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Transit lt1

Manufactured by Geneflow
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TransIT-LT1 is a transfection reagent designed for efficient delivery of DNA, RNA, and other nucleic acids into a variety of cell types. It facilitates the transfer of genetic material into cells, enabling various research and experimental applications.

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Lab products found in correlation

Glutamax, by Thermo Fisher Scientific (4 mentions) Gibco tryple express, by Thermo Fisher Scientific (2 mentions) 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions) Rneasy micro kit, by Qiagen (1 mentions) Reverse transcription system, by Promega (1 mentions) Short tandem repeat profiling, by Eurofins (1 mentions) P35g 1.0 14 c, by MatTek (1 mentions) Viafect, by Promega (1 mentions)

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TransIT-LT1 transfection reagent (5 citations) TransIT-LT1 reagent (4 citations)

5 protocols using transit lt1

1

Lentiviral and Retroviral Production

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HEK293T cells were seeded at 2 × 106 cells per 10‐cm diameter dish and were cultured in DMEM, 10% FCS, 1% penicillin/streptomycin overnight. For lentiviral production, the cells were transfected with a mix containing an 8:1 ratio of packaging: envelope vectors, that is, 4.4 μg of pCMV delta R8.9, 0.6 μg of pCMV‐VSV‐G (McKay et al. 2011), and 5 μg of the appropriate lentiviral vector (empty or BMI‐1 insert) with 36 μL of TransIT‐LT1 (Geneflow). For retroviral production, the cells were transfected with 5 μg of pKAT (Addgene) and 5 μg of retroviral vector (empty or with hTERT insert) and 36 μL of TransIT‐LT1 (Geneflow). Cells were grown for 48–72 h and the supernatant containing packaged virus was harvested and ultracentrifuged (48,384 g for 3.5 h at 4°C) to produce a concentrated viral stock which was stored at ‐80°C.
Torr E., Heath M., Mee M., Shaw D., Sharp T.V, & Sayers I. (2016). Expression of polycomb protein BMI‐1 maintains the plasticity of basal bronchial epithelial cells. Physiological Reports, 4(16), e12847.
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2

Plasmids and RT-qPCR Analysis

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Plasmids containing PBF, PTTG, PBF M1 and PTTG BD- cDNA have been described elsewhere (22 (link), 24 (link)). Transfections were performed with TransIT-LT1 (Geneflow) using standard protocols. Total RNA was extracted using the RNeasy Micro Kit (Qiagen) prior to reverse transcription using the Reverse Transcription System (Promega). Expression of specific mRNAs was determined on a 7500 Real-time PCR system (Applied Biosystems) using primers listed in Supplementary Table S6.
Read M.L., Modasia B., Fletcher A., Thompson R.J., Brookes K., Rae P.C., Nieto H.R., Poole V.L., Roberts S., Campbell M.J., Boelaert K., Turnell A.S., Smith V.E., Mehanna H, & McCabe C.J. (2018). PTTG and PBF functionally interact with p53 and predict overall survival in head and neck cancer. Cancer research, 78(20), 5863-5876.
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3

Culturing and Transfecting HeLa and COS-7 Cells

Cited 1 time
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HeLa and COS-7 cells (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium/F-12 with GlutaMAX (ThermoFisher) supplemented with foetal bovine serum (FBS, 10%, Sigma). Cells were maintained at 37 °C in humidified air with 5% CO2 and passaged every 3–4 days using Gibco TrypLE Express (ThermoFisher). For imaging, cells were grown on 35-mm glass-bottomed dishes (#P35G-1.0-14-C, MatTek) coated with human fibronectin (10 μg.ml−1). Cells were transfected, according to the manufacturer’s instructions, using TransIT-LT1 (GeneFlow) (1 μg DNA per 2.5 μl reagent). Short tandem repeat profiling (Eurofins, Germany) was used to authenticate the identity of HeLa cells [7 (link)]. Screening confirmed that all cells were free of mycoplasma infection.
Prole D.L, & Taylor C.W. (2019). A genetically encoded toolkit of functionalized nanobodies against fluorescent proteins for visualizing and manipulating intracellular signalling. BMC Biology, 17, 41.
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4

Cell Culture and Transfection Protocol

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HEK293 and HeLa cells (both from ATCC) were cultured in Dulbecco’s modified Eagle’s medium/F-12 with GlutaMAX (ThermoFisher) supplemented with fetal bovine serum (FBS, 10%, Sigma). The cells were maintained at 37 °C in humidified air with 5% CO2, and passaged every 3–4 days using Gibco TrypLE Express (ThermoFisher). For imaging, cells were grown on 35-mm glass-bottomed dishes (#P35G-1.0-14-C, MatTek) coated with human fibronectin (10 µg ml−1). HeLa cells and EGFP-IP3R1 HeLa cells were transfected, according to the manufacturer’s instructions, using TransIT-LT1 (GeneFlow) and ViaFect (Promega) reagents, respectively (1 µg DNA per 2.5 µl reagent). Short tandem repeat profiling was used to authenticate HeLa cells (Eurofins, Germany) and HEK293 cells (Public Health England). Screening confirmed that all cells were free of mycoplasma infection.
Thillaiappan N.B., Chavda A.P., Tovey S.C., Prole D.L, & Taylor C.W. (2017). Ca2+ signals initiate at immobile IP3 receptors adjacent to ER-plasma membrane junctions. Nature Communications, 8, 1505.
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5

Generation and Validation of PBF-HA Mutant

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Plasmids containing PBF cDNA with a hemagglutinin (HA) tag have been described (37 (link)). The QuikChange Site-Directed Mutagenesis Kit (Stratagene) was used to generate Y174A PBF-HA. Plasmids expressing human Myc-CTTN (No. RC223259) and GFP-PBF (No. RG202109) were purchased from Origene (Rockwell). CTTN siRNAs (s4666 and s4667) and control siRNA (AM4635) were purchased from Invitrogen. Plasmid DNA and siRNA transfections were performed with TransIT-LT1 (Geneflow) and siPORT (Invitrogen) using standard protocols.
Watkins R.J., Imruetaicharoenchoke W., Read M.L., Sharma N., Poole V.L., Gentilin E., Bansal S., Bosseboeuf E., Fletcher R., Nieto H.R., Mallick U., Hackshaw A., Mehanna H., Boelaert K., Smith V.E, & McCabe C.J. (2016). Pro-invasive Effect of Proto-oncogene PBF Is Modulated by an Interaction with Cortactin. The Journal of Clinical Endocrinology and Metabolism, 101(12), 4551-4563.
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