Es qualified fbs

Manufactured by Thermo Fisher Scientific

Sourced in United States, France

The ES-qualified FBS is a high-quality fetal bovine serum (FBS) product that has been specifically qualified for use in embryonic stem (ES) cell culture applications. It is subjected to rigorous testing and quality control measures to ensure consistent performance and suitability for this specialized cell culture requirement.

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8 protocols using es qualified fbs

Cell lines and lentiviral transduction protocol

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Prostate cancer cell lines namely, DU145, VCaP and LNCaP were grown in the 10% FBS (Invitrogen, Carlsbad, CA) containing MEM, DMEM with Glutamax and RPMI1640 (Invitrogen) respectively, under 5% CO₂ in a cell culture incubator. Lentiviruses were generated by the University of Michigan Vector Core. DU145 and LNCaP cells were infected with lentiviruses expressing either scramble shRNA or EZH2, EED specific shRNA duplexes containing GFP reporter. Stable cell lines were generated by selection with 1 μg/ml puromycin and monitored with GFP fluorescence