High five

Manufactured by Thermo Fisher Scientific

Sourced in United States

The High Five is a laboratory centrifuge designed for high-speed separation of samples. It features adjustable speed and time settings to accommodate a variety of sample types and volumes. The High Five is a versatile and reliable tool for research and diagnostic laboratories.

Automatically generated - may contain errors

Lab products found in correlation

Spelling variants of this product

High Five cells (74 citations) High Five insect cells (62 citations) Trichoplusia ni High Five cells (7 citations)

26 protocols using high five

Optimized Insect and Mammalian Cell Culture

Check if the same lab product or an alternative is used in the 5 most similar protocols

Insect Sf-9 (Invitrogen) and High Five (Invitrogen) cells were routinely subcultured at 0.4–1 × 10⁶ cell/mL every 3–4 days when cell density reached 2–3x10⁶cell/mL, as described elsewhere (Fernandes et al., 2020 (link)). Insect-XPRESS^TM (Sartorius) and Sf-900^TM II SFM (Thermo Fisher Scientific) media were used to culture Sf-9 and High Five cells, respectively. Human HEK293-E6 cells (NRC) (Durocher, 2002 (link)) were routinely subcultured to 0.5–0.6 × 10⁶ cells/mL every 3–4 days when cell density reached 2–3 × 10⁶ cells/mL in 125- or 500-mL shake flasks (20% working volume, w/v) in an Innova 44R incubator (orbital motion diameter of 2.54 cm Eppendorf) at 37°C with 5% CO₂ and stirring rates of 75 or 90 rpm. FreeStyle F17 (Thermo Fisher Scientific) media, supplemented with 4 mM GlutaMAX™ (Thermo Fisher Scientific) and 0.1% of Pluronic™ F-68 (Life Technologies), and 25 µg/mL of Geneticin were used to culture the HEK293 cells.

Fernandes B., Sousa M., Castro R., Schäfer A., Hauser J., Schulze K., Amacker M., Tamborrini M., Pluschke G., Alves P.M., Fleury S, & Roldão A. (2022). Scalable Process for High-Yield Production of PfCyRPA Using Insect Cells for Inclusion in a Malaria Virosome-Based Vaccine Candidate. Frontiers in Bioengineering and Biotechnology, 10, 879078.

+ Open protocol

+ Expand

Production and Purification of QUAD 3.0 Protein

Check if the same lab product or an alternative is used in the 5 most similar protocols

QUAD 3.0 protein consists of IL-13.E13K in the N-terminal end, CH₂-CH₃ regions of the human IgG1, and eA5 followed by a cysteine at the C-terminal end of the protein. QUAD 3.0 was produced in High Five^TM (Thermo Fischer, Waltham, MA) cells as described previously [46 (link),85 (link)]. Briefly, the gene for QUAD 3.0 was cloned into pMIB V5 His A vector (Thermo Fischer, Waltham, MA, USA). The modified plasmid was introduced in High Five^TM cells using Cellfectin II reagent (Thermo Fischer, Waltham, MA, USA) as described by the manufacturer. Blasticidin was used to select the transfected insect cells. The protein was secreted and the QUAD 3.0-containing media, which was collected. QUAD 3.0 was purified on a HiTrap^® Protein G HP (GE, Boston, MA, USA) using fast protein liquid chromatography (FPLC) (GE, Boston, MA, USA) system.

Sharma P., Roberts C., Herpai D., Fokt I.D., Priebe W, & Debinski W. (2020). Drug Conjugates for Targeting Eph Receptors in Glioblastoma. Pharmaceuticals, 13(4), 77.

+ Open protocol

+ Expand

GTPγS Binding Assay for β2AR

Check if the same lab product or an alternative is used in the 5 most similar protocols

Membranes were prepared from High Five (Invitrogen) or Sf9 cells expressing β₂AR or mutants and Gsαβγ. Typically, membranes (2–5 μg) were pretreated with guanosine diphosphate (final assay concentration of 10 μM) in assay buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM MgCl₂ and 1 mM ascorbic acid) and different concentrations of AS408 for 20 min at room temperature before adding [³⁵S]GTPγS (for a final concentration of 0.1 nM) with a range of concentrations of agonist (epinephrine or norepinephrine). For most cases the assays were incubated at room temperature for a period of 1 h before stopping by rapid filtration through GF/B membranes and washing with ice-cold assay buffer. To determine the K_B for AS408 on β₂AR and mutants, assay times were reduced to 10 min at 30 °C, to avoid saturating [³⁵S]GTPγS binding to Gs. Assays were performed in a 96-well microplate format and radioactivity was measured using a TopCount (Packard). All data were analyzed using GraphPad Prism 6.0 (GraphPad).

