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Translt lt1 transfection reagent

Manufactured by Mirus Bio
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TransIT-LT1 Transfection Reagent is a lipid-based transfection agent for efficient delivery of DNA, RNA, and proteins into a variety of cell lines. It is designed to facilitate the transfer of genetic material into cells for various applications, such as gene expression analysis and protein production.

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Lab products found in correlation

Tristar lb 941 luminometer, by Berthold Technologies (1 mentions) Luciferase assay system, by Promega (1 mentions) Phusion polymerase master mix, by New England Biolabs (1 mentions) Superscript 4 reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Direct zol rna purification kit, by Zymo Research (1 mentions) Geltrex, by Thermo Fisher Scientific (1 mentions) Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

LT1 transfection reagent (38 citations) LT1 reagent (11 citations) Transfection reagent (10 citations) TranslT-LT1 (5 citations)

6 protocols using translt lt1 transfection reagent

1

Luciferase Reporter Assay in BPH-1 Cells

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For the luciferase reporter assay, BPH-1 cells were plated in 48-well plates 24 h before transfection; then, the cells were transfected with the luciferase reporter using the TranslT-LT1 transfection reagent (Mirus Bio, Madison, WI, USA) according to the manufacturer’s instructions. For reporter quantitation, cell lysates were assayed using the Luciferase Assay System (Promega, Madison, WI, USA) and a TriStar LB941 Luminometer (Berthold technologies, Bad Wildbad, Germany) according to the manufacturer’s instructions.
Song K.H., Seo C.S., Yang W.K., Gu H.O., Kim K.J, & Kim S.H. (2021). Extracts of Phyllostachys pubescens Leaves Represses Human Steroid 5-Alpha Reductase Type 2 Promoter Activity in BHP-1 Cells and Ameliorates Testosterone-Induced Benign Prostatic Hyperplasia in Rat Model. Nutrients, 13(3), 884.
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2

Reverse Genetics-Derived LAIV Vaccine and Challenge Viruses

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The LAIV vaccine was initially generated by reverse genetics from H3N2 A/Swine/Texas/4199-2/1998 (TX98) influenza virus as described previously (Solórzano et al. 2005 (link)). Briefly, the LAIV encodes a truncated NS1 protein with four stop codons introduced after 126 reading codons, resulting in a 3’ truncation of the wild-type NS1 protein from 219 to 126 amino acids. The remaining genetic material from wild-type TX98 was used to encode PB2, PB1, PA, HA, NP, NA, M1, M2 and NS2. Plasmids encoding each gene segment were used to transfect HEK 293T human embryonic kidney cells expressing a temperature-sensitive mutant of SV40 large T antigen using the TranslT®-LT1 transfection reagent (Mirus Bio LLC, Madison, WI). The HEK 293T cells were subsequently co-cultured with Madin-Darby Canine Kidney (MDCK) cells, after which virus particles recovered from the culture supernatant were further propagated through MDCK cells. For the current studies, the LAIV vaccine and challenge viruses were propagated through MDCK cells from in-house stocks. The challenge viruses included the wild-type TX98 containing an intact NS1 gene, the H3N2 A/Swine/Colorado/23619/1999 (CO99), and the H1N1 pandemic A/California/04/2009 (CA04) viruses. The identity of the virus subtypes was confirmed by Sanger sequencing.
Artiaga B.L., Morozov I., Ransburgh R., Kwon T., Balaraman V., Indran S.V., De Carvalho Madrid D.M., Gu W., Henningson J., Ma W., Richt J.A, & Driver J.P. (2022). Evaluating α-galactosylceramide as an adjuvant for live attenuated influenza vaccines in pigs. Animal Diseases, 2(1), 19.
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3

Optimized Retroviral and Lentiviral Transduction

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3 × 106 Plat-E cells were seeded in 10-cm dishes in media (DMEM with 10% FBS, 1% L-glutamine, 1% penicillin/streptomycin) the day before transfection, and the medium was changed just before transfection. For retroviral transduction, we used a mixture of 10 μg retroviral plasmid + 3.4 μg pCL-Eco packaging vectors or PCL10A1; for lentiviral transduction, the mixture contained 10 μg Lentiviral plasmid +7.5 μg Gag pol + 5 μg Rev + 2.5 μg VSV-G packaging vectors. The plasmid mixtures were incubated with 40 μl TranslT-LT1 Transfection Reagent (Mirus Bio LLC) at ~22 °C for 20 min in 1.5 ml Opti-MEM media and then added to the PlatE cells, after which the cells were incubated at 37 °C in a 10% CO2 incubator for 30–40 h. The supernatant was filtered through a 40 μm filter before being used for transduction of CD8+ T cells.
Seo H., González-Avalos E., Zhang W., Ramchandani P., Yang C., Lio C.W., Rao A, & Hogan P.G. (2021). BATF and IRF4 cooperate to counter exhaustion in tumor-infiltrating CAR T cells. Nature immunology, 22(8), 983-995.
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4

