Individual genotypes were obtained from AgABB51 via plaque purification [44 ]. A concentration of 108 OB/mL was used to inoculate fifth instar A. gemmatalis larvae using the droplet feeding method [45 ]. At 48 h post infection (hpi), infected larvae were bled and hemolymph containing budded virions was collected and stored at −20 °C. Hemolymph was then passed through a 0.45 µm filter and used to prepare six serial dilutions in Sf-900 II (Gibco) medium with antibiotics. A 200 µL volume from each dilution was used to inoculate 5 × 105 Sf9 cells (Gibco) and, at 10 d post infection (dpi), clearly separated individual plaques containing individual clones were collected with a sterile Pasteur pipette and diluted in 300 µL Sf-900 II medium. Clones were then injected into the hemocoel of A. gemmatalis fifth instar larvae that were individually placed in the wells of a cell culture plate with a semisynthetic diet and incubated at 25 °C until death or pupation.
Sf900 2
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SF900 II is a cell culture media preparation system designed for the efficient and consistent production of cell culture media. It features automated mixing, heating, and dispensing capabilities to streamline the media preparation process.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions)
Sf9 cells, by Thermo Fisher Scientific (2 mentions)
Cellfectin 2, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Hycell transfx h, by Cytiva (1 mentions)
Vi cell xr cell counter, by Beckman Coulter (1 mentions)
Ni nta superflow resin, by Qiagen (1 mentions)
Nah2po4, by Merck Group (1 mentions)
Igepal, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Sf 900 2 sfm, by Thermo Fisher Scientific (1 mentions)
12 well plate, by Corning (1 mentions)
Bac to bac system, by Thermo Fisher Scientific (1 mentions)
Flexstation 2, by Molecular Devices (1 mentions)
Fluo 8 am, by AAT Bioquest (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Prism v6, by GraphPad (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
23 protocols using sf900 2
1
Isolation and Propagation of Baculovirus Clones
2
Sf9 and HEK293SF Cells for rAAV Production
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Stably transformed rep2cap5 packaging Sf9 cells (B8) and suspension adapted mammalian cells (HEK293SF) used for rAAV viral vector production were provided by Dr. Zolotukhin (University of Florida, Gainesville, FL, USA) and Dr. Chahal (National Research Council of Canada, Montreal, QC, Canada), respectively. The Sf9/B8 and HEK293SF-cells were maintained in serum-free suspension cultures at appropriate cell-culture conditions (27°C for Sf9 and 37°C, 5% CO2, and 85% relative humidity for HEK293 cells) in a shaker incubator (Infors, Basel, Switzerland) at 120 rpm speed of agitation. The maintenance and production medium for insect cells and mammalian cells were Sf900-II (Thermo Fisher Scientific, Waltham, MA, USA) and Hycell TransFx-H (Cytiva Life Sciences, Chicago, IL, USA) (additionally supplemented with 0.1% w/v of Kolliphor P188 and 4 mM Glutamax), respectively. The cell density analysis for routine maintenance flasks and virus production run was performed using the Vi-Cell XR cell counter (Beckman Coulter, Brea, CA, USA). The recombinant baculovirus carrying the AAV transgene expression cassette (Bac-gfp) consisted of an AAV2-ITR flanking egfp under the control of chicken β-actin-CMV hybrid promoter. The recombinant baculovirus stock used for rAAV5 production was generated using naive Sf9 cells following a standard protocol, as published in our previous report.54 (link)
3
Recombinant Expression and Purification of RSV L+P
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RSV L + P complexes were prepared as previously described (53 (link), 54 (link)). Briefly, codon-optimized sequences of RSV L and a 6×HIS-tagged P were coexpressed in SF9 cells in serum-free medium SF900-II (Thermo Fisher Scientific) from a recombinant baculovirus vector generated with the pFastBac dual system. Cells were harvested at 78 hours after infection and lysed in 50 mM NaH2PO4 (pH 8.0), 150 mM NaCl, 20 mM imidazole, and 0.5% Igepal (MilliporeSigma). After purification through immobilized metal affinity chromatography with Ni-NTA Superflow resin (Qiagen), cells were dialyzed into storage buffer: 20 mM tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, and 1 mM dithiothreitol.
4
Cell Culture Conditions for Huh7, ExpresSf+, and Sf9 Cells
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Huh7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Waltham, MA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA) at 37°C, 5% CO2. ExpresSf+ and Sf9 cells were maintained in Sf-900 II (Thermo Fisher Scientific, Waltham, MA) medium alone or supplemented with 10% FBS (v/v) (Thermo Fisher Scientific, Waltham, MA), respectively, in shaker flasks at 28°C, 135 rpm.
