Sf 900 medium

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Sf-900 medium is a serum-free, protein-free insect cell culture medium designed for the growth and maintenance of insect cells. It is a complete, defined medium that supports the growth of various insect cell lines, including Spodoptera frugiperda (Sf) cells, Trichoplusia ni (Tn) cells, and others. The Sf-900 medium provides the necessary nutrients, growth factors, and supplements required for optimal insect cell growth and productivity.

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Sf-900 II serum-free medium (60 citations) Sf-900 III medium (37 citations) Sf-900 III SFM medium (34 citations) Sf-900 III serum-free medium (16 citations) Sf-900 II medium (15 citations) Sf-900TM II medium (4 citations) Sf-900TM III SFM (1x) Serum Free Medium (2 citations) SF-900 II SFM serum-free medium (2 citations) Sf-900 II insect medium (2 citations) Sf-900 serum-free medium (2 citations)

14 protocols using sf 900 medium

Cell Culture and Influenza Virus Propagation

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Spodoptera frugiperda 9 (Sf9) insect cells (Invitrogen, USA, Carlsbad, CA, Catalog No. 11496–015) were maintained at 27°C in Sf-900 medium (Invitrogen) supplemented with 1% antibiotics/antimycotics (Gibco-BRL, CA, USA).
HEK 293T cells (American Type Culture Collection [ATCC], Manassas, VA, USA, Catalog No. CRL-3216) were cultured in Dulbecco’s modified Eagle’s medium (D MEM; Gibco-BRL) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL) and 1% penicillin-streptomycin. Madin-Darby canine kidney (MDCK) cells (ATCC, Catalog No. PTA-6500) were grown in Eagle’s minimum essential medium (MEM; Gibco-BRL) containing 10% FBS and 1% penicillin and streptomycin. The cells were maintained in a humidified 5% CO₂ atmosphere at 37°C.
Mouse-adapted influenza virus type A/CA/04/2009 (ma-pH1N1) was kindly provided by the International Vaccine Institute (IVI, Seoul, Republic of Korea). Influenza virus type A/PR/8/1934 (H1N1) was kindly provided by the Centers for Disease Control and Prevention (CDC, Osong, Republic of Korea). The viruses were amplified in 10-day-old embryonated eggs. After incubating at 37°C for 3 days and chilling at 4°C for 12 hours, the allantoic fluid was harvested, aliquoted, and stored at -80°C until use.

Gwon Y.D., Kim S., Cho Y., Heo Y., Cho H., Park K., Lee H.J., Choi J., Poo H, & Kim Y.B. (2016). Immunogenicity of Virus Like Particle Forming Baculoviral DNA Vaccine against Pandemic Influenza H1N1. PLoS ONE, 11(5), e0154824.

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Cell Culture Protocols for Sf9, 293TT, and MDCK Cells

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Sf9 (Invitrogen, CA, USA) cells were maintained at 27°C in Sf-900 medium (Invitrogen, CA, USA) supplemented with 1% antibiotics/antimycotics (Gibco-BRL, CA, USA). 293TT cells (kindly donated by Dr. Schiller, National Cancer Institute, NIH, USA) were cultured in Dulbecco’s modified Eagle’s medium (D MEM; Gibco-BRL) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL) and 400 μg/mL hygromycin B (Invitrogen) [27 (link)].
Madin-Darby canine kidney (MDCK; American Type Culture Collection, Manassas, VA, USA) cells were grown in Eagle’s minimum essential medium (MEM; Gibco-BRL) containing 10% FBS and 1% penicillin and streptomycin. The cells were maintained in a humidified 5% CO₂ atmosphere at 37°C.

Choi H.J., Gwon Y.D., Jang Y., Cho Y., Heo Y.K., Lee H.J., Kim K.C., Choi J., Lee J.B, & Kim Y.B. (2015). Effect of AcHERV-GmCSF as an Influenza Virus Vaccine Adjuvant. PLoS ONE, 10(6), e0129761.