Optimized Retroviral and Lentiviral Transduction

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3 × 106 Plat-E cells were seeded in 10-cm dishes in media (DMEM with 10% FBS, 1% L-glutamine, 1% penicillin/streptomycin) the day before transfection, and the medium was changed just before transfection. For retroviral transduction, we used a mixture of 10 μg retroviral plasmid + 3.4 μg pCL-Eco packaging vectors or PCL10A1; for lentiviral transduction, the mixture contained 10 μg Lentiviral plasmid +7.5 μg Gag pol + 5 μg Rev + 2.5 μg VSV-G packaging vectors. The plasmid mixtures were incubated with 40 μl TranslT-LT1 Transfection Reagent (Mirus Bio LLC) at ~22 °C for 20 min in 1.5 ml Opti-MEM media and then added to the PlatE cells, after which the cells were incubated at 37 °C in a 10% CO2 incubator for 30–40 h. The supernatant was filtered through a 40 μm filter before being used for transduction of CD8+ T cells.
Seo H., González-Avalos E., Zhang W., Ramchandani P., Yang C., Lio C.W., Rao A, & Hogan P.G. (2021). BATF and IRF4 cooperate to counter exhaustion in tumor-infiltrating CAR T cells. Nature immunology, 22(8), 983-995.
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5

Investigating RAD51D Isoforms in U2OS Cells

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Human U20S and U20S SCR (Sister chromatid recombination) #18 Wild-Type (WT) [26 (link)] and RAD51D CRISPR Knock-out (Clone D4) cell lines (both gifted from Mauro Modesti; Garcin et al., in preparation) were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin and streptomycin. Cells were transfected using TranslT®-LT1 transfection reagent (Mirus Bio) diluted in OptiMEM® serum free media and following the manufacturer’s instructions. RAD51D cDNAs were expressed from mammalian expression plasmids pCDNA3 FLAG-RAD51D isoform 1 (NM_002878.3) (gifted from Paul Russell [27 (link)]), pCDNA3.1 3xFLAG-RAD51 D isoform 4 (NM_133629.2) and isoform 6 (NM_001142571.1) (supplied by Novoprolabs). pCBAScel was a gift from Maria Jasin (Addgene plasmid # 26477) [28 (link)], Mutations were introduced into the cDNA of RAD51D by site-directed mutagenesis using Phusion polymerase master mix (M0531S, NEB) and Dpnl (R0176S, NEB). All mutations were verified by DNA sequencing (Genewiz).
Baldock R.A., Pressimone C.A., Baird J.M., Khodakov A., Luong T.T., Grundy M.K., Smith C.M., Karpenshif Y., Bratton-Palmer D.S., Garcin E.B., Gon S., Modesti M, & Bernstein K.A. (2019). RAD51D splice variants and cancer-associated mutations reveal XRCC2 interaction to be critical for homologous recombination. DNA repair, 76, 99-107.
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6

Optimized SSO/ASO Transfection in Brain Organoids

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SSOs were synthesized by Integrated DNA Technologies (IDT). For SSO transfection, 4×105 cells were seeded in 12-well plates coated with GelTrex (Thermo Fisher, Gibco, A1413301). 200nM SSOs were transfected into cells using TranslT-LT1 Transfection Reagent (Mirus, MIR2300) according to the manufacturer’s instructions. Brain organoids derived from iPSC 28126 were treated by three doses of SSOs (300nM) from day 133 to 137. On day 139, RNA and protein were extracted from brain organoids for PCR and western blot analyses. For brain organoids derived from iPSC 21792, they were treated by five doses of ASOs (200nM) from day 169 to 173. RNA and protein were extracted on day 174. Total RNA was extracted using TRIzol reagent (Thermo Fisher, 15596018) and Direct-zol RNA Purification Kit (Zymo Research, R2060) 24 hours after transfection. cDNA was synthesized by Superscript IV Reverse Transcriptase kit (Thermo Fisher, 18090050). Quantitative PCR (Q-PCR) was performed using SYBR Green PCR Master Mix (Thermo Fisher, 4344463) in QuanStudio Real-Time PCR Systems (Thermo Fisher, ZG11CQS3STD) according to manufacturers’ instructions.
Marra A, & Brigham D. (2001). Streptococcus pneumoniae Causes Experimental Meningitis following Intranasal and Otitis Media Infections via a Nonhematogenous Route. Infection and Immunity, 69(12), 7318-7325.
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