Viruses were propagated in Sf21 cells (Invitrogen). Protein expression was carried out in either Sf21 or High Five cells (Invitrogen). Both cell lines were maintained as suspension cultures in ExCell 420 serum-free medium (Sigma-Aldrich) at densities between 0.8 × 106 and 4 × 106 (Sf21) or between 0.6 × 106 and 4 × 106 cells per milliliter (High Five). Viral titers were determined by plaque assays. Transfection of Sf21 cells with bacmid DNA was performed with the CellFectin II transfection reagent (Invitrogen) according to the manufacturer’s instructions. For virus propagation, cells were infected with a multiplicity of infection (MOI) <0.01. For expression cultures, each virus was used at a MOI between 0.1 and 1. Expression cultures were harvested between 72 and 96 h post-infection.
Ex cell 420 serum free medium
Manufactured by Merck Group
Sourced in Germany
EX-CELL 420 serum-free medium is a liquid cell culture medium designed for the growth and maintenance of a variety of mammalian cell lines. It is a chemically-defined, protein-free, and animal-component-free formulation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Bac to bac baculovirus expression system, by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Sf 900 2 sfm, by Thermo Fisher Scientific (2 mentions)
Mccoy s 5a modified medium, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Wisent (2 mentions)
Streptomycin, by Wisent (2 mentions)
Sf21 cells, by Thermo Fisher Scientific (1 mentions)
High five cells, by Thermo Fisher Scientific (1 mentions)
Cellfectin 2 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Drosophila s2 cells, by Thermo Fisher Scientific (1 mentions)
Spodoptera frugiperda sf9 cells, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Tcs spe, by Leica (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
13 protocols using ex cell 420 serum free medium
1
Baculovirus-Based Protein Expression
2
Cell Culture of Drosophila S2 and HEK293T
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Drosophila S2 cells were purchased from Invitrogen and cultured in EX-CELL® 420 Serum-Free Medium (Sigma). HEK293T (or 293 T) cells from ATCC were confirmed to be negative for mycoplasma contamination. 293T cells were cultured at 37 °C with 5% CO2 in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% FBS (Gibco) and 1X penicillin-streptomycin (Invitrogen).
3
Cpo_CPRQ Expression in Baculovirus
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The expression construct for Cpo_CPRQ in the baculovirus expression vector system (BEVS) donor vector pDEST8_CPRQ was made by LR reaction. Recombinant bacmids were made according to instructions for the Bac-to-Bac® Baculovirus expression system given by the manufacturer (Invitrogen) using DH10EMBacY (Geneva Biotech). Baculovirus generation was done using Spodoptera frugiperda Sf9 cells (Thermo Fisher Scientific), Ex-Cell 420 serum-free medium (Sigma), and baculoFECTIN II (OET). The virus was then amplified once to generate a P2 virus stock using Sf9 cells and Ex-Cell 420 medium. Viral titer in the P2 stock was determined using the BaculoQUANT all-in-one qPCR kit (OET) and found to be 3 × 108 pfu/mL for Cpo_CPRQ.
Insect cell lines Sf9 were diluted to 2 × 106 cells/mL. Heterologous expression was performed in 20-mL cultures in Ex-Cell 420 medium and the cells infected at an MOI of 1. The cultures were incubated in 125-mL Erlenmeyer flasks (100 rpm, 27 °C), with fatty acid methyl-ester substrates supplemented at a final concentration of 0.25 mM after 1 day. After 3 days, 7.5-mL samples were taken from the culture and centrifuged for 15 min at 4500g at 4 °C. The pellets were stored at − 80 °C until fatty acid analysis. Sf9 expression experiments were conducted in three replicates.
Insect cell lines Sf9 were diluted to 2 × 106 cells/mL. Heterologous expression was performed in 20-mL cultures in Ex-Cell 420 medium and the cells infected at an MOI of 1. The cultures were incubated in 125-mL Erlenmeyer flasks (100 rpm, 27 °C), with fatty acid methyl-ester substrates supplemented at a final concentration of 0.25 mM after 1 day. After 3 days, 7.5-mL samples were taken from the culture and centrifuged for 15 min at 4500g at 4 °C. The pellets were stored at − 80 °C until fatty acid analysis. Sf9 expression experiments were conducted in three replicates.
4
Recombinant hERG(S1-coil) Expression in Sf9 Cells
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The hERG(S1-coil) sequence optimized for expression in insect cells was subcloned into pFB1. A double-streptavidin tag and 3C cleavage site were added at the 5′ end of the construct and a TEV (Tobacco Etch Virus) cleavage site and 6-histidine tag added at the 3′extremity (Table 1 ). Following the Bac-to-BacTM method (Invitrogen, Life Technologies, ThermoFischer Scientific), the recombinant vector was transferred to competent E. coli DH10Bac cells to produce recombinant bacmid by homologous recombination. Viral stock was obtained by transfection of Sf9 cells with the recombinant bacmid. Finally, a culture of Sf9 insect cells at a density of 4 × 106 cells/mL in suspension in EX-CELL®420 Serum-Free medium (SigmaAldrich) was infected by the viral stock encoding hERG(S1-coil) at a concentration of 1:500 (v/v) for 48 h at 28 °C with shaking. Cells were harvested by centrifugation and stored at −80 °C until purification.
