Sf9 cells (Life Technologies) were cultured in SF900 III SFM medium (Invitrogen) at 28 °C. For transfection, 106 cells were plated in 1.5 ml of TC100 medium per well of a six-well plate. After one hour at RT, the cells were transfected using 5 μl of Fugene HD (Roche) with 200 ng of flashBAC plasmid and 1 μg of pIG252-1. After overnight incubation, another 0.5 ml of SF900 III medium was added. Virus was harvested 7 days later and serially expanded in Sf9 cells cultured in suspension culture. Titres were determined using a FITC-labelled antibody directed against baculovirus envelope gp64 protein (eBiosciences) and recombinant protein production was initiated by infecting exponentially growing Sf9 cells with a multiplicity of infection of 20. Cells were harvested 48 h later and lysed in Tris-buffered saline containing Triton X-100 (150 mM NaCl, 20 mM Tris-HCl, pH 7.4, 0.2% Triton X-100). After sonication and clarification of the lysate by centrifugation (27,000g, 4 °C, 15 min), histidine-tagged ISPD was purified from cell lysates by metal affinity chromatography as described before64 (link), and eluted with a gradient of imidazole. Addition of 50 μM CTP was crucial for the stability and activity of the recombinant protein.
Sf 900 3 sfm medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Sf-900 III SFM medium is a serum-free, protein-free cell culture medium designed for the growth and maintenance of insect cells in suspension culture. The medium is optimized to support the growth of various insect cell lines without the need for additional supplementation.
Automatically generated - may contain errors
Lab products found in correlation
Sf9 insect cells, by Thermo Fisher Scientific (6 mentions)
Freestyle 293 f, by Thermo Fisher Scientific (5 mentions)
Freestyle 293 medium, by Thermo Fisher Scientific (5 mentions)
Sf9 cells, by Thermo Fisher Scientific (4 mentions)
Dmem basic, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Fetal calf serum (fcs), by Merck Group (4 mentions)
Ad293 cells, by Agilent Technologies (3 mentions)
Esf 921 medium, by Expression Systems (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Smm 293 ti, by Sino Biological (2 mentions)
Dmem high glucose medium, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by US Biological (2 mentions)
Anti flag beads, by Merck Group (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
Sybr green 1 master, by Roche (1 mentions)
Expifectamine transfection reagent, by Thermo Fisher Scientific (1 mentions)
34 protocols using sf 900 3 sfm medium
1
Expression and Purification of Recombinant ISPD
2
Isolation of Sf9-derived Extracellular Vesicles
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Sf9‐derived EVs were isolated using a previously reported method [22 (link)]. Briefly, 4.0 × 105 Sf9 cells·mL−1 were maintained in Sf‐900 III SFM medium (Invitrogen) overnight at 27 °C. The budded virus (BV) suspension was added at multiplicity of infection of 0.5 and incubated at 27 °C for 96 h. The culture medium was centrifuged at 500 g for 5 min and 2000 g for 10 min at 4 °C, followed by 0.22‐µm filtration. The supernatant was ultra‐centrifuged at 100 000 g for 70 min at 4 °C and the resultant pellet was resuspended in phosphate‐buffered saline. The suspension was ultra‐centrifuged at 40 000 g for 30 min at 4 °C in a stepwise sucrose density gradient [10%, 15%, 20%, 25%, and 30% sucrose (w/v) in phosphate‐buffered saline buffer]. The upper fraction (containing EVs) and lower fraction (containing BVs) were collected separately. The EV protein concentration was estimated using a Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA).
3
Purification of p300 Deacetylase and FLAG-tagged
4
RNA Extraction, RT-qPCR, and Protein Purification
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RNA was isolated from cells using RNeasy Mini Kit (QIAGEN). Reverse transcription was carried out using random primers and SuperScript II Reverse Transcriptase (Invitrogen) using standard protocol. qRT-PCR was performed using the LightCycler 480 (Roche Life Science) with the SYBR Green I Master (Roche) hot start reaction mix. Experiments were done in technical triplicates normalized to GAPDH and b Actin. Graphs show mean across R3 independent biological experiments ± SEM. See Table S3 for primers.
Insect Cell Culture and FLAG Purification SF9 cells were grown in Sf-900 III SFM medium (Invitrogen) with 1% PSG in suspension. For protein expression, cells were seeded at 2 million/ml, infected with baculovirus, and incubated for 72 hr. Cells were harvested by centrifugation at 4 C, 400 3 g for 3 min.
For FLAG purification, cells were lysed in lysis buffer (50 mM Tris-HCl [pH 7.5], 300 mM NaCl, 1 mM EDTA, 5 mM DTT, 1% Triton X-100, and Roche Complete Protease Inhibitor Cocktail tablet) and homogenized with a Dounce homogenizer (15 strokes). Lysate was cleared by centrifugation at 10,000 3 g, 4 C for 30 min, and incubated at 4 C for 1 hr with anti-FLAG beads (Sigma). Beads were washed three times each with lysis and wash buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 1 mM EDTA), and proteins were eluted twice at RT with 2 mg/ml 3xFLAG peptide in wash buffer rotating for 20 min.
Insect Cell Culture and FLAG Purification SF9 cells were grown in Sf-900 III SFM medium (Invitrogen) with 1% PSG in suspension. For protein expression, cells were seeded at 2 million/ml, infected with baculovirus, and incubated for 72 hr. Cells were harvested by centrifugation at 4 C, 400 3 g for 3 min.
