Sf 900 3 media

Manufactured by Thermo Fisher Scientific

The Sf-900 III media is a serum-free, insect cell culture medium designed for the growth and maintenance of insect cells. It provides a defined, optimized environment to support the culture of various insect cell lines.

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Lab products found in correlation

Reach in co2 incubator, by Thermo Fisher Scientific (2 mentions) Lr clonase 2, by Thermo Fisher Scientific (1 mentions) Effectene, by Qiagen (1 mentions) Avitag, by Avidity (1 mentions) Pen strep, by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Merck Group (1 mentions) Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions) Gentamicin sulfate, by Thermo Fisher Scientific (1 mentions) Kg 1 cells, by American Type Culture Collection (1 mentions) Fugene hd, by Promega (1 mentions) Strep tactin xt column, by IBA Lifesciences (1 mentions) Benzonase, by Merck Group (1 mentions) Complete edta free protease inhibitor, by Roche (1 mentions)

Spelling variants of this product

Sf-900 III SFM media (11 citations) SF-900 III Serum Free Media (2 citations)

5 protocols using sf 900 3 media

Bait and Prey Protein Expression

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Bait expression vectors were modified from the pECIA14 vector (Özkan et al., 2013 (link)). An Avitag (Avidity) was added between the hexahistidine and FLAG tags at the C-terminus of the vector with standard cloning procedures to make a new Gateway (Thermo Fisher, Waltham, MA) destination vector. ECD sequences were moved from entry vectors for Beats and Sides, described in (Özkan et al., 2013 (link)), into the modified pECIA14 vector using LR Clonase II (Thermo Fisher). Prey proteins were expressed from the pECIA2 vector (Özkan et al., 2013 (link)).
All proteins, excepting the unpurified prey, were expressed in Drosophila Schneider 2 cells grown in S2 media with 10% fetal bovine serum, 50 units/mL penicillin and 50 μg/mL streptomycin. The unpurified prey proteins were expressed in Sf-900 III media (Thermo Fisher). Proteins were transfected using Effectene (Qiagen, Hilden, Germany), following manufacturer’s instructions. Copper (0.5 mM CuSO₄) was added the day after transfection to induce expression of protein. For the baits, 3 mM biotin was also added to the media to facilitate in vivo biotinylation. Prey proteins were purified using Ni-NTA resin, following standard procedures.

Li H., Watson A., Olechwier A., Anaya M., Sorooshyari S.K., Harnett D.P., Lee H.K., Vielmetter J., Fares M.A., Garcia K.C., Özkan E., Labrador J.P, & Zinn K. (2017). Deconstruction of the beaten Path-Sidestep interaction network provides insights into neuromuscular system development. eLife, 6, e28111.

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Cell Culture Conditions for Various Cell Lines

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SKW3 T cells (DSMZ) were cultured in RPMI-1640+GluMax (Thermo Fisher Scientific) complemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), 10 mM HEPES and 50 U/mL Pen-Strep (Thermo Fisher Scientific) at 37 °C and 5% CO₂.
LentiX cells and 293T cells were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS, 2 mM L-Glutamine, 10 mM HEPES and 50 U/mL Pen-Strep (Thermo Fisher Scientific) at 37 °C and 5% CO₂.
KG-1 cells (ATCC) were cultured in IMDM (Thermo Fisher Scientific) supplemented with 10% FBS and 50 U/mL Pen-Strep (Thermo Fisher Scientific) at 37 °C and 5% CO₂.
SF9 cells were cultured in SF900-III media (Thermo Fisher) supplemented with 10% FBS and 10 mg/mL gentamicin sulfate (Thermo Fisher) at 27 °C and atmospheric CO₂.
Hi5 cells were grown in insect cell culture medium (Expression Systems) supplemented with 10 mg/mL gentamicin sulfate (Thermo Fisher) at 27 °C and atmospheric CO₂.
Jurkat cell lines were cultured in RPMI 1640 supplemented with 10% FBS, 2 mM L-Glutamine, 50 U/mL Penicillin, 50 μg/mL Streptomycin, and 50 μM β-mercaptoethanol at 37 °C and 5% CO₂.
HEK293T cell line was cultured in DMEM supplemented with 10% FBS, 2 mM L-Glutamine, and 18 mM HEPES at 37 °C and 5% CO₂.

