TransIT-LT1 (Mirus Bio LLC, Madison, WI, USA) was used for plasmid DNA transfection. For optimum transfection efficiency cells were seeded at 70–80% confluency. TransIT-LT1 was mixed with OptiMEM (Gibco) at a ratio of 1:33 and incubated at room temperature for 5 min, DNA was then added at a ratio of 1 μg DNA:3 μL TransIT-LT1, and incubated for a minimum of 15 min. The transfection mix was added to the cells directly into the culture medium.
Transit lt1
Manufactured by Thermo Fisher Scientific
Sourced in United States
TransIT-LT1 is a non-liposomal lipid-based transfection reagent designed for efficient delivery of nucleic acids, such as plasmid DNA, into a variety of mammalian cell lines. It is a liquid formulation that can be directly added to cell culture media for simple and effective transfection.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (5 mentions)
Pspax2, by Thermo Fisher Scientific (2 mentions)
Pspax2, by Addgene (2 mentions)
Transit lt1, by Mirus Bio (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Transit lt1, by Takara Bio (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Glomax multi detection system, by Promega (1 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Pcmv vsv g envelope plasmid, by Addgene (1 mentions)
Pspax2 packaging plasmid, by Addgene (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Cytiva (1 mentions)
Dulbecco modified eagle medium (dmem), by Cytiva (1 mentions)
Hek 293t cells, by Cytiva (1 mentions)
9 protocols using transit lt1
1
Plasmid DNA Transfection with TransIT-LT1
2
Signaling Pathway Analysis in Tumorigenesis
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Analysis of various key signaling pathways implicated in human tumorigenesis was performed using Cignal Finder Cancer 10-pathway Reporter Array kit (SABiosciences: Qiagen, Hilden, Germany). Cellular transfection was performed with TransIT-LT1 (MirusBio, Madison, WI, USA). Plasmid DNA for each signaling pathway provided in the kit (Cat. #: CCA-001L; Invitrogen, Carlsbad, CA, USA) was mixed with TransIT-LT1 to form TransIT-LT1/DNA complexes before being added to the cell suspension and incubated at 37°C overnight. The transfected cells were then incubated with extracts of the four Phyllanthus species for another 24 h. Dual-Glo luciferase reagent was added to each well and incubated at room temperature for 10 min to obtain firefly luminescence readings using the GloMax Multi-detection System (Promega, Durham, NC, USA). This was followed by addition of Dual-Glo Stop & Glo reagent to obtain renilla-luminescence readings. Firefly constructs visualized modulation of key transcription-factor activity, usually representing a downstream target of a particular signaling pathway. The renilla construct functions as an internal control to normalize transfection efficiencies and to monitor cell viability. Luminescence for each sample was calculated based on the firefly-to-renilla luminescence ratio.
3
Lentiviral sgRNA Library Production and Titration
4
Lentiviral sgRNA Library Production and Titration
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For virus production of sgRNA libraries, HEK293FT cells were seeded into 15cm dishes to reach confluency of 60% the following day. Cells were transfected with 14.3 μg library plasmid, 10.9 μg psPAX2 (Addgene ID #12260) and 7.1 μg pMD2.G (Addgene ID #12259) per plate using 119 μl TransIT-LT1 (Mirus Bioscience) in 850 μl OptiMEM (Gibco). 48h and 72h post transfection, supernatant was collected and filtered (0.45 μm). For other constructs, HEK293FT were seeded in 10 cm plates and transfected with 2 μg viral plasmid, 1.25 μg psPAX2 and 0.75 μg pMD2.G per plate using 18 μl TransIT-LT1 in 270 μl OptiMEM (Gibco) the next day.
Lentiviral sgRNA libraries were functionally titrated by spinfection (2h, 1000g, 33°C) of target cells with varying amounts of lentiviral supernatant in 12-well plates with 3x10^6 cells per well as described in syngeneic68 (link)
. Amount of lentivirus needed per 12-well for a target MOI of 0.3 was calculated as survival of 0.25 relative to unselected control.
Lentiviral sgRNA libraries were functionally titrated by spinfection (2h, 1000g, 33°C) of target cells with varying amounts of lentiviral supernatant in 12-well plates with 3x10^6 cells per well as described in syngeneic68 (link)
. Amount of lentivirus needed per 12-well for a target MOI of 0.3 was calculated as survival of 0.25 relative to unselected control.
5
Transient Transfection of HEK 293 Cells
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Transient transfection of HEK 293 cells was performed with TransIT-LT1 (Takara). For transfection, 14 μg of expression plasmid (3MST/pCI)9 (link) was mixed with 45 μL of TransIT-LT1 in 1.5 mL of Opti-MEM (Invitrogen) and then added to HEK 293 cells at 60–70% confluency in a 9 cm dish. Cells were harvested at 48 h post-transfection and washed with ice-cold PBS. After precipitation by centrifugation, cell pellets were resuspended in the ice-cold buffer and sonicated.