Liu X., Kaindl J., Korczynska M., Stößel A., Dengler D., Stanek M., Hübner H., Clark M.J., Mahoney J., Matt R.A., Xu X., Hirata K., Shoichet B.K., Sunahara R.K., Kobilka B.K, & Gmeiner P. (2020). An allosteric modulator binds to a conformational hub in the β2 adrenergic receptor. Nature chemical biology, 16(7), 749-755.

+ Open protocol

+ Expand

Heterotrimeric G-protein and Nanobody Purification

Cited 3 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

Gs and Go heterotrimer were expressed in HighFive™ (Invitrogen) insect cells using recombinant baculovirus and purified by chromatography on Ni-NTA, MonoQ, and Superdex 200 resin, as previously described^{31 (link)}. Nanobodies were expressed in Escherichia coli and purified as previously described^{11 (link),14 (link),27 (link)}.

DeVree B.T., Mahoney J.P., Vélez-Ruiz G.A., Rasmussen S.G., Kuszak A.J., Edwald E., Fung J.J., Manglik A., Masureel M., Du Y., Matt R.A., Pardon E., Steyaert J., Kobilka B.K, & Sunahara R.K. (2016). Allosteric coupling from G protein to the agonist binding pocket in GPCRs. Nature, 535(7610), 182-186.

+ Open protocol

+ Expand

Purification of GGTase3 and FBXL2-SKP1 Complexes

Check if the same lab product or an alternative is used in the 5 most similar protocols

The DNAs encoding human GGTase3 heterodimer (PTAR1-RabGGTB) and FBXL2-SKP1 were subcloned into pFastBac vectors for protein co-expression in High Five (Invitrogen) monolayer insect cells. Among all four recombinant proteins, PTAR1 was expressed as a glutathione S-transferase (GST) N-terminal fusion protein and the tetrameric complex of GGTase3-FBXL2-SKP1 was isolated by glutathione affinity chromatography using buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl and 1.0 mM DTT (dithiothreitol). The eluted tetrameric complex was further purified by anion exchange and gel filtration chromatography, after overnight TEV (tobacco etch virus) protease cleavage, in a final buffer containing 20 mM HEPES pH7.5, 150 mM NaCl and 2.0 mM DTT. The protein sample was concentrated and flash frozen for crystallization. For testing the binding activity of FBXL2-SKP1 with GGTase3 and its geranylgeranylation by GGTase3, the two individual protein complexes of FBXL2-SKP1 and GGTase3 were expressed in E. coli with the affinity tags fused on PTAR1 and FBXL2, respectively. The proteins were isolated and purified similarly as the tetrameric complex by three steps of affinity, anion exchange, and gel filtration chromatography. The affinity tag may be left on the proteins for the purposes of assays.

Kuchay S., Wang H., Marzio A., Jain K., Homer H., Fehrenbacher N., Philips M.R., Zheng N, & Pagano M. (2019). GGTase3 is a Newly Identified Geranylgeranyltransferase Targeting a Ubiquitin Ligase. Nature structural & molecular biology, 26(7), 628-636.

+ Open protocol

+ Expand

MiRNA Regulation of Baculovirus Genes

Check if the same lab product or an alternative is used in the 5 most similar protocols

At temperature of 27°C, Trichoplusia ni cell line High Five (Invitrogen, USA) was cultured in Express Five medium (Invitrogen, USA) with 1.8 mM/mL l-glutamine (Invitrogen, USA) were cultured. The cells were co-transfected with a synthesized miRNA mimic (300 nM) and pIZ/EGFP consisting of 3′UTR of wsv459 or wsv322 (6 µg/mL) at about 70% confluence. The miRNA mimic was synthesized by T7 Kit siRNA Synthesis (TaKaRa, Japan). All experiments of transfection were accomplished with Cellfectin (Invitrogen) according to Cellfectin’s manual in triplicate. 12 h for after culture, cells were planked with cell density of 2.0 × 10⁴ cells/well to 96-well plates. The fluorescence density of High Five was detected using a Flex Station II microplate reader (Molecular Devices, USA) with condition of Ex (490 nm)/Em (510 nm) at 48 h post-transfection. The fluorescence value was revised by subtracting the spontaneous fluorescence of non-EGFP cells.

