Express five sfm media

Manufactured by Thermo Fisher Scientific

Express Five SFM media is a serum-free, chemically defined culture medium designed for the expansion of human mesenchymal stem cells (hMSCs) in suspension culture. It provides a high-quality, consistent, and standardized environment for the growth and maintenance of hMSCs.

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8 protocols using express five sfm media

Cultivation and RNA Extraction Protocols for Diverse Cell Lines

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Human HEK293 epithelial cells (ATCC CRL1573) were cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin solution. Murine NIH3T3 fibroblasts (ATCC CRL1658) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% donor calf serum (DCS) and 1% penicillin–streptomycin solution. Both mammalian cell lines were cultured at 37°C in a humidified incubator with 5% CO₂. Drosophila melanogaster Schneider 2 (S2) cells (Thermo Fisher Scientific R69007) were cultured in ExpressFive SFM media (Thermo Fisher Scientific), supplemented with 10% heat-inactivated FBS and 20 mM L-Glutamine, at 28°C.
S. cerevisiae USY006 was grown in YPD liquid or plates at 30°C. RNA was isolated using the standard hot phenol method (Rissland and Norbury 2009 (link)). Synchronized populations of L1 C. elegans were grown on NGM plates for 60 h until adult staged. Worms were washed off of plates with PBS buffer and resuspended in ultrapure water. RNA was extracted using TRI reagent (Molecular Research Center), according to manufacturer's instructions.

Lugowski A., Nicholson B, & Rissland O.S. (2018). DRUID: a pipeline for transcriptome-wide measurements of mRNA stability. RNA, 24(5), 623-632.

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S2 Cell Culture Conditions

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S2 cells (gift from the Butros Laboratory, Heidelberg) were cultured in Express Five SFM media (Thermo Fisher) supplemented with 10% (v/v) Glutamax (Thermo Fisher). Cultures were maintained adherent or in shaking incubators at 27 °C at a speed of 80 rpm. Cells were kept at a density of 1–16 million/mL.

Bhardwaj V., Semplicio G., Erdogdu N.U., Manke T, & Akhtar A. (2019). MAPCap allows high-resolution detection and differential expression analysis of transcription start sites. Nature Communications, 10, 3219.

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Culturing Drosophila S2 Cells

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Drosophila melanogaster Schneider 2 (S2) cells (Cat #R69007, Thermo Fisher Scientific) were cultured in ExpressFive SFM media (Thermo Fisher Scientific) supplemented with 10% heat-inactivated FBS (Wisent) and 20mM l-Glutamine (Thermo Fisher Scientific) at 28°C.

Narula A., Ellis J., Taliaferro J.M, & Rissland O.S. (2019). Coding regions affect mRNA stability in human cells. RNA, 25(12), 1751-1764.

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Culturing HEK293T, SF21, and Hi5 Cells

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HEK293T cells (ATCC) were propagated in DMEM medium (GIBCO) supplemented with 10% FBS (Thermo Fisher) and Pen Strep (GIBCO).
SF21 Insect cells were cultured in SF-900™ II SFM media (GIBCO). Hi5 cells were grown in Express Five™ SFM media (GIBCO) supplemented with L-Glutamine (GIBCO).

Pfleiderer M.M, & Galej W.P. (2021). Structure of the catalytic core of the Integrator complex. Molecular Cell, 81(6), 1246-1259.e8.

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Bacterial and Insect Cell Culture

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Bacterial strains (Escherichia coli DH5α, BL21 pRil, DH10MultiBac) were grown in Luria Bertani (LB) medium in the presence of appropriate antibiotics.
Spodoptera frugiperda strain Sf21 insect cells were grown in suspension at 27°C in Sf-900 III SFM (1x) Serum Free Medium (Gibco). High Five cells were grown in suspension at 27°C in ExpressFive SFM media (Gibco) supplemented with 16 mM L-glutamine (Gibco). Absence of mycoplasma in insect cell cultures was confirmed regularly.

Roostalu J., Thomas C., Cade N.I., Kunzelmann S., Taylor I.A, & Surrey T. (2020). The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability. eLife, 9, e51992.