6
METTL3 and METTL16 Knockdown in MOLM-13 Cells
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MOLM-13 cells were transfected using Lipofectamine
2000 reagent (Invitrogen) according to the manufacturer’s instructions
using pLKO-TETon-Puro lentiviral vectors expressing shRNAs against
the coding sequence of human METTL3, METTL16, or a scrambled control. Twenty-four hours after spinfection, the
cells were replated in fresh medium containing 1 μg mL–1 of puromycin and kept in selection medium for 7 days. Anti-METTL3
and scrambled shRNAs were induced by treating the cells with 200 ng
mL–1 tetracycline for 3 days, anti-METTL16 was induced
by identical treatment for 2 days.
gRNA assays were performed
using dual gRNA vectors as reported previously.44 (link) Viral supernatants were collected 48 h after transfection.
All transfections and viral collections were performed in 15 cm plates
as mentioned below. For virus production, 5 μg of the above
plasmids and 5 μg of psi-Eco packaging vector were transfected
dropwise into the 293T cells using 47.5 μL TransIT LT1 (Mirus)
and 600 μL of Opti-MEM (Invitrogen). The resulting viral supernatant
was harvested, and transduction of cells was performed in 6-well plates.
After transduction, transduced cells were sorted for BFP (for gRNA).
The gRNA sequences are listed inTable S12 .
2000 reagent (Invitrogen) according to the manufacturer’s instructions
using pLKO-TETon-Puro lentiviral vectors expressing shRNAs against
the coding sequence of human METTL3, METTL16, or a scrambled control. Twenty-four hours after spinfection, the
cells were replated in fresh medium containing 1 μg mL–1 of puromycin and kept in selection medium for 7 days. Anti-METTL3
and scrambled shRNAs were induced by treating the cells with 200 ng
mL–1 tetracycline for 3 days, anti-METTL16 was induced
by identical treatment for 2 days.
gRNA assays were performed
using dual gRNA vectors as reported previously.44 (link) Viral supernatants were collected 48 h after transfection.
All transfections and viral collections were performed in 15 cm plates
as mentioned below. For virus production, 5 μg of the above
plasmids and 5 μg of psi-Eco packaging vector were transfected
dropwise into the 293T cells using 47.5 μL TransIT LT1 (Mirus)
and 600 μL of Opti-MEM (Invitrogen). The resulting viral supernatant
was harvested, and transduction of cells was performed in 6-well plates.
After transduction, transduced cells were sorted for BFP (for gRNA).
The gRNA sequences are listed in
7
Shuttle-GFP Reporter Transfection and Validation
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The plasmid for expression of shuttle-GFP (Woerner et al., 2016 (link)), a reporter for nucleocytoplasmic transport, was a gift of Ulrich Hartl and Nadine Wischnewski (Max Planck Institute of Biochemistry, Martinsreid, Germany). The shuttle-GFP plasmid was transfected into human primary myogenic cells or SV40-immortalized MEFs using X-treme GENE 9 HP (cat. XTGHP-RO, Roche Diagnostics, Indianapolis, USA), Turbofect (cat. R0533, Thermo Fisher Scientific), or TransIT-LT1 (cat. MIR2304, Mirus Bio, Madison, WI, USA) following the manufacturer's instructions. At 24 h after transfection with the shuttle-GFP plasmid, the transfected cells were exposed to BacMam-DUX4 viruses for an additional 48 h, at which time the cells were fixed and immunostained for the V5 epitope tag as above. As a positive control for shuttle-GFP function, we confirmed that GFP accumulated in the nucleus when cultures were treated with a known inhibitor of nuclear export, Leptomycin B (cat. 9676, Cell Signaling Technology), used at 10 nM for 3 h at 37°C.
8
Lentiviral Production in HEK293T Cells
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Lentivirus was produced in a 10 cm2 plate by transient transfection of ∼70% confluent HEK293T cells in 10 ml of media using 63 μl TransIT-LT1 (Mirus, MIR2304) in 600 μl Opti-MEM reduced serum medium (Thermo Fisher Scientific, 31985088) containing 10.5 μg lentiviral expression plasmid, 14 μg psPAX2 packaging plasmid (Addgene, 12260), and 5.25 μg pCMV-VSV-G envelope plasmid (Addgene, 8454). Lentiviral supernatant was harvested at 48 h post-transfection and passed through a 0.45 μm syringe filter (Pall, 4614).
9
Cell Culture and Transfection Protocol
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HEK-293T cells (ATCC Cat# CRL-3216), Vero cells (ATCC Cat# CCL-81) and DF-1 cells (ATCC Cat# CRL-12203) were maintained in the Dulbecco modified Eagle medium (DMEM, Hyclone Laboratories, Logan, UT, USA), supplemented with 10% fetal bovine serum (FBS, Gibco, Billings, MT, USA) and 1% penicillin/streptomycin, and maintained at 37 °C with 5% CO2. Chicken HD11 macrophages (BCRJ Cat# 0099) were cultured in the Roswell Park Memorial Institute medium (RPMI) 1640 medium (Hyclone Laboratories, USA), supplemented with 10% FBS and 1% penicillin/streptomycin, and maintained at 37 °C with 5% CO2. The gene knockout HD11 cells (STING−/− cells and IRF7+/− cells) were as described previously [26 (link)]. Transfection was performed by using Lipofectamine 2000 or TransIT-LT1 (Thermo Fisher Scientific), following the manufacturer’s instructions.
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