The PA, PB1 and PB2 genes of Influenza A virus RNA polymerase were amplified by reverse-transcription and PCR from H1N1 strain A/PR/8. The 3x FLAG tag was added to the N-terminal full-length PA and cloned into pFastBac1 vector (Life Technologies, Carlsbad, CA). Full-length PB1 and PB2 genes were cloned into pFastBac Dual vector (Life Technologies) as described previously[31 (link)]. Recombinant baculovirus was generated for PA and PB1/PB2 using Sf9 cells and was used to co-infect Trichopulsia ni cells (Hi5) at 1.5 x 106 cells/ml in ESF-921 medium (Expression Systems, David, CA). Purification and biochemical characterization of the polymerase heterotrimer was described previously [31 (link)]. The purified polymerase complex was stored at -80°C prior use.
Esf 921 medium
Manufactured by Expression Systems
Sourced in United States
ESF 921 medium is a specialized cell culture medium designed for the growth and maintenance of insect cells. It provides the necessary nutrients and growth factors to support the optimal proliferation of insect cells in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Cellfectin 2 reagent, by Thermo Fisher Scientific (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Sf 900 3 sfm medium, by Thermo Fisher Scientific (3 mentions)
Bac to bac baculovirus expression system, by Thermo Fisher Scientific (3 mentions)
Dh10bac e coli competent cells, by Thermo Fisher Scientific (2 mentions)
Pei max, by Polysciences (2 mentions)
25 cm2 suspension cell flasks, by Sarstedt (2 mentions)
Fetal calf serum (fcs), by Merck Group (2 mentions)
Dmem high glucose medium, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by US Biological (2 mentions)
Dulbecco modified eagle medium (dmem), by Corning (2 mentions)
Bac to bac system, by Thermo Fisher Scientific (2 mentions)
Ni nta, by Qiagen (2 mentions)
Sf 900 2 sfm medium, by Thermo Fisher Scientific (2 mentions)
Fugene hd transfection reagent, by Promega (2 mentions)
Pfastbac1 vector, by Thermo Fisher Scientific (1 mentions)
Pfastbac dual vector, by Thermo Fisher Scientific (1 mentions)
Freestyle 293 f expression medium, by Thermo Fisher Scientific (1 mentions)
Expicho s cells, by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
58 protocols using esf 921 medium
1
Influenza A Virus RNA Polymerase Reconstitution
2
Recombinant Bacmid Generation for ATP13A2 Expression
3
Culturing Diverse Mammalian Cell Lines
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All mammalian cell lines were incubated at 37 °C in a humidified atmosphere containing 5–8% CO2. FreeStyle™ 293-F suspension cells (Gibco, R79007) were maintained in FreeStyle 293F expression medium (Gibco, 12338-018), ExpiCHO-S™ cells (Gibco, A29127) were maintained in ExpiCHOTM Expression Medium (Gibco, A29100-01), TZM-bl cells (NIH AIDS Research and Reference Reagent Program, Cat. no. 8129) were maintained in DMEM medium (Gibco,11995-065). Sf9 (Gibco, 11496015) and High Five Cells (Gibco, B85502) were maintained in Sf-900TM II SFM medium (Gibco,10902-088) and ESF 921 medium (Expression Systems, 96-001-01) at 27 °C respectively.
4
Purification and Characterization of ERC1 Protein
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Sf9 cells in ESF921 medium (Expression Systems) were co-transfected with linearized viral genome and the expression plasmid and selected for high infectivity. Viruses were produced and used to infect Sf9 cells and to obtain lysates for protein purification as described44 (link),45 (link). The 6xHis-MBP-ERC1 fusion protein was purified as previously described for extended coiled-coils in 20 mM HEPES pH7.4, 250 mM NaCl, 0.5 mM TCEP (46). Briefly, amylose resin was used to affinity isolate the dimeric ERC1 protein, subsequently eluted with 10 mM maltose, and subjected to size-exclusion chromatography. Protein concentration was determined by UV280 and Bradford assay.
The light-scattering from purified ERC1 was analyzed by an autosampler equipped Viskotek TDAMax system as described45 (link). The data obtained were averaged across the protein elution volume and molecular masses determined by OmniSEC software package.
Samples for rotary shadowing were prepared as described45 (link). Briefly, samples diluted in spraying buffer (100 mM ammonium acetate, 30% glycerol) were sprayed via a capillary onto freshly cleaved mica chips, which were then mounted in a high vacuum evaporator (MED 020, Baltec) and dried. Specimens were platinum coated (5–7.5 nm) and carbon was evaporated. Replicas were examined and imaged onto a CCD (Morgagni 268D, FEI; Morada G2, Olympus).
The light-scattering from purified ERC1 was analyzed by an autosampler equipped Viskotek TDAMax system as described45 (link). The data obtained were averaged across the protein elution volume and molecular masses determined by OmniSEC software package.
Samples for rotary shadowing were prepared as described45 (link). Briefly, samples diluted in spraying buffer (100 mM ammonium acetate, 30% glycerol) were sprayed via a capillary onto freshly cleaved mica chips, which were then mounted in a high vacuum evaporator (MED 020, Baltec) and dried. Specimens were platinum coated (5–7.5 nm) and carbon was evaporated. Replicas were examined and imaged onto a CCD (Morgagni 268D, FEI; Morada G2, Olympus).