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Cell Culture Methods for Diverse Cell Lines

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HEK-293FT and HeLa cells were grown in DMEM (Gibco) supplemented with 2 mM Glutamine, 10,000 units/mL Penicillin/Streptomycin, and 10 % FBS at 37°C and 5% CO₂. Cultures were passaged every 5–6 days by adding TrypLE (Gibco) and re-plating at 1:10 ratio.
Drosophila melanogaster S2 cells were maintained in Express Five SFM media (Gibco) at room temperature in the dark and passaged once a week.
Sptlc2^tm2.1Jia (Li et al., 2009 (link)), Prox1^CreERT2 (Bazigou et al., 2011 (link)) and Cdh5^CreERT2 (Wang et al., 2010 (link)) mice were obtained from the Cancer Research Consortium (UK) under an MTA. Nr2f2^f/f mice were re-derived from frozen sperm (B6;129S7-Nr2f2^tm2Tsa/Mmmh) received from the Mutant Mouse Regional Resource Center (MMRRC) at the University of Missouri. Mice were maintained in the Yale Animal Resources TAC facility and used under the approved IACUC protocols.
H9 human embryonic stem cells (WA09, NIH approval number NIHhESC-10–0062) were grown in feeder-free conditions in plates coated with Matrigel (BD Biosciences) in E8 media (Thermo Fisher) or mTeSR1 media (Stem Cell Technologies) at 37°C and 5% CO₂. Cultures were passaged every 4 days by adding 1mg/mL Dispase (Stem Cell Technologies) for 5 minutes at 37°C and re-plating at a 1:10 ratio.

Wang T., Wang Z., de Fabritus L., Tao J., Saied E.M., Lee H.J., Ramazanov B.R., Jackson B., Burkhardt D., Parker M., Gleinich A.S., Wang Z., Seo D.E., Zhou T., Xu S., Alecu I., Azadi P., Arenz C., Hornemann T., Krishnaswamy S., van de Pavert S.A., Kaech S.M., Ivanova N.B, & Santori F.R. (2021). 1-deoxysphingolipids bind to COUP-TF to modulate lymphatic and cardiac cell development. Developmental cell, 56(22), 3128-3145.e15.

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Baculovirus Rescue and Influenza Virus Growth

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Sf9 cells (CRL-1711, ATCC) for baculovirus rescue were grown in Trichoplusia ni medium-formulation Hink insect cell medium (TNM-FH, Gemini Bioproducts) supplemented with 10% fetal bovine serum (FBS; Sigma) and penicillin (100 U/ml)-streptomycin (100 μg/ml) solution (Gibco). BTI-TN-5B1–4 (High Five, ATCC) cells for protein expression were grown in serum-free Express Five SFM media (Gibco) supplemented with penicillin (100 U/ml)-streptomycin (100 μg/ml) solution. Madin Darby canine kidney (MDCK, ATCC) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% FBS and penicillin (100 U/ml)-streptomycin (100 μg/ml) solution. B/Malaysia/2506/04 virus was grown in 10-day-old embryonated chicken eggs (Charles River) for 72 hours at 33°C. Eggs were then cooled overnight at 4°C before harvesting the allantoic fluid. Harvested allantoic fluid was centrifuged at 4,000 g for 10 minutes at 4°C to pellet debris. Viruses were then aliquoted and stored at −80°C prior to determining stock titers via plaque assay.

McMahon M., Tan J., O’Dell G., Roubidoux E.K., Strohmeier S, & Krammer F. (2022). Immunity induced by vaccination with recombinant influenza B virus neuraminidase protein breaks viral transmission chains in guinea pigs in an exposure intensity-dependent manner. bioRxiv.

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Propagation and Maintenance of HEK-293T and Drosophila S2 Cells

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HEK-293T (human embryonic kidney cells expressing simian virus 40T antigen; ATCC CRL-3216) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS). Cells were maintained at 37° C in a humidified incubator with 5% CO2. Drosophila Schneider 2 (S2) cell line (Invitrogen) were grown in synthetic Express Five SFM media (Gibco) supplemented with 20 mM L-Glutamine and 50 U/mL of penicillin and 50 μg/mL of streptomycin. Cells were maintained at 28° C.
VSVΔG-eGFP is a recombinant VSV which was derived from a full-length cDNA clone of the VSV genome (Indiana serotype) in which the coding region of the G protein was replaced by a modified version of the GFP gene and pseudotyped with the VSV G protein [34 (link),46 (link),47 (link)]. VSVΔG-eGFP is propagated on HEK-293T cells that have been previously transfected with pCAGGS-VSV G.

Belot L., Ouldali M., Roche S., Legrand P., Gaudin Y, & Albertini A.A. (2020). Crystal structure of Mokola virus glycoprotein in its post-fusion conformation. PLoS Pathogens, 16(3), e1008383.

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