Reverse transfection procedures were set up using Lipofectamine® RNAiMAX (Invitrogen). The final siRNA concentration was 20 nM, and SEMG cells were resuspended in DMEM/F12 supplemented with 10% FBS and then seeded on Lab-Tek™ 8-well chamber slides (Nunc). The siRNAs were diluted in Transfectagro™ (Corning) and mixed with Lipofectamine® RNAiMAX in Transfectagro™ at the 1:1 ratio by volume. The medium was refreshed for the transfected cells after 6 h of incubation, and the cells were assayed after 48–72 h, according to the experimental conditions.
Transfectagro
Manufactured by Corning
Transfectagro is a laboratory equipment product designed for cell transfection. It facilitates the introduction of genetic material into cells for various research and experimental purposes.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (6 mentions)
Opti mem, by Thermo Fisher Scientific (4 mentions)
Hek293f cell, by Thermo Fisher Scientific (2 mentions)
Expi293 cell, by Thermo Fisher Scientific (2 mentions)
Fectopro reagent, by Polyplus Transfection (2 mentions)
Steriflip sterile disposable vacuum filter units, by Merck Group (2 mentions)
Dsirna duplexes, by Integrated DNA Technologies (2 mentions)
Transfectagro, by Integrated DNA Technologies (2 mentions)
Transfectagro, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Corning (1 mentions)
Poly 1 c, by InvivoGen (1 mentions)
Pei max 40000, by Polysciences (1 mentions)
Sepharose 4b beads, by GE Healthcare (1 mentions)
Galanthus nivalis lectin, by Vector Laboratories (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Lab tek 8 well chamber slide, by Thermo Fisher Scientific (1 mentions)
Pierce fab preparation kit, by Thermo Fisher Scientific (1 mentions)
10 protocols using transfectagro
1
Reverse Transfection Optimization in SEMG Cells
2
Poly(I:C) Transfection in MRC5 and Efk3 Cells
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MRC5 and Efk3 cells were seeded at a concentration of 3 × 105 cells/well in six-well plates and transfected with 750 ng/mL poly(I:C) (InvivoGen, San Diego, CA, USA) using Lipofectamine 2000 (Invitrogen, Camarillo, CA, USA) as previously described [38 (link)]. Briefly, 750 ng/mL poly(I:C) was mixed in a total volume of 250 μL of TransfectaGro (Corning) and 12 μL of Lipofectamine 2000. This mixture was incubated at room temperature for 15 min and added to cells in complete medium. Cells were harvested 16 h post-transfection and RNA was extracted.
3
SOSIP Trimer and gp120 Purification
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SOSIP trimers and gp120 monomer were expressed in HEK293F cells and purified as described previously [49 (link)]. Briefly, SOSIP and gp120 were transfected with 150 μg of Furin DNA (SOSIP only), 300 μg of trimer DNA, and 1.5 mL of PEI MAX 40000 (Polysciences) or 1 mL of 293fectin in Transfectagro (Corning). Five days after transfection, supernatants were harvested by spinning at 3500xg and filtered with 0.22 or 0.45 μM filters. Supernatants were run over Galanthus nivalis lectin (Vector Labs) column or PGT145 affinity columns. As described previously, PGT145 affinity columns were made by PGT145 antibody coupled to CnBr-activated Sepharose 4B beads (GE) [49 (link)]. Purified proteins were run on size exclusion chromatography columns, specifically Superdex 200 10/300 GL column (Cytvia) in TBS or PBS. Some SOSIPs were biotinylated using Pierce Biotinylation Kit using the manufacturer’s protocol. Trimers were aliquoted into small volumes and frozen at −80°C. Antigenicity was tested for binding to known antibodies using ELISA or BLI.
4
Mitochondrial Calcium Imaging in OPA1 Mutants
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Cells were plated on glass 25 mm coverslips and then transfected with specific constructs using Lipofectamine 2,000–3,000 (Invitrogen). MEF cells were transfected pCCEY plasmid encoding for human OPA1 isoform 1. OPA1 GTPase or GED mutants were built in the same backbone. For mitochondrial matrix calcium analysis, we used mitochondrial matrix-targeted Ca2+-sensitive proteins mtRCaMP (Kd ∼ 1 µM) and CEPIA3mt (Kd ∼ 10 µM). The transfection was performed with OPTI-mem (Thermofisher) or Transfectagro (Corning) and Lipofectamine 2,000 or 3,000 (Invitrogen, Cat#11668019 or Cat#L300015, respectively) according to the manufacturer´s protocol. For optimal expression, the cells were grown for 48 h after transfection. One μg of DNA per plasmid per 35 mm dish was used for each experiment.