He Y., Ma T, & Zhang X. (2017). The Mechanism of Synchronous Precise Regulation of Two Shrimp White Spot Syndrome Virus Targets by a Viral MicroRNA. Frontiers in Immunology, 8, 1546.

+ Open protocol

+ Expand

Heterotrimeric G-protein and Nanobody Purification

Cited 3 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

+ Open protocol

+ Expand

Insect Cell Culture for Baculovirus

Check if the same lab product or an alternative is used in the 5 most similar protocols

Sf9 (Invitrogen) and High Five (Invitrogen) insect cells were employed for baculovirus generation and protein expression, respectively. Both cell lines were cultured in suspension cultures in serum-free HyQ media (Hyclone) at 25°C under shaking conditions.

Jiménez-Dalmaroni M.J., Radcliffe C.M., Harvey D.J., Wormald M.R., Verdino P., Ainge G.D., Larsen D.S., Painter G.F., Ulevitch R., Beutler B., Rudd P.M., Dwek R.A, & Wilson I.A. (2014). Soluble human TLR2 ectodomain binds diacylglycerol from microbial lipopeptides and glycolipids. Innate immunity, 21(2), 175-193.

+ Open protocol

+ Expand

Purification of Murine CRY2 and PER2 Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

The mouse CRY2 (amino acids 1–512) was expressed as a glutathione S-transferase (GST) fusion protein in High Five (Invitrogen, Carlsbad, CA) suspension insect cells and isolated by glutathione affinity chromatography using buffer containing 20 mM Tris–HCl pH 8, 200 mM NaCl, 10% glycerol, 5 mM DTT (dithiothreitol). The protein was cleaved on-column by tobacco etch virus (TEV) protease then purified further by cation-exchange chromatography. Proteolytically stable murine PER2 (amino acids 1095–1215) was expressed as a GST-fusion protein in Escherichia coli expression system and isolated through glutathione affinity chromatography using buffer containing 20 mM Tris–HCl pH 8, 300 mM NaCl, 5 mM DTT. The protein was cleaved on-column by TEV protease then purified further by anion-exchange and size-exclusion chromatography. Both proteins were combined, concentrated, and further purified by size-exclusion chromatography using buffer containing 20 mM Tris–HCl pH 8, 300 mM NaCl, 5 mM DTT, 10% glycerol to establish stoichiometric binding.

Nangle S.N., Rosensweig C., Koike N., Tei H., Takahashi J.S., Green C.B, & Zheng N. (2014). Molecular assembly of the period-cryptochrome circadian transcriptional repressor complex. eLife, 3, e03674.

+ Open protocol

+ Expand

Cell Culture Protocols for Insect and Mammalian Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Insect Sf9 (Invitrogen) and High Five (Invitrogen) cells were routinely sub-cultured at 0.4–1 × 10⁶ cell/mL every 3–4 days when cell density reached 2–3 × 10⁶ cell/mL in the Insect-XPRESS^TM (Sartorius) and Sf-900^TM II SFM (Thermo Fisher Scientific) medium, respectively. HEK293-E6 cells (NRC) (Durocher et al., 2002 (link)) were routinely sub-cultured at 0.5–0.6 × 10⁶ cell/mL every 3–4 days, when cell density reached 2–3 × 10⁶ cell/mL in the Free Style F17 (Thermo Fisher Scientific) medium supplemented with 4 mM GlutaMAX™ (Thermo Fisher Scientific), 0.1% Pluronic™ F-68 (Life Technologies) and 25 μg/ml of Geneticin (Thermo Fisher Scientific). Cells were cultured using 125–500 ml shake-flasks (Corning) with 10%–20% working volume. Insect Sf9 and High Five cells were maintained at 27°C in a shaking incubator (Inova 44R—Eppendorf) set to 100 rpm and with 2.54 cm shaking diameter. HEK 293 cells were maintained in a similar incubator at 37°C with 5% CO₂ and the stirring rate set to 75 rpm.

Correia R., Fernandes B., Castro R., Nagaoka H., Takashima E., Tsuboi T., Fukushima A., Viebig N.K., Depraetere H., Alves P.M, & Roldão A. (2022). Asexual Blood-Stage Malaria Vaccine Candidate PfRipr5: Enhanced Production in Insect Cells. Frontiers in Bioengineering and Biotechnology, 10, 908509.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!