5
Shrimp Intestine pH Response Assay
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Intestines were removed from shrimp under sterile conditions. The excrements were discarded, and intestines were washed in PBS containing 1 mg/ml streptomycin and 1000 IU/ml penicillin for 5 min. After rinsing 5 times with PBS, intestines were cut into 3-mm pieces. The fragments from 10 shrimp were mixed together, and 12 ml of Sf-900 II cell culture medium (Sf-900 II SFM, ThermoFisher) containing 5% fetal bovine serum (Gibco), 1 mg/ml streptomycin and 1000 IU/ml penicillin was added. The fragments were sequentially passed through 100 μM and 40 μM cell sieves, plated in 12-well plates (Corning) and cultured at 28°C for 12 h [26 , 27 (link)]. Then, the intestinal fragments were collected by centrifugation at 1000 rpm for 3 min, and the supernatants were discarded. The fragment resuspensions were treated with gradient pH 6.4–8.0 medium for 2, 6 and 12 h. In this case, the fragments were independently cultured in three wells for each pH at each time point, and the mRNA levels of LvNHE were detected by real-time PCR as described above.
6
Ciona GPCR-mediated Calcium Signaling
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Sf9 cells (Thermo Fisher Scientific) were grown in Sf900 II (Thermo Fisher Scientific) containing 10% FBS (Sigma) at 28 °C. Ciona GPCR-Gαq16-recombinant baculoviruses were generated in Sf9 cells transfected with the above bacmids using Cellfectin II, titrated, isolated, and transiently transfected into Sf9 cells using the Bac-to-Bac system according to the manufacturer’s instruction (Thermo Fisher Scientific). Forty-eight hours after transfection, Sf9 cells were loaded for 30 min with 2.5 μM of Fluo-8 AM (AAT Bioquest) diluted in loading buffer [HBSS supplemented with 1.25 mM of probenecid and 0.04% (wt/vol) of pluronic F-127]. Each Ciona GPCR-fused human Gαq16 expression at cell membrane was confirmed by immunostaining using the anti-Gαq16 antibody (Ori Gene TA318890). Various concentrations of peptides were administrated to Sf9 cells in a FlexStation II-automated apparatus (Molecular Devices). Real-time fluorescent kinetics for Fluo-8 were observed at excitation/emission wavelengths of 490/514 nm. The calcium accumulation data were analyzed using Prism v6 (GraphPad) to fit to a sigmoidal concentration-response curve, and the means ± SEMs of EC50 were calculated.
7
Culturing Spodoptera frugiperda Sf9 Cells
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Spodoptera frugiperda (Sf9 cell line, ATCC CRL-1711) were maintained in a modified Grace’s medium (Gibco, Thermo Fisher Scientific, Inc., USA) supplemented with 10% fetal bovine serum (FBS). Sf9 cells were added to a flask with 20 ml of fresh complete medium for a final density of 3 × 105 viable cells/ml and placed in an incubator shaker set at 27 °C with the shaking speed at 120 rpm for 3 days. Sub-culture was done when the cell reached the mid-log phase of growth. In this study, the Sf9 cells were also gradually adapted to grow in a serum-free medium, Sf900-II (Thermo Fisher Scientific, Inc., USA), using similar culture conditions.
8
Bm5 Cell Culture and Silkworm Rearing
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Bm5 cells were maintained at 27 °C in Sf-900II (Thermo Fisher Scientific K. K., Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific K. K.) and Antimycotic-Antibiotic (Thermo Fisher Scientific K. K.). In addition, Bm5 cells were also cultivated in non-FBS Sf900II medium. Fourth-instar silkworm larvae were purchased from Ehime Sansyu (Ehime, Japan). Silkworm larvae were reared on an artificial diet, Silkmate 2S (Nosan, Yokohama, Japan) at 25 °C.
9
Baculovirus Expression of Recombinant AFP
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AFP and AFP fragments were expressed using the Bac‐to‐Bac Baculovirus Expression System (Invitrogen Inc.). The process was as follows: (1) Sf9 insect cells were cultured in SFX‐Insect (Cytiva Ltd.), SF900 II and Grace's medium (Thermo Fisher Ltd.) containing 10% foetal bovine serum (FBS) or without serum at a density of 1 × 106 cells/ml. (2) P1 baculoviruses were amplified to obtain P2 baculoviruses. The amplification process was as follows: 10 ml of P1 baculovirus was added to 100 ml of Sf9 cells cultured in SFX‐Insect, SF900 II and Grace's medium and harvested at 72 h after infection. (3) Fifty milliliters of P2 baculovirus was added to 1 L of Sf9 cells, and secreted AFP and AFP fragments were harvested in the medium at 72 h after infection, as described previously.10
10
Recombinant PEDV S1 Subunit Production
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Recombinant PEDV S1 subunit was prepared as previously described by us [38 (link)]. Briefly, recombinant PEDV S1 subunit was expressed by the Drosophila S2 cells in Sf-900 II serum-free medium (Invitrogen, Carlsbad, USA), and purified by HisTrap excel prepacked column (GE Healthcare, Fairfield, USA) and HiTrap Q HP prepacked column (GE Healthcare). The purified S1 subunit was applied to 15% SDS-PAGE for analysis.
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