5
Spodoptera frugiperda Cell Culture Protocol
6
Insect Cell-Based Baculovirus Amplification
7
Generation of RFP-GFP-ATG8a Fusion Construct
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ATG8a was amplified using the primers 5′-TGACCTAGCTAGATCTAAGTTCCAATACAAGGAGGA-3′ and 5′-TGACCTAGCTGAATTCTTAGTTAATTTTGGCCATGCC-3′. Atg8a was cloned into the BglII–EcoRI sites of mRFP-EGFP-LC3 (Kimura et al., 2007 (link)). The cytomegalovirus promoter from this construct was removed using AseI–NheI digestion and replaced with the actin promoter amplified from pAFW (Drosophila Genomics Resource Center) using the primers 5′-TGACGATCGCATTAATCAGCATGCAATTCTATATTCT-3′ and 5′-TGACGATCGCGCTAGCGGCCTCGATATCTGGATCCGG-3′. The Pactin-RFP-GFP-ATG8a fragment (AseI–MluI digest) was then subcloned in the HindIII site of the previously described p2ZOp2F vector (Hegedus et al., 1998 (link)), which contains Zeocin as a selection agent for transfection in Drosophila cell lines. For transfections, 1 µg Pactin-RFP-GFP-ATG8a was added to 10 µl Cellfectin (Invitrogen) plus 100 µl Grace’s medium (Invitrogen) and incubated for ≥30 min. 3.75 × 106 S2 cells in 400 µl Grace’s media were incubated with transfection medium overnight. 1 ml ESF921 medium (Expression Systems) was added back to the cells, and the cells were incubated for an additional 3 d before adding Zeocin at a concentration of 0.6–0.8 mg/ml. The media were replaced every 4 d until the negative control showed 0% viability (∼3 wk). Transfected cells were maintained in ESF921 + 0.1 mg/ml Zeocin.
8
Drosophila Cell Culture Protocols
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Drosophila l(2)mbn cells were grow in Schneider’s medium (Invitrogen) supplemented with 10% FBS in 25-cm2 suspension cell flasks (Sarstedt) at 25°C. Drosophila S2-RFP-GFP-Atg8a cells were grown in ESF921 medium (Expression Systems) in the presence of 50 µg/ml Zeocin. All experiments were performed 3–4 d after passage.
9
Bacterial Expression and Cell Culture Protocol
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All bacteria were cultured in Luria–Bertani medium with appropriate antibiotics at 37°C. Plasmids were maintained in E. coli XL-1 Blue (Stratagene, San Diego, CA) and purified using standard miniprep kits (Machery-Nagel, Bethlehem, PA) before transfection. During bacterial expression, GST-fusion proteins were produced in E. coli Rosetta(DE3)pLysS (EMD Millipore, Billerica, MA). Sf9 insect cells were grown in ESF921 medium (Expression Systems, Davis, CA) at 28°C and infected with baculoviruses encoding MBP- or GST-fusion proteins at a multiplicity of infection of ∼1. Cos7, HeLa, and HFF cells were cultured in DMEM plus 10% fetal bovine serum and antibiotic-antimycotic (Invitrogen) at 37°C in 5% CO2. Transfections with DNA or RNA used Lipofectamine-LTX or RNAiMAX (Invitrogen), respectively (Campellone et al., 2008 (link)). Control siRNAs, siRNAs to glyceraldehyde-3-phosphate dehydrogenase, and siRNA pair #3 to Rab1a+b were from Ambion, and siRNA pairs #1 and 2 to Rab1a+b and siRNAs #1 and 2 to RhoD were from Sigma-Aldrich (St. Louis, MO). For selection of HFF clones stably transfected with LAP or LAP-WHAMM, media were supplemented with 750 μg/ml G418. To induce LAP expression, 7.6 mM sodium butyrate was added for 12–18 h.
10
Expression of Codon-Optimized LRRC8A in Sf9 Cells
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The coding sequence for LRRC8A from Mus musculus was codon optimized for Spodoptera frugiperda and synthesized (Gen9, Cambridge, MA). The sequence was then cloned into a custom vector based on the pACEBAC1 backbone (MultiBac; Geneva Biotech, Geneva, Switzerland) with an added C-terminal PreScission protease (PPX) cleavage site, linker sequence, superfolder GFP (sfGFP) and 7xHis tag, generating a construct for expression of mmLRRC8A-SNS-LEVLFQGP-SRGGSGAAAGSGSGS-sfGFP-GSS-7xHis. MultiBac cells were used to generate a Bacmid according to manufacturer’s instructions. Spodoptera frugiperda (Sf9) cells were cultured in ESF 921 medium (Expression Systems, Davis, CA) and P1 virus was generated from cells transfected with Cellfectin II reagent (Life Technologies, Carlsbad, CA) according to manufacturer’s instructions. P2 virus was then generated by infecting cells at 2 million cells/mL with P1 virus at an MOI ~ 0.1, with infection monitored by fluorescence of sfGFP-tagged protein and harvested at 72 hr. P3 virus was generated in a similar manner to expand the viral stock. The P3 viral stock was then used to infect 1 L of Sf9 cells at 4 million cells/mL at an MOI ~ 2–5. At 72 hr, infected cells containing expressed LRRC8A-sfGFP protein were harvested by centrifugation at 2500 x g and frozen at −80°C.
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