Recombinant baculoviruses containing influenza M1 (from A/California/06/2009 H1N1 strain) and HA (from A/Brisbane/59/2007 strain) genes were kindly provided by Redbiotec AG (Schlieren, Switzerland). An amplification of baculovirus stocks was performed as described elsewhere [25 (link)]. Briefly, insect Sf-9 cells (cultivated in Sf-900TM II medium (Gibco, Waltham, MA, USA)) were infected at a concentration of 1 × 106 cell/mL using a multiplicity of infection of 0.1 plate-forming units per viable cell (pfu/cell). When a cell viability of approximately 80% was reached, the supernatant was harvested by centrifugation at 200× g and 4 °C for 10 min and centrifugation at 2000× g and 4 °C for 20 min. The clarified supernatant was aliquoted appropriately and stored at 4 °C until further use.
Sf 900tm 2 medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Sf-900TM II medium is a serum-free cell culture medium formulated for the growth of insect cells. It is designed to support the optimal growth and viability of insect cell lines, such as Sf9 and High Five, in suspension culture. The medium provides the necessary nutrients and growth factors to maintain insect cell health and productivity.
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Lab products found in correlation
6 well tissue culture plate, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bti tn 5b1 4, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Tnm fh medium, by GE Healthcare (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Complete protease inhibitor cocktail, by Merck Group (1 mentions)
4 protocols using sf 900tm 2 medium
1
Amplification of Baculovirus Influenza Stocks
2
Alphanodavirus Infection Dynamics in Tn-Hi5 Cells
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Alphanodavirus-free Tn-Hi5 cell line (BTI-TN-5B1-4) was purchased from Thermo Fisher Scientific (United States). Cells were cultured in TNM-FH medium (GE Healthcare Life Sciences) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco) containing 0.5% penicillin-streptomycin solution (Gibco) at 27°C. ApNPV-Δph/egfp+ (Wang et al. 2010 (link)) was propagated in A. pernyi pupae and used to infect the cell line. Viral titer was determined by an end-point dilution assay with Tn-Hi5 cells according to a published method (Cha et al. 1997 (link)). For the infection, 1 × 106 Tn-Hi5 cells were seeded into wells of a 6-well tissue culture plate (Falcon) and infected with ApNPV-Δph/egfp+ (multiplicity of infection: 10). The ApNPV-Δph/egfp+ inoculum was removed after 1 h. The cells were rinsed with SF-900TM II medium (Gibco) and cultured in TNM-FH medium supplemented with 10% FBS at 27°C. The time at which the inoculum was removed was considered as 0 hpi. Cells were collected at 6, 12, 18, 24, 36, and 48 hpi. Uninfected cells (mock infection) were collected at 48 h as the control. Three independent biological replicates were prepared at each time point.
3
MERS-CoV S1 Protein Expression
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Baby Syrian hamster kidney (BHK-21) cells were cultured in serum-free medium (VirusPro®, Shanghai, China) at 37 °C with 5% CO2 on an orbital shaker at 130 rpm in suspension culture for virus infection. Mouse neuroblastoma (NA) cells were cultured with Dulbecco’s modified eagle medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) plus 10% fetal bovine serum (FBS, Biological Industries, Kibbutz Beit Haemek, Israel) for determination of viral titer and verification of virus inactivation. Spodoptera frugiperda 9 (Sf9, Gibco, Grand Island, NY, USA) insect cells were grown in Sf-900TM II medium (Life Technologies, San Diego, CA, USA) for protein expression. The recombinant RV expressing MERS-CoV S1 protein (RV/MERS) was constructed and stored in our laboratory.
4
Production and Purification of HPV16 VLPs
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HPV16 L1 virus-like particles (VLPs) were produced as we previously reported [1 (link),22 (link)]. Briefly, Spodoptera frugiperda Sf21 cells (Life Technologies Corporation, Carlsbad, CA, USA) in Sf900TMII medium (Life Technologies) were infected with L1-producing recombinant baculovirus for 72 h at 26 °C. Infected cells were pelleted by centrifugation and resuspended in 1X Dulbecco’s phosphate-buffered saline (D-PBS) (2.67 mM KCl, 1.47 mM KH2PO4, 137.93 NaCl mM and 8.06 mM Na2HPO4-7H2O pH 7.2–7.7) complemented with cOmpleteTM protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MI, USA) in siliconized microtubes and lysed by sonication in a GEX 130 PB ultrasonic processor (Cole-Parmer Instrument Co., Vernon Hills, IL, USA) at 60% cycle duty 3 times (5 s each). Total lysates were incubated overnight at 37 °C for VLP maturation; digested at 37 °C for 1 h with Serratia marcescens nuclease (Sigma-Aldrich) at 0.1% final concentration; and immediately chilled on ice, mixed with a 0.17 volumes of 5 M NaCl and clarified by centrifugation at 2000× g for 15 min. The resulting supernatant was purified through CsCl isopycnic centrifugation and extensively dialyzed against 1X D-PBS at 4 °C. VLP production was verified by negative stain in TEM and immunoblotting, and VLP stock was stored in 1X D-PBS at 4 °C until use.
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