5
Visualizing Midgut RNAi in Western Corn Rootworm
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Fifty diet-raised 2nd instar WCR were treated and surface-sterilized as previously described [15 (link)], with modifications (S1 Method ). Twenty midguts were dissected and rinsed in sterile 1x PBS to eliminate gut contents. Pretreated midguts were incubated with Cy3-labeled dvssj1 dsRNA and siRNAs at 10 ng/μl in insect medium (EX-CELL 420 Serum-Free Medium; Sigma) for 15 hours at 25°C protected from light. Unconjugated Cy3 dye was used as a control at the same concentration. Midguts were washed twice with 1x PBS and fixed in 4% paraformaldehyde for 1 hour at room temp. Then midguts were washed three times with 1x PBST (PBS +0.1% Tween) for 5 minutes each. Samples were counterstained with DAPI (Sigma, 10 μg/μl of stock diluted to 1:1000) for 5 min and washed once in 1x PBST. Individual guts were placed on glass slides with 2x SlowFade concentrated Slow Fade antifade reagent in PBS, then coverslipped and imaged on a Leica TCS SPE. The 405 nm laser line was used for excitation of DAPI staining and the 532 nm laser line was used for excitation of Cy3 staining. Image analyses were carried out as described in S1 Method .
6
Maintenance of Cell Lines for Research
7
Cell Culture Conditions for Insect Cell Lines
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Heliothis zea MG cells line is maintained at 28 °C and cultured with EX-Cell420 Serum-Free Medium (Sigma-Aldrich) that containing 10% fetal bovine serum and 0.5% Penicillin-Streptomycin Liquid as description in previous paper (53 (link)). Spodoptera frugiperda ovary cell line (Sf9 cells) was cultured with Sf-900 Ⅱ Serum-Free Medium (containing 10% fetal bovine serum, 0.5% Penicillin-Streptomycin Liquid) at 28 °C. fetal bovine serum (Gibco). Penicillin-Streptomycin Liquid (HyClone).
8
Culturing FreeStyle™ 293F and Schneider 2 Cells
9
Recombinant Monoclonal Insect Cell Lines
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We used recombinant monoclonal D. melanogaster S2 cell lines expressing either gloverin from the greater wax moth Galleria mellonella (GmGlv) or BR021 from the harlequin ladybird Harmonia axyridis [30 (link),32 (link),33 (link)]. The genes encoding both antimicrobial peptides were controlled by the D. melanogaster metallothionein promoter. The constructs included a secretion signal and a V5/His6 or V5/GFP tag. Stable monoclonal cell lines were prepared as previously described [29 ,30 (link)]. The cells were grown in suspension at 27 °C in ExCell 420 serum-free medium (Sigma Aldrich, Munich, Germany) supplemented with 8–10 mM L-glutamine (Biochrom, Berlin, Germany). Selection was achieved by adding 10 µg/mL Blasticidin S or 300 µg/mL Hygromycin B (Invivogen, Toulouse, France) during subculture, depending on which selection marker was used. For maintenance, cultures were split every 3–4 days to obtain 1.5 × 106 cells/mL.
10
Expression and Purification of Chlamydomonas HAP2
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cDNA encoding Chlamydomonas HAP2, residues 23–582, codon optimized for mammalian cell expression, was cloned into ET15S2 vector, a ligation-independent cloning variant of the pExpreS2-2 vector (ExpreS2ion Biotechnologies) that includes N-terminal secretion signal from Hspa5 and C-terminal His8 tag. Drosophila melanogaster Schneider S2 cells (ExpreS2 cells, ExpreS2ion Biotechnologies), grown in EX-CELL 420 Serum-Free Medium (Sigma), were transfected using EXpreS2 transfection reagent. Stable transfectants were selected in the same medium supplemented with 4 mg/ml G418 and expanded in EX-CELL 420 medium at 25°C. After centrifugation at 5,000 g for 20 min, 1 L culture supernatant was filtered (0.22 μm pore) and made 2 mM in NiCl2 and 300 mM in NaCl. Protein was purified using a 10 ml Ni2+-nitrilotriacetate column (Qiagen) followed by size exclusion chromatography using a Superdex 200 10/300 GL column in 20 mM Tris-HCl, pH 7.5, 500 mM NaCl with a yield of 1 – 1.5 mg per L supernatant.
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