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Transit 293

Manufactured by Mirus Bio
Sourced in United States

TransIT-293 is a transfection reagent designed for the efficient delivery of nucleic acids, such as plasmid DNA, into 293 and 293T cell lines. It is a proprietary, non-liposomal formulation that facilitates the uptake of DNA into cells, enabling effective gene expression.

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155 protocols using transit 293

1

Generation and Validation of Inducible Cell Lines

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All cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose, GlutaMAX, and sodium pyruvate (ThermoFisher 10569) with 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Parent HEK293T, Flp-In T-REx 293, and HeLa T-REx cell lines were authenticated by STR profiling. ATP13A1 knockout cell lines were generated by transfecting Flp-In T-REx HeLa (Fig. 2D and fig. S5-9) or Flp-In T-REx 293 (Fig. 2A-C and fig. S3-4) cells with pX459 plasmids containing target guide RNAs using Lipofectamine 2000 or TransIT293 (Mirus Bio), respectively, according to manufacturer’s instructions. After 24 hr, transfected cells were selected with 2 mg/mL puromycin for 48 hr. Single clones were isolated and knockouts validated by immunoblotting and amplicon sequencing.
To establish stable doxycycline-inducible cell lines expressing either FLAG-tagged tail-anchored proteins or FLAG-tagged ATP13A1, wildtype or ATP13A1 knockout Flp-In T-REx HeLa or 293 cells were co-transfected with a 1:1 ratio of pOG44 and pcDNA5/FRT/TO vector containing the desired insert using Lipofectamine 2000/Lipofectamine 3000 or TransIT293 (Mirus Bio), respectively. After 24 hr, cells were selected with 10 μg/mL Blasticidin and 150-300 μg/mL Hygromycin for at least two weeks, and expression was validated by induction with 10 ng/mL doxycycline for 24-48 hr and immunoblotting.
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2

Transient and Stable Transfection of HEK293 Cells

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All cells were cultured at 37°C and 5% CO2 in DMEM with high glucose (4.5 g/l), GlutaMAX, and pyruvate (110 mg/l) (Life Technologies 10569044) supplemented with 10% fetal bovine serum (FBS). Transient transfections of HEK293 cells were performed using TransIT-293 (Mirus) according to the manufacturer’s instructions. To generate stable cell lines, Flp-In T-REx 293 cells were cotransfected with the pcDNA5/FRT/TO plasmid containing the reporter gene and pOG44 encoding for the Flp recombinase in a 1:1 ratio using TransIT-293 (Mirus) following the manufacturer’s instructions. Two days after transfection, cells were placed under selection and maintained with 10 μg/ml blasticidin S HCl (Life Technologies) and 100 μg/ml hygromycin B (Life Technologies). Reporter expression was induced with 10 ng/ml doxycycline hyclate (Sigma-Aldrich) for 4 h for fluorescent reporters or 24 h for FVβ reporters prior to analysis, unless otherwise indicated.
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3

Recombinant Antibody Production in 293T Cells

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Plasmids were expanded and transfected into 293T cells. Cells were plated at a density of 5.0 x 106 cells in 10 ml in a 10 cm dish. 10 ug of total DNA were transfected onto cells using 293 TransIT (Mirus Bio, Madison, US) following manufacturer’s protocols. Cells were washed 16 h post transfection and cultured with FreeStyle™ 293 Expression media (Gibco, Thermo Fisher Scientific). Cell culture supernatants containing protein were harvested at 7 days post transfection. Antibodies within the supernatants were bound to magnetic protein G beads (Merck, St. Louis, MO), washed three times with PBS, eluted from the beads, and neutralized using 1 M Tris, pH 8.0.
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4

Lentiviral Transduction and RNAi Knockdown

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HCT116 cells were transfected with jetPRIME (Polyplus Transfection), whereas HEK-293T cells were transfected with 293 TransIT (Mirus Bio LLC). Lentiviral expression and packaging plasmids were cotransfected into HEK-293T cells to produce lentiviral particles encoding HA-US2, HA-US11, Cre recombinase, GFP, hepatitis C virus core F130E, SPPwt myc KKEK, or SPP D/A myc KKEK. These particles were used to transduce cells, and when appropriate, stable clones were isolated after selection with puromycin (EMD Millipore). RNAi knockdown of SPP in HeLa cells was performed using an ON-TARGETplus siRNA pool against human HM13 (Thermo Fisher Scientific) transfected using Oligofectamine (Invitrogen).
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5

Overexpression and Detection of scAZ1-avitag

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HEK293T cells were plated at approximately 0.5×106 cells/well in a 6-well plate and cultured overnight at 37 °C under 5% CO2 before transfection. The cells were transfected with a plasmid encoding scAZ1-avitag using TransIT-293 (Mirus Bio) following the manufacturer’s procedure. The cells were further incubated at 37 °C for 48 h. The cells were washed with PBS and lysed with M-PER mammalian protein extraction reagent (Thermo Scientific) supplemented with Complete™ protease inhibitor cocktail (Roche) at 4 °C for 10 minutes. Immunoblotting was performed using an anti-AviTag antibody (GenScript mouse mAb, A01738).
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6