For FLAG purification, cells were lysed in lysis buffer (50 mM Tris-HCl [pH 7.5], 300 mM NaCl, 1 mM EDTA, 5 mM DTT, 1% Triton X-100, and Roche Complete Protease Inhibitor Cocktail tablet) and homogenized with a Dounce homogenizer (15 strokes). Lysate was cleared by centrifugation at 10,000 3 g, 4 C for 30 min, and incubated at 4 C for 1 hr with anti-FLAG beads (Sigma). Beads were washed three times each with lysis and wash buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 1 mM EDTA), and proteins were eluted twice at RT with 2 mg/ml 3xFLAG peptide in wash buffer rotating for 20 min.
5
Spodoptera frugiperda Sf9 Cell Baculovirus Production
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Log-phase Spodoptera frugiperda Sf 9 (ATCC® CRL-1711) cells were seeded at 1 × 106 cells/well in Sf-900™ III SFM medium (Life Technologies, Scoresby, Victoria, Australia), in each well of a 6-well dish (Nunc, Life Technologies, Scoresby, Victoria, Australia), and allowed to adhere (1 h, 27°C). Bacmid DNA (1 μg) was added to prediluted ExpiFectamine™ Transfection Reagent (Life Technologies, Scoresby, Victoria, Australia) prepared according to manufacturer's instructions. Following incubation of the ExpiFectamine™/Bacmid DNA complex [room temperature (RT), 5 min], the DNA–lipid mixture was added dropwise onto the Sf9 cells and incubated for 72 h at 27°C. Once the cells appeared infected, the virus was harvested from the culture medium by centrifugation (500 g, 5 min). The cleared supernatant was transferred to a sterile tube and stored at 4°C, protected from light.
6
Culturing Human and Monkey Cell Lines
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HEK293T (human embryonic kidney) and Vero E6 (monkey kidney) cells were obtained from American Type Culture Collection. Huh-7 (human hepatoma) cells were kindly provided by Dr Charles M. Rice at Rockefeller University. These cell lines were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units ml−1 penicillin, and 100 μg ml−1 streptomycin (Life Technologies Inc.). Sf9 insect cells were purchased from Life Technologies Inc., and cultured in Sf-900 III SFM medium supplemented with 100 units ml−1 penicillin and 100 μg ml−1 streptomycin (Life Technologies Inc.)
7
Cultivation of Sf9 Insect Cells
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The S. frugiperda (Sf9) insect cell line (Thermo Fisher Scientific) was maintained in suspension cultures in a sterile Erlenmeyer flask and grown in Sf-900™ III SFM medium (Thermo Fisher Scientific) supplemented with 1% (v/v) antibiotic-antimycotic solution (Thermo Fisher Scientific) at 27 °C under continuous shaking at 100 rpm. Additionally, the suspension volume did not exceed 10% of the total volume of the Erlenmeyer flask.
8
Bacterial and Insect Cell Expression of Immune Proteins
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Human β2m and the ectodomains of mouse H2-Db and human HLA-A*02:01 were expressed as inclusion bodies in Escherichia coli BL21(DE3) as described before (Rodenko et al., 2006 (link); Thomas and Tampé, 2017a (link)). TAPBPR proteins were expressed in Spodoptera frugiperda (Sf21 or Sf9) insect cells according to standard protocols for the Bac-to-Bac system (Thermo Fisher Scientific, Waltham, MA). A high-titer recombinant baculovirus stock was used to infect the insect cells at a density of 1.5–2.0 × 106 cells/mL, which were cultivated in Sf-900 III SFM medium (Thermo Fisher Scientific) at 28°C. The cell culture medium containing secreted TAPBPR was harvested 72 hr after infection.
9
Recombinant Baculovirus Expression of OfCad in Sf9 Cells
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Spodoptera frugiperda Sf9 cell lines [36 (link)] were cultured in Sf-900™ III SFM medium (Thermo Fisher Scientific, Waltham, MA, USA) at 27 °C with a density of 5 × 106 cells/mL and subcultured every 3 d. Log phase cells were used for infection and were seeded at a density of 2 × 106 cells mL−1, and transfected with P3 recombinant baculovirus stocks. By following the operation manual of Bac-to-Bac baculovirus expression system (Thermo Fisher Scientific, Waltham, MA, USA), purified pFastBac™ HTA-OfCad plasmids were transformed into E. coli DH10Bac cells to generate recombinant bacmids. Purified recombinant bacmids were used to infect Sf9 by using the FuGENE HD transfection reagent (Promega, Madison, WI, USA). P3 viral stocks were stored at 4 °C for further use. Negative control used in the current study includes cells transfected with empty bacmid (EB) and the non-transfected cells (Sf9).
10
Cell Culture Protocols for Protein Expression
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FreeStyle 293 F (Thermo Fisher Scientific) suspension cells were cultured in 293 Expression Medium (Gibco) supplemented with 1% fetal bovine serum (FBS) at 37 °C, with 6% CO2 and 70% humidity. Sf9 insect cells (Thermo Fisher Scientific) were cultured in Sf-900 III SFM medium (Thermo Fisher Scientific) at 27 °C. HEK293T cells (ATCC) were cultured in DMEM basic (Thermo Fisher Scientific) supplemented with 10% FBS at 37 °C, with 6% CO2 and 70% humidity.
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