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Calcium Imaging of Transfected Sf9 Cells

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Sf9 cells were plated into 12-well plates and left to settle for 20 min before being transfected by 500 ng of plasmid (pIB/V5-His vector as control or pIB/V5-His-HarmGR vector) and 3 μL of Fugene HD transfection reagent (Promega, USA) in 100 μL per well of Sf-900 medium (Invitrogen, USA). Forty-eight hours after transfection, cells were prepared for calcium imaging and data analysis as described previously26 (link)40 47 . Graphpad Prism5 and Microsoft Excel 2012 were utilized for data analysis.

Xu W., Papanicolaou A., Zhang H.J, & Anderson A. (2016). Expansion of a bitter taste receptor family in a polyphagous insect herbivore. Scientific Reports, 6, 23666.

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Culturing Insect and Mammalian Cells for CHIKV Infection

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Spodoptera frugiperda Sf21 cells were cultured in Grace’s medium (Invitrogen) with 10 % fetal bovine serum (FBS; Invitrogen) and Sf9 cells in Sf900 medium (Invitrogen) with 5 % FBS. Aedes albopictus U4.4 cells and C6/36 cells were cultured in Leibovitz’s medium (Invitrogen) supplemented with 10 % FBS, 2 % tryptose phosphate (Invitrogen) and 1 % non-essential amino acids (Invitrogen). All insect cells were cultured at 27 °C. Vero E6 and HEK293t mammalian cells were cultured in Dulbecco’s modified Eagle medium (Invitrogen) supplemented with 10 % FBS at 37 °C and 5 % CO₂. Infections in cell culture were performed with CHIKV isolate S27 and mosquitoes were infected with CHIKV 06-021 strain.

Fros J.J., Geertsema C., Zouache K., Baggen J., Domeradzka N., van Leeuwen D.M., Flipse J., Vlak J.M., Failloux A.B, & Pijlman G.P. (2015). Mosquito Rasputin interacts with chikungunya virus nsP3 and determines the infection rate in Aedes albopictus. Parasites & Vectors, 8, 464.

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Drosophila-Expressed Soluble DR4 Purification

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Expression of soluble DR4 was performed using stable transfectants of Drosophila S2 Schenider cells, as described previously for DR1 [41] (link). Stable transfected cell lines were established by selection under 1.0 mg/L geneticin (Invitrogen Life technologies – California, USA) and grown in SF900 medium (Invitrogen Life technologies – California, USA) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin (Invitrogen Life technologies – California, USA), 250 µg/L amphotericin B and 2 mM L-glutamine (Invitrogen Life technologies – California, USA), at 22–24°C. Cell cultures were induced at a density of 5–10×10⁶ per mL by the addition of 0.5 mM CuSO₄ and culture supernatant was collected 4–6 days later by centrifugation at 4000xg. Supernatant was concentrated 10-fold in a 10,000 molecular weight cut-off spiral filtration device (Millipore, Massachusetts, USA). DR4 was purified by immunoaffinity with LB3.1-conjugated protein A column, as described [42] (link). The final yield of DR4 was in the range of 0.2–0.5 mg/L of cultured cells.

Parra-López C.A., Bernal-Estévez D., Vargas L.E., Pulido-Calixto C., Salazar L.M., Calvo-Calle J.M, & Stern L.J. (2014). An Unstable Th Epitope of P. falciparum Fosters Central Memory T Cells and Anti-CS Antibody Responses. PLoS ONE, 9(7), e100639.

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Silkworm Cell Culture and Viral Infection

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B. mori cells, originating from the ovary of silkworm, are maintained in our laboratory. Cells were grown at 27 °C in Sf-900 medium (Thermo Fisher Scientific, USA) complemented with 10% fetal bovine serum. The BmN cells were cultured at a density of 1 × 10⁶ cells in 25 cm² flask. In the present study, two categories of cells were examined: control cells (normal cell) and those that were infected with the preserved virus, B. mori nucleopolyhedrovirus (BmNPV), in 24 h using 10 MOI (multiplicity of infection). The cell culture and viral infection experiments were done as biological triplicates. After infection, the cells were collected and centrifuged at 3000 rpm. The control cells were treated similarly. Both pellets were washed twice with PBS after the supernatant was discarded.

Shobahah J., Xue S., Hu D., Zhao C., Wei M., Quan Y, & Yu W. (2017). Quantitative phosphoproteome on the silkworm (Bombyx mori) cells infected with baculovirus. Virology Journal, 14, 117.