Overexpression and Detection of scAZ1-avitag

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HEK293T cells were plated at approximately 0.5×106 cells/well in a 6-well plate and cultured overnight at 37 °C under 5% CO2 before transfection. The cells were transfected with a plasmid encoding scAZ1-avitag using TransIT-293 (Mirus Bio) following the manufacturer’s procedure. The cells were further incubated at 37 °C for 48 h. The cells were washed with PBS and lysed with M-PER mammalian protein extraction reagent (Thermo Scientific) supplemented with Complete™ protease inhibitor cocktail (Roche) at 4 °C for 10 minutes. Immunoblotting was performed using an anti-AviTag antibody (GenScript mouse mAb, A01738).
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7

Transient AAV Vector Expression in 293T Cells

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Example 8

Each individual AAV expression vectors were transfected into 293T cells with TransIT-293 (Mirus, Inc.) to test their function before being packaged into AAV viral vectors. 293T cells were transfected with the same amount of plasmid and harvested at the same time points. SaCas9 protein expression was assessed by western blotting with primary antibody probing for the triple Flag tag at the C-terminus of saCas9, while loading control was demonstrated by αTubulin expression. Deletion events at IVS26 mutation could be determined by PCR amplification followed by Sanger sequencing or ddPCR. The results are shown in FIG. 15.

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8

Reverse Genetics for Influenza A Virus

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Reverse genetics was performed using pPol I plasmids containing cDNA sequences of the A/WSN/1933 (WSN; H1N1) viral genes between the human Pol I promoter and mouse Pol I terminator as described previously (47 (link)). Briefly, eight pPol I plasmids and pCAGGS protein-expression plasmids for PB2, PB1, PA, and NP were mixed with TransIT-293 (Mirus Bio, Madison, WI) and added to HEK293T cells. At 48 h posttransfection, the cells were treated with 1 μg/mL TPCK-trypsin (Worthington Biochemical, Lakewood, OH) for 30 min and centrifuged at 1,750 × g for 15 min at 4°C, and the supernatant was collected and stored at −80°C. PB2-FLAG virus was generated by replacing pPol I/PB2 wt with pPol I/PB2-FLAG plasmid. For subsequent viral amplification, MDCK cells were infected at an MOI of 10−5, followed by incubation for 2 days in BSA/MEM containing 1 μg/mL TPCK-trypsin.
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9

Production and Characterization of HIV Variants

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Recombinant R5 HIV (HIVAD8) and X4 HIV (HIVNL4.3) were prepared by transfecting plasmid encoding full-length of each HIV gene (pAD8 [45 (link)] and pNL4.3 [46 (link)]) into HEK293T cells, respectively. A pseudotyped HIV-1 virus (HIVLuc-VSVG) expressing the luciferase gene (HIVLuc) was created by transfection of pHIV-1NL4/3-Luciferase-ΔEnv and p-VSV-G into HEK293T cells in 10 cm dishes [42 (link), 47 (link), 48 (link)]. The HIV-1NL4/3-EnvAD.8-ΔVpr with Vpr-BLAM was produced by transfection of pHIV-1NL4/3-EnvAD.8-ΔVpr and pVpr-BLAM [49 (link), 50 (link)] (a kind gift from Dr. Warner Greene) into HEK293T cells. All plasmid DNA transfections were conducted using TransIT-293 (Mirus, Houston, TX, USA) and Opti-MEM I medium (Thermo Fisher Scientific) following a method previously reported [42 (link)]. Supernatants were then ultracentrifuged at 100,000 × g for 2 hours at 4°C onto a 20% sucrose in 10 mM HEPES-150 mM NaCl cushion [51 (link)]. Pelleted particles were resuspended in D10 medium and the concentration of HIV p24 was quantitated by using an HIV-1 p24 ELISA Kit (Perkin Elmer, Boston, MA, USA) [44 (link), 51 (link)]. Infection titer (50% tissue culture infectious dose, TCID50) of each virus was determined by an endpoint assay [52 (link)].
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10

Dual-Luciferase Assay for Transfection Efficiency

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Using TransIT-293 (Mirus, Madison, WI, USA) for HEK293 and Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) for miR-1266-expressing A549, cells were cotransfected with the internal pGL control vector, pRL-based reporter vectors, or miRNA expression vectors at a molar ratio of 1:5 or 1:5:25 (total 300 or 1550 ng vectors), respectively. After 48 h, relative luciferase activities were determined using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Relative activities were calculated by dividing the value of pRL by the value of pGL.
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