg/mL polybrene followed by further incubation for 72 h.
Opti mem
Manufactured by OriGene
Sourced in United States
Opti-MEM is a cell culture medium designed to support the growth and maintenance of a variety of mammalian cell lines. It is optimized to provide essential nutrients and reduce the need for serum supplementation, which can help reduce costs and minimize variability in experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
L3000015, by Thermo Fisher Scientific (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Lipofectamin 2000, by Addgene (1 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
Am17010, by Thermo Fisher Scientific (1 mentions)
Am17000, by Thermo Fisher Scientific (1 mentions)
Sr30004, by OriGene (1 mentions)
Lipofectamine 2000, by OriGene (1 mentions)
Lipofectamine 3000, by OriGene (1 mentions)
8 protocols using opti mem
1
Mitochondrial Labeling with Lentiviral Transduction
2
Setd2 Silencing in NIH/3T3 Cells
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On day one, NIH/3T3 cells were seeded at 15 625 cells/cm2 in a well of a six-well plate. After 24 h, 3.75 μl Lipofectamine 3000 (Invitrogen, L3000008) was diluted in 125 μl Opti-MEM (Gibco, 31985070). Setd2-specific or Scrambled siRNAs (OriGene, SR423523) were diluted in 125 μl Opti-MEM to a final concentration of 10 nM. Diluted Lipofectamine 3000 and siRNAs were mixed and incubated for 20 min at room temperature. The siRNA–lipid complex was added to the cells and incubated for 48 h. Transfections were carried out in triplicate and gene expression was assayed by RT-qPCR.
3
Transfection of 3T3-L1 Preadipocytes with DNAJC6
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3T3-L1 preadipocytes were transfected with DNAJC6 using a chemical method (L3000015, Thermo Fisher, Waltham, MA, USA). To increase the transfection efficiency, serum-free DMEM was used. A DNA master mix was prepared by mixing Opti-MEM, 2500 ng DNAJC6 DNA (OriGene, Rockville, MD, USA), P3000 reagent, and lipofectamine 3000 reagent. After reacting at room temperature for 15 min, the DNA master mix was dispensed into cells and cultured for 3–4 h. To remove untransfected cells during the transfection process, cells was cultured in DMEM containing 500 μg/mL of G418 (Cat #10131035, Thermo Fisher, Waltham, MA, USA) for 20 h under the same conditions described above. Thereafter, TgHsp cells were cultured using the same process and conditions of differentiation as those of the control group (Days 0–8).
4
Overexpression of Mouse Frataxin in HEK293T Cells
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HEK 293 T cells were plated in 35 mm collagen coated plates at a density of 3·105 cells/plate. After 20 h, cells were washed once with optiMEM™ medium (Gibco, 31,985–047) and transfected with a pCMV6-Entry vector containing untagged mouse Fxn ORF (OriGene, NM_008044) using 10 µM of Polyethylenimine in optiMEM™ medium. Cells were incubated for 1 h at 37ºC and then the plasmid was removed and cells were cultured with DMEM (Gibco, 41,966–029) supplemented with 10% Fetal Bovine Serum. After 24 h, cells were washed once with cold PBS and lysed in 200 µl lysis buffer consisting of 125 mM TRIS–HCl pH 7.5, 2% SDS and protease inhibitor cocktail (Roche). Lysates were heated at 100 °C for 3 min and loaded on SDS-PAGE gels.