5
Transient Expression and Purification of Antibodies and Viral Proteins
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To express Abs, the corresponding heavy chain and light chain plasmids were transiently transfected into the Expi293 cell (Life Technologies) at 3 × 106 cells/ml with FectoPRO PolyPlus transfection reagent (Polyplus, catalog no. 116–040). To boost the protein yield, we fed cells with 300 mM sodium valproic acid solution and glucose solution 1 day after transfection. To purify mAbs, the supernatants were harvested 4 days after transfection. To express the soluble S ectodomain proteins from SARS-CoV-1, SARS-CoV-2, and their truncations, plasmids were transfected into human embryonic kidney (HEK) 293F cells (Life Technologies) at 1 million cells/ml. For 1 liter of HEK293F transfection, we combined 350 μg of plasmids with 16 ml of transfectagro (Corning) and filtered with 0.22-μm Steriflip Sterile Disposable Vacuum Filter Units (Millipore Sigma). The 1.8 ml of 40K polyethylenimine (PEI) (1mg/ml) was mixed into 16 ml of transfectagro in another conical tube. The filtered plasmid solution was gently combined with the mixed PEI solution by inverting the tube several times. After resting at room temperature for 30 min, the mixture was poured into 1 liter of HEK293F cells. The supernatant was centrifuged and filtered with 0.22-μm membrane to a glass bottle 5 days after transfection.
6
Transient Monoclonal Antibody Expression and Fab Purification
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Monoclonal antibodies were transiently expressed in 293F cells. Heavy and light chain DNA was added in a 1:1 ratio with 1.5 mL PEI: 1mL cell volume as a transfection reagent and diluted in Opti-MEM (Thermo Fisher) or Transfectagro (Corning) media. Five days after transfection, supernatants were harvested and sterile filtered through a 0.22 or 0.45 μm filter. IgG containing supernatant was batch bound overnight at 4°C using Praesto AP (Purolite). The next day, beads were washed 10x with PBS, and the antibody was eluted using 0.2 M citric acid and neutralized to pH 7 with 2M Tris pH 9.0. Antibodies were buffer exchanged into PBS.
Fabs were papain digested using Pierce Fab Preparation Kit (Thermo Fisher) following the manufacturer’s protocol for human Fab digest. Polyclonal IgG from sera were also prepared using Pierce Fab Preparation Kit with each digest at 0.5 mg/mL and a digest time of 10–16 hr.
Fabs were papain digested using Pierce Fab Preparation Kit (Thermo Fisher) following the manufacturer’s protocol for human Fab digest. Polyclonal IgG from sera were also prepared using Pierce Fab Preparation Kit with each digest at 0.5 mg/mL and a digest time of 10–16 hr.
7
Transient Expression and Purification of Antibodies and Viral Proteins
8
Plasmid and siRNA Transfections in Cell Culture
9
Plasmid and siRNA Transfections in Cell Culture
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Plasmid transfections were performed using Lipofectamine 2000 (Thermo Fisher) according to the manufacturer’s protocol but using Transfectagro (Corning) instead of Opti-MEM. Cells were incubated with transfection mix in Transfectagro supplemented with 10 % FBS for 6–8 h, following by a change of media to regular growth media and analysis after 18–20 h.
DsiRNA duplexes were obtained from Integrated DNA Technologies. Transfections with siRNA were performed with the appropriate duplexes (seeKey Resources Table ) using Lipofectamine RNAiMAX (Thermo Fisher) following the manufacturer’s protocol except using Transfectagro in place of Opti-MEM. Cells were incubated wit transfection mix in Transfectagro supplemented with 10% FBS for 12–16 h, followed by exchange with fresh media. NC1 (negative control 1, IDT) was used as the control siRNA duplex for all experiments. Forty-eight h post transfection, cells were subjected to analysis via western blot, microscopy or flow cytometry.
DsiRNA duplexes were obtained from Integrated DNA Technologies. Transfections with siRNA were performed with the appropriate duplexes (see
10
Plasmid and siRNA Transfection Protocol
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Plasmid transfections were performed using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol but replacing Opti-MEM with Transfectagro (Corning). Cells were incubated with the transfection mix in Transfectagro with 10% FBS for 7-8 h, then the media was exchanged back to regular growth medium. DsiRNA duplexes were purchased from IDT. siRNA transfections were achieved using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's procedures except using Transfectagro instead of Opti-MEM. Twelve to 16 h after incubation with the transfection mix in Transfectagro with 10% FBS, cells were supplemented with the regular growth medium.
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