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Bti tn 5b1 4

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The BTI-TN-5B1-4 is a laboratory equipment product from Thermo Fisher Scientific. It is designed to perform a specific core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation. Therefore, the description for this product is not available.

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12 protocols using bti tn 5b1 4

1

Baculovirus Production in Insect Cells

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Baculoviruses were produced in Sf21 insect cells (EMBL Protein Expression and Purification Core Facility, Heidelberg Germany) using cellfectin II reagent (Thermo Fisher Scientific). The second virus generation was amplified in 50 ml (1 × 106 cells/ml) culture. Afterwards, baculoviruses were harvested and diluted 1:100 in 100−400 ml (1−2 × 106 cells/ml) expression culture in Sf21 or High Five cell line (BTI-TN-5B1-4, cat no. B855-02, Invitrogen) using Sf-900 III medium (Thermo Fisher Scientific) supplemented with 100 units/ml penicillin/100 µg/ml streptomycin (Thermo Fisher Scientific). Expression was done shaking at 27 °C for 60 h. Cells were harvested via centrifugation (800×g for 5 min), flash frozen in liquid N2 and stored at −80 °C until further usage.
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2

Insect Cell Line Cultivation

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Spodoptera frugiperda and Trichoplusia ni insect cell lines, Sf-9 and BTI-TN-5B1-4 (also called High-five), respectively, (Invitrogen, Carlsbad, USA) were used. Sf-9 cells were maintained in TNM-FH medium (Sigma Aldrich, St. Louis, USA) containing 10 % fetal bovine serum (FBS, Invitrogen), 0.1 % Pluronic F-68 solution (Sigma Aldrich), and 10 µg/mL gentamycin sulfate (Sigma Aldrich) (complete TMN-FH medium). High-five cells were cultured in Express-five serum-free medium (SFM) (Invitrogen) supplemented with 16 mM of l-glutamine (Invitrogen), and 10 µg/mL gentamycin (complete Express-five SFM medium). Cell cultures were carried out at 27 °C either as a monolayer or in suspension in shaker flasks, according to the manufacturer’s instructions.
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3

Generation of AgMNPV-I-PpoI Baculovirus

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AgMNPV-2D isolate (wild type) [10 (link)] was used as the parental virus for the generation of AgMNPV-I-PpoI. UFLAg-286 (kindly supplied by Dr. Bergmann Ribeiro; Universidade de Brasília) and High-FiveTM (BTI-TN-5B1-4, InvitrogenTM, Carlsbad, CA, USA) cells were grown at 28 °C in TC-100 or Grace’s (InvitrogenTM) media containing 10% fetal bovine serum (Internegocios S.A., Mercedes, Argentina). A. gemmatalis were reared at IMYZA, (INTA, Castelar, Argentina). Larvae were maintained in the laboratory on an artificial diet [11 ] under controlled temperature (26 ± 1 °C), photoperiod (14L/10D) and 80% relative humidity.
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4

High Five Cell Culture for Baculovirus Expression

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A suspension adapted High Five cell line (BTI-Tn-5B1-4, Invitrogen) was prepared in a 60 ml culture in Express Five Serum Free Medium supplemented with 20 mMl-glutamine (Thermo Fisher Life Technologies). The culture was incubated at 28°C and 87 rev min−1 for ∼24 h. When a critical cell density (>2 × 106 cells ml−1) had been reached, the culture was successively passaged into 100 ml, 600 ml, 1.8 l and 3.6 l Express Five Serum Free Medium. The 3.6 l culture (∼1–2 × 106 cells ml−1) was split into 6 × 600 ml cultures and infected with 750 µl of baculovirus P2 stock. The cultures were incubated at 28°C and 87 rev min−1 until YFP fluorescence was observed in 95% of the cells (∼2–3 days). The supernatant was harvested by centrifugation at 400g for 15 min at 4°C, followed by further clearing of debris by centrifugation at 4000g for 60 min at 4°C. DTT and PMSF were added to achieve final concentrations of 1 and 0.1 mM, respectively.
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5

Recombinant Protein Expression in Insect Cells

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Sf9 cells (Gibco™ Sf-900™ III SFM, Gibco™ 12659017, Cat. No. 15236025, Carlsbad, CA, USA) and high five cells (BTI-TN-5B1-4, Invitrogen™ B85502, Cat. No. 10747474, Carlsbad, CA, USA) were purchased by Xi’an Lihe Biotechnology Co., LTD (Xi’an, China) from Thermo Fisher Scientific (Waltham, MA, USA). Sf9 cells were used to isolate and propagate recombinant baculoviral stocks, and high five cells were used to express the recombinant protein. All cells were cultured in IB905 insect serum-free medium (world-medium, Suzhou, China).
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6