5
Assessing NKCC1, NKCC2, and KCC2 Activity in HEK293 Cells
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HEK293 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1% L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin, and maintained at 37°C in a 5% CO2 humidified atmosphere. To assess NKCC1 and NKCC2 activity (Cl− influx assay), 3 million HEK cells were plated in a 10 cm cell-culture dish and transfected with a transfection mixture comprising 5 mL of DMEM, 4 mL Opti-MEM, 8 μg of DNA plasmid coding for NKCC1 (PRK-NKCC1 obtained from Medical Research Council and the University of Dundee), NKCC2 (OriGene plasmid #RC216145) subcloned in PRK5 plasmid, or mock control (empty vector), together with 8 μg of a plasmid coding for the Cl−-sensitive variant of the mbYFPQS (Addgene plasmid #80742),24 (link) and 32 μL of Lipofectamin 2000. To assess KCC2 activity (Tl influx assay), cells were transfected with 16 μg of KCC250 (link) subcloned in PRK5 plasmid or mock control (empty vector) and 32 μL of Lipofectamin 2000. After 4 h, the cells were collected and plated in 96-well black-walled, clear-bottomed plates at a density of 2.5 × 104. After 48 h, cells were used for the Cl− or Tl influx assays. All reagents were purchased from Life Technologies, unless otherwise specified.
6
Depleting EZH2 in Human Endothelial Cells
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To deplete EZH2, HUVECs at 75-80% confluence were used for transfection. In brief, Lipofectamine 2000 or RNAi Max (6 μL; Thermo Fisher Scientific) 42 (link) was mixed with Opti-MEM (250 μL; Thermo Fisher Scientific), and then two independent siRNA oligos against human EZH2 (20 nM; #SR301494-A, -B, Origene, Rockville, MD) or non-target control siRNA (25 nM; #SR30004, Origene) diluted in 250 μL Opti-MEM was added to the solution, mixed gently, and incubated at room temperature for 20 min. After 3-4 h of transfection, the medium was changed to M200 complete medium and cells were treated 48 h after transfection. Additional Silencer® Select EZH2 siRNA (#s4916, Thermo Fisher Scientific) or Silencer® Select Negative Control #1 siRNA (#S25503, Thermo Fisher Scientific) were used for RNA sequencing. The same method was used to transfect HUVECs with Anti-miRTM negative control (100 nM; #AM17010, Ambion) or Anti-miRTM hsa-miR-101-3p miRNA inhibitor (100 nM; #AM17000, Ambion) using RNAi Max as the transfection reagent.
7
siRNA Transfection in Cell Cultures
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Cells were seeded in media without antibiotics. After 24 hours, 10 μL Lipofectamine 2000 (Invitrogen) was incubated for 5 minutes with 240 μL Opti-MEM (Invitrogen). Next, 0.2 nmol (100 nM) of non-specific (Invitrogen), p53-specific siRNA (sc-45917, Santa Cruz biotechnology, CA), or 14-3-3σ-specific siRNA (SR503571, OriGene Technologies, Rockville, MD) was resuspended in 250 μL Opti-MEM and combined with Lipofectamine 2000. After 20 minutes, 500 μL of the siRNA-Lipofectamine 2000 mix was added to cells with 1.5 mL Opti-MEM. Six hours later, complete growth media was added.
8
Setd2 Knockdown in NIH/3T3 Cells
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On day one, NIH/3T3 cells were seeded at 15,625 cells/cm2 in a well of a 6-well plate.
After 24 hours, 3.75 l Lipofectamine 3000 (Invitrogen, L3000008) was diluted in 125 l Opti-MEM (Gibco, 31985070). Setd2-specific or Scrambled siRNAs (OriGene, SR423523) were diluted in 125 l Opti-MEM to a final concentration of 10 nM. Diluted Lipofectamine 3000 and siRNAs were mixed and incubated for 20 minutes at room temperature. The siRNA-lipid complex was added to the cells and incubated for 48 hours. Transfections were carried out in triplicate and gene expression was assayed by RT-qPCR.
After 24 hours, 3.75 l Lipofectamine 3000 (Invitrogen, L3000008) was diluted in 125 l Opti-MEM (Gibco, 31985070). Setd2-specific or Scrambled siRNAs (OriGene, SR423523) were diluted in 125 l Opti-MEM to a final concentration of 10 nM. Diluted Lipofectamine 3000 and siRNAs were mixed and incubated for 20 minutes at room temperature. The siRNA-lipid complex was added to the cells and incubated for 48 hours. Transfections were carried out in triplicate and gene expression was assayed by RT-qPCR.
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