Zhao X., Kolawole E.M., Chan W., Feng Y., Yang X., Gee M., Jude K.M., Sibener L.V., Fordyce P.M., Germain R.N., Evavold B.D, & Garcia K.C. (2022). Tuning T cell receptor sensitivity through catch bond engineering. Science (New York, N.Y.), 376(6589), eabl5282.

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Culturing Sf9 and HEK293S GnTI- Cells

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The Sf9 insect cells were obtained from the ATCC (Cat# CRL-1711) and cultured at 27°C at 130 rpm in Erlenmeyer flasks with smooth bottoms with Sf900 III media (Thermo Fisher Scientific), in an InnovaR shaking incubator. HEK293S GnTI⁻ suspension cells were obtained from the ATCC (Cat# CRL-3022) and cultured at 37°C in baffle-bottomed Erlenmeyer flasks with 8% CO₂ at 130 rpm, in a Thermo Scientific Reach-In CO₂ Incubator. All cell lines used in this study are female in origin.

Noviello C.M., Gharpure A., Mukhtasimova N., Cabuco R., Baxter L., Borek D., Sine S.M, & Hibbs R.E. (2021). Structure and gating mechanism of the α7 nicotinic acetylcholine receptor. Cell, 184(8), 2121-2134.e13.

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Culturing Sf9 and HEK293S Cell Lines

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The Sf9 insect cells were obtained from the ATCC (Cat# CRL-1711) and cultured in an InnovaR shaking incubator at 27°C at 130 rpm with Sf900 III media (Thermo Fisher Scientific). HEK293S GnTI-suspension cells were obtained from the ATCC (Cat# CRL-3022) and cultured in a Thermo Scientific Reach-In CO₂ Incubator at 37°C with 8% CO2 at 130 rpm. All cell lines used in this study are female in origin.

Noviello C.M., Kreye J., Teng J., Prüss H, & Hibbs R.E. (2022). Structural mechanisms of GABAA receptor autoimmune encephalitis. Cell, 185(14), 2469-2477.e13.

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Purification of Hop1-Red1 Complex

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DNA sequences corresponding to Hop1 (Uniprot ID P20050) and Red1 (Uniprot ID P14291) were amplified from yeast genomic DNA (SK1 strain), and cloned into vectors from the InteBac suite 66 . Hop1 was cloned with a 3C cleavable N-terminal 2xStrep-II tag for insect cell expression (plasmid ID pWL661), and Red1 with a C-terminal MBP fusion, also for insect cell expression (plasmids ID pWL1157). Expression plasmids were used to generate bacmids via the EMBacY cell line 67 . Bacmids were used to transfect SF9 cells using FuGene HD (Promega). Baculovirus was generated through three rounds of amplification in SF9 cells grown in Sf-900 III media (ThermoFisher). For expression of the Hop1-Red1 complex, Hi5 cells were infected at a 1:100 (v/v ratio) amplified virus of both pWL661 and pWL1157 and the cells were cultured for 72 hours post infection. Cells were harvested, washed once in PBS and resuspended in lysis buffer (50 mM Na-HEPES pH 7.5, 300 mM NaCl, 10% Glycerol, 1 mM MgCl2 10 µM ZnSO4) the buffer was also supplemented with both cOmplete EDTA-free protease inhibitor (Roche) and Benzonase (Merck). Cells were lysed by sonication, cleared by ultracentrifugation, and passed over a Streptactin-XT column (IBA Lifesciences). Samples were eluted in lysis buffer supplemented with 50 mM biotin. Sample purity was analysed on SDS-PAGE stained with Coomassie Brilliant Blue.

Nivsarkar V., Chen L., Funk S.K., Weir J.R., & Vader G. (2022). Studying meiosis in mitosis: Activating the meiosis-specific Red1-Hop1-Mek1 complex in mitotic budding yeast cells.

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