Cell Culture Conditions for ED, 293T, Sf9, and High Five Cells

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ED cells were grown in Iscove’s modified Dulbecco’s medium (IMDM; Pan, Biotech, Aidenbach, Germany) containing 20% fetal bovine serum (FBS; Biochom GmBH, Berlin, Germany), 1 mM sodium pyruvate (Pan Biotech, Aidenbach, Germany), 1% nonessential amino acids (NEAA; Biochom GmBH, Berlin, Germany), and P-S solution (100 U/ml penicillin: Panreac, AppliChem GmBH, Darmstadt, Germany); 100 μg/ml streptomycin: Alfa Aesar, Thermo Fisher Scientific, Kandel, Germany (P-S) at 37°C under a 5% CO2 atmosphere.
Human embryonic kidney (293 T, ATCC CRL-11268) cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM; Biochom GmBH, Berlin, Germany), supplemented with 10% FBS and P-S. Sf9 cells (IPLB-Sf21-AE, Invitrogen, Germany) were propagated in serum free Sf-900 III medium (Gibco, Thermo Fisher Scientific, New York, USA) and High Five™ cells (BTI-TN-5B1-4, Invitrogen, Germany) in serum free Express Five medium (Gibco, Thermo Fisher Scientific, New York, USA) at 27°C on orbital shaker.
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7

Sf9 and Hi5 Cell Culture

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Sf9 cells [17] and T. ni High Five TM (Hi5; BTI-Tn-5B1-4; Invitrogen, Waltham, MA, USA) [18] were maintained in suspension cultures in ESF921 media (Expression Systems, Davis, CA, USA) using standard methods [19] . 1.25 × 10 5 Sf9 cells/well and 6.25 × 10 4 Hi5 cells/well were seeded in 96-well plates for the detection of eGFP reporter gene expression, while 10 6 Sf9 cells/well and 0.5 × 10 6 Hi5 cells/well were seeded in 12-well plates for the β-galactosidase enzyme assay and protein gel analysis.
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8

Propagation and DNA Extraction of AnpeNPV

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AnpeNPV L2 (GenBank accession number: EF207986) was propagated in A. pernyi pupae and viral DNA was extracted as previously described (Fan et al. 2007 (link)). Tn-Hi5 cells (BTI-TN-5B1-4) purchased from Thermo Fisher Scientific (Waltham, MA) were cultured in TNM-FH medium (GE Healthcare Life Sciences, Chicago, IL) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, Billings, MT) containing 0.5% penicillin–streptomycin solution (Gibco) at 27°C. Restriction enzymes were purchased from TaKaRa (Dalian, China).
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9

Baculovirus-Mediated Recombinant Protein Production

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Baculovirus generation and amplification were performed in Sf9 cells (CRL-1771, American Type Culture Collection), a clonal isolate of Spodoptera frugiperda Sf21 cells, and grown in Trichoplusia ni medium-formulation Hink insect cell medium (Gemini Bioproducts) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco), penicillin (100 U/ml)–streptomycin (100 μg/ml) solution (Gibco), and 0.1% (v/v) Pluronic F-68 (Gibco). High Five cells (BTI-TN-5B1-4, B85502, Thermo Fisher Scientific) were grown in Express Five SFM (Gibco) supplemented with 16 mM l-glutamine (Gibco) and used for recombinant HA and NA production (58 (link)). Both adherent cell lines were grown at 27°C.
Adherent Madin-Darby canine kidney (MDCK) and human embryonic kidney 293T cells were grown in modified Eagle’s medium (MEM) containing 10% (v/v) FBS and penicillin (100 U/ml)–streptomycin (100 μg/ml) solution in a humidified incubator at 37°C and 5% CO2.
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10

Alphanodavirus Infection Dynamics in Tn-Hi5 Cells

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Alphanodavirus-free Tn-Hi5 cell line (BTI-TN-5B1-4) was purchased from Thermo Fisher Scientific (United States). Cells were cultured in TNM-FH medium (GE Healthcare Life Sciences) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco) containing 0.5% penicillin-streptomycin solution (Gibco) at 27°C. ApNPV-Δph/egfp+ (Wang et al. 2010 (link)) was propagated in A. pernyi pupae and used to infect the cell line. Viral titer was determined by an end-point dilution assay with Tn-Hi5 cells according to a published method (Cha et al. 1997 (link)). For the infection, 1 × 106 Tn-Hi5 cells were seeded into wells of a 6-well tissue culture plate (Falcon) and infected with ApNPV-Δph/egfp+ (multiplicity of infection: 10). The ApNPV-Δph/egfp+ inoculum was removed after 1 h. The cells were rinsed with SF-900TM II medium (Gibco) and cultured in TNM-FH medium supplemented with 10% FBS at 27°C. The time at which the inoculum was removed was considered as 0 hpi. Cells were collected at 6, 12, 18, 24, 36, and 48 hpi. Uninfected cells (mock infection) were collected at 48 h as the control. Three independent biological replicates were prepared at each time point.
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