S2 cells (2 × 106) were soaked with 10 μg dsRNA for 1 hr in ExpressFive SFM medium (Invitrogen) supplemented with 200 mM glutamine (Invitrogen) in the absence of FBS, followed by the addition of medium containing 10% FBS. Soaking was repeated after 4 days, and cells were harvested and processed at day 7.
Express five sfm medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Express Five SFM Medium is a serum-free, animal-component-free medium designed for the expansion of mammalian cells, including stem cells, in suspension culture.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (6 mentions)
Fugene hd transfection reagent, by Promega (5 mentions)
Effectene reagent, by Qiagen (3 mentions)
Grace s insect medium, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (2 mentions)
Neon transfection system, by Thermo Fisher Scientific (2 mentions)
Glutamine, by Thermo Fisher Scientific (2 mentions)
Ham s f12k medium, by American Type Culture Collection (2 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (2 mentions)
Complete schneider s drosophila medium, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Corning (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
25 cm2 cell culture flask, by Greiner (1 mentions)
Amicon ultra 4 centrifugal filter device, by Merck Group (1 mentions)
Cellfectin reagent, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
21 protocols using express five sfm medium
1
Efficient RNA Interference in S2 Cells
2
Recombinant FREP1 Protein Expression
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To generate recombinant FREP1 similar to the endogenous FREP1, the complete coding sequence of FREP1 was PCR-cloned into pIB/V5-His plasmid (Life Tech, Grand Island, NY) with the primer pair 5′-TCAAAGCTTCACCATGGTGAATTCATTCGTGTCG-3′ and 5′-ACTCTAGATTACGCGAACGTCGGCACAGTCGTG-3′, to generate the plasmid pIB-FREP1. After being amplified in E. coli DH5α, the plasmid was purified with an endotoxin-free plasmid preparation kit (Sigma-Aldrich). The cabbage looper ovarian cell-derived High Five cell line47 (link) was used to express the recombinant FREP1 protein according to the user manual48 . In brief, endotoxin-free recombinant pIB-FREP1 plasmid was mixed with Cellfectin® Reagent (1 μL Cellfectin/μg plasmid, Invitrogen, Grand Island, NY) in 5–6 mL Express Five® SFM medium (Invitrogen). The cells were cultured in 25 cm2 cell culture flasks (Greiner Bio-One, Monroe, NC) for 48 hrs at 27 °C. Medium and cells were separated by centrifugation at 300 × g for 5 min. The proteins in the medium were concentrated using Amicon® ULTRA-4 Centrifugal Filter Devices (Milipore, Billerica, MA) by centrifugation at 5,000 × g for 10 min and the protease inhibitors (Mini Tablets, EDTA-free, Thermo Scientific, Waltham, MA) were added to protect FREP1 from being degraded. Expression of FREP1 protein was determined by western blotting assays.
3
Cell Culture and Transfection Protocols
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Cell culture. HDF cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) and supplemented with 10% fetal bovine serum, 5% minimum essential medium nonessential amino acids (100×, Gibco), 5% penicillin and streptomycin (Gibco), and l -glutamine (Gibco). T-Rex-CHO cells were grown in Ham’s F12K medium (American Type Culture Collection) with the same supplements. Drosophila S2 cells were cultured in Express Five SFM Medium (Invitrogen) supplemented with penicillin (100 U/ml), streptomycin (100 U/ml) (Gibco), and 45 ml of 200 mM l -glutamine (Gibco) per 500 ml of medium.
Plasmids and mRNA were introduced to the cells by the Neon Transfection System (Invitrogen) with 100-μl tips according to cell-specific protocols (www.lifetechnologies.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-protocols-cell-line-data.html ). Cells electroporated with DNA plasmids were harvested after 48 hours if not indicated differently. Cells electroporated with mRNA were harvested after 4 hours, if not indicated differently. All transfections in S2 cells were performed using Effectene reagent (Qiagen).