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Baculovirus Production in Silkworm Ovary Cells

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BmN cells that were derived from silkworm ovary were cultured at 27°C in Sf-900 medium
(Thermo Fisher Scientific, America) supplemented with 10% fetal bovine serum (Corning,
America). The BmNPV genome was extracted from the E.
coli strain DH10Bac and transfected into BmN cells to propagate the
virus. Viral titers were determined by end-point dilution assay (Reed and Muench, 1938)
[14 (link)].

Lei J., Hu D., Xue S., Mao F., Obeng E., Quan Y, & Yu W. (2019). HN1L is essential for cell growth and survival during nucleopolyhedrovirus infection in silkworm, Bombyx mori. PLoS ONE, 14(5), e0216719.

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Cell Line Maintenance Protocols

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Spodopterafrugiperda Sf9, Drosophila melanogaster S2 and D. melanogaster S2[Cas9] (kind gift from Klaus Förstemann, Munich) cell lines were maintained in Sf-900 medium (Gibco) and Schneider's medium (Gibco), respectively, supplemented with 10% (v/v) Fetal calf serum (Sigma) and 1% (v/v) Penicillin-Streptomycin (Gibco) under standard conditions (26°C).

Mačinković I., Theofel I., Hundertmark T., Kovač K., Awe S., Lenz J., Forné I., Lamp B., Nist A., Imhof A., Stiewe T., Renkawitz-Pohl R., Rathke C, & Brehm A. (2019). Distinct CoREST complexes act in a cell-type-specific manner. Nucleic Acids Research, 47(22), 11649-11666.

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Baculovirus Protein Expression in Insect Cells

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S2 and Sf9 cell lines (kind gift from Peter Becker, Munich) were maintained at 26 °C in Schneider medium (Gibco) and Sf-900 medium (Gibco), respectively, supplemented with 10% fetal calf serum. RNA interference, baculovirus generation and infection are described in ref. 40 (link). Briefly, double-stranded RNA was generated by T7 Polymerase in vitro transcription from PCR amplimers generated with T7 promotor-containing primers (Supplementary Table 1). Double-stranded RNAs were transfected into S2 cells using Effectene (Qiagen). Baculoviruses were generated using the Bac-to-bac system (Invitrogen). Baculoviruses were amplified twice and then used to infect Sf9 cells for protein production. Cells were then harvested 48–72 h after infection. EcR (ER33854) and USP (LD09973) cDNAs were obtained from BDGP. Vectors for generation of baculoviruses expressing untagged EcR, N-terminally FLAG-tagged EcR and N-terminally HA-tagged USP were generated by PCR-cloning of the respective open-reading frames into pFastBac or pVL1392 using appropriate sets of primers. Baculoviruses and expression vectors for dLint-1 and dMi-2 were constructed in the same manner46 (link)47 (link).

Kreher J., Kovač K., Bouazoune K., Mačinković I., Ernst A.L., Engelen E., Pahl R., Finkernagel F., Murawska M., Ullah I, & Brehm A. (2017). EcR recruits dMi-2 and increases efficiency of dMi-2-mediated remodelling to constrain transcription of hormone-regulated genes. Nature Communications, 8, 14806.

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Cultivation of Pv11 and Sf9 Insect Cells

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Pv11 cells, originally isolated from egg masses of P. vanderplanki, were cultivated in accordance with a previously published protocol [12 (link)]. Briefly, we cultivated the cells in IPL-41 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 2.6 g/L tryptose phosphate broth. We obtained insect Sf9 cells derived from S. frugiperda, the fall armyworm (Lepidoptera), from Merck, and cultivated them in Sf900 medium (Gibco, Grand Island, NY, USA) without supplements. We maintained both Pv11 and Sf9 cultures in non-humidified incubators at 25 and 28 °C, respectively.

Kondratyeva S.A., Voronina T.A., Nesmelov A.A., Miyata Y., Tokumoto S., Cornette R., Vorontsova M.V., Kikawada T., Gusev O.A, & Shagimardanova E.I. (2022). Intracellular Localization and Gene Expression Analysis Provides New Insights on LEA Proteins’ Diversity in Anhydrobiotic Cell Line. Biology, 11(4), 487.

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