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Plasmids and mRNA were introduced to the cells by the Neon Transfection System (Invitrogen) with 100-μl tips according to cell-specific protocols (
4
Culturing Hi-5 and Sua5B Insect Cell Lines
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We used the BTI-Tn-5B1-4 cell line [High Five Cells (Hi-5)], which is derived from ovarian cells of the cabbage looper (Trichoplusia ni) and is the standard insect cell line commonly used in many laboratories. We also chose to use the An. gambiae Sua5B cell line as these cells are immunocompetent and hemocyte-like. The Hi-5 and Sua5B cells were cultured in Express Five SFM medium (Invitrogen) and Grace’s Supplemented Insect Medium (Gibco, Waltham, MA), respectively, at 27 °C, as described previously [22 (link)]. The Sua5B cell medium also contained 10% heat-inactivated fetal bovine serum (Gibco); the cell lines were generally passaged twice per week upon reaching 90% confluency.
5
Drosophila Melanogaster S2 Cell Cultivation
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Drosophila melanogaster S2 cells were firstly thawed at passage 12 with Schneider's Drosophila Medium (Bio&Sell, supplemented with 10% FBS (Fetal Bovine Serum, Biochrom)) and later cultivated in Express Five SFM medium (protein‐free and serum‐free, Invitrogen). One bottle of the Express Five medium (1 l) was supplemented with 90 ml of L‐Glutamine (200 mM, Invitrogen). During cultivation, cells were grown at 25°C without CO2 in tissue culture flasks (75 cm2, Corning) and were split into fresh flasks when 90% confluent. The cells in passage 18 were seeded into 96‐well plates (Falcon 96 Well Tissue Culture Plates, Corning) with 40,000 cells per well in 100 µl using an electronic multichannel pipette VIAFLO (1,250 µl, INTEGRA) on a pipetting robot ASSIST (INTEGRA). The cells 24 h growth rate and viability were monitored in the culture dishes (in duplicate; 100 mm, Corning) with 12 × 106 cells in 14 ml medium. Cell counting and assessment of cell viability were performed using the Cell Counter and Analyzer System (CASY, Roche).
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Drosophila melanogaster S2 cells were firstly thawed at passage 12 with Schneider's Drosophila Medium (Bio&Sell, supplemented with 10% FBS (Fetal Bovine Serum, Biochrom)) and later cultivated in Express Five SFM medium (protein‐free and serum‐free, Invitrogen). One bottle of the Express Five medium (1 l) was supplemented with 90 ml of L‐Glutamine (200 mM, Invitrogen). During cultivation, cells were grown at 25°C without CO2 in tissue culture flasks (75 cm2, Corning) and were split into fresh flasks when 90% confluent. The cells in passage 18 were seeded into 96‐well plates (Falcon 96 Well Tissue Culture Plates, Corning) with 40,000 cells per well in 100 µl using an electronic multichannel pipette VIAFLO (1,250 µl, INTEGRA) on a pipetting robot ASSIST (INTEGRA). The cells 24 h growth rate and viability were monitored in the culture dishes (in duplicate; 100 mm, Corning) with 12 × 106 cells in 14 ml medium. Cell counting and assessment of cell viability were performed using the Cell Counter and Analyzer System (CASY, Roche).
6
Cell Culture and Transfection Protocols
7
Cultivation of Insect and Myeloma Cells
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Spodoptera frugiperda (Sf9; ATCC, Manassas, VA, USA) and High Five (ATCC, Manassas, VA, USA) insect cells were grown at 27 °C under an air atmosphere in Grace’s insect medium (Gibco, New York, NY, USA) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Flowery Branch, GA, USA) and 1% antibiotic-antimycotic solution (Gibco, New York, NY, USA), and Express Five SFM medium (Gibco, New York, NY, USA), respectively. Murine myeloma cell line Sp2/0Ag14 was purchased from the American Type Culture Collection (ATCC-CRL-1581, Rockville, MD, USA) and was maintained in RPMI-1640 (Gibco, New York, NY, USA) supplemented with 10% FBS at 37 °C with 5% CO2.
8
Ebola Virus Protein Expression in Insect Cells
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Spodoptera frugiperda Sf9 cells (ATCC, CRL-1711) were cultured in Grace’s insect medium (Gibco) supplemented with 10% FBS, 1% penicillin-streptomycin, and 0.1% Pluronic F68 solution (Gibco) at 28 °C without CO2. High Five Cells (BTI-TN-5B1-4, Invitrogen) were maintained in Express Five SFM medium (Gibco) supplemented with l -Glutamine and 1% penicillin-streptomycin. We used Grace’s insect medium (Gibco) supplemented with 3% FBS, 1% penicillin-streptomycin, and 0.1% Pluronic F68 solution to amplify the recombinant baculoviruses.
Rabbit antisera against GP (C2023) and VP40, which have been described previously [32 (link),33 (link)], and mouse monoclonal antibodies against Ebola GP protein (mAb 42/3.71 or mAb 12/1.1; kindly provided by Dr. Ayato Takada, Hokkaido University, Hokkaido, Japan) were used for Western blot analysis. A monoclonal antibody against Ebola GP protein (mAb 42/3.71) was also used as an ELISA positive control.
The GP and VP40 cDNAs sequences were consistent with those of Zaire ebolavirus Ebola virus/H.sapiens-wt/SLE/2014/Makona-G3750.2, which was isolated from a patient during the 2014–2016 Ebola outbreak in West Africa (GenBank accession No. KM233059).
Rabbit antisera against GP (C2023) and VP40, which have been described previously [32 (link),33 (link)], and mouse monoclonal antibodies against Ebola GP protein (mAb 42/3.71 or mAb 12/1.1; kindly provided by Dr. Ayato Takada, Hokkaido University, Hokkaido, Japan) were used for Western blot analysis. A monoclonal antibody against Ebola GP protein (mAb 42/3.71) was also used as an ELISA positive control.
The GP and VP40 cDNAs sequences were consistent with those of Zaire ebolavirus Ebola virus/H.sapiens-wt/SLE/2014/Makona-G3750.2, which was isolated from a patient during the 2014–2016 Ebola outbreak in West Africa (GenBank accession No. KM233059).
9
Culturing Drosophila Cell Lines
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S2 GFP-α-tubulin cells were a kind gift from E. Griffis (University of Dundee, UK) and S2 cells were from ATCC (CRL-1963) (a kind gift from R. Palmer, Umeå University, Sweden). Drosophila D.Mel-2 (Dmel) cells (a kind gift from P.P d’Avino and D. Glover, University of Cambridge, Cambridge, UK) were grown in Express Five SFM medium (Gibco) containing 2 mM L-glutamine, 100 U/ml penicillin and 100µg/ml streptomycin and Drosophila Schneider 2 (S2) cells were cultured in Schneider’s Drosophila Medium (Gibco) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100µg/ml streptomycin.
10
Cultivation of CSFV and Recombinant E2
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Classical swine fever virus isolate Honduras/1997 (a field isolate from Honduras) was kindly provided by Dr. Sabrina Swenson from the Animal and Plant Health Inspection Service (APHIS), United States Department of Agriculture (USDA). This CSFV isolate was passaged four times in swine testicle cells (ST; ATCC) cultured in DMEM (Gibco) supplemented with 10 % fetal bovine serum (FBS; Atlanta Biologicals) and 1 % Penicillin-streptomycin solution (Gibco). For recombinant E2 production in insect cells, Spodoptera frugiperda insect cells (Sf9; ATCC) were grown in Grace’s insect medium (Gibco) supplemented with 10 % FBS and 1 % antibiotic-antimycotic solution (Gibco), and High Five insect cells (Invitrogen) were grown in Express Five SFM medium (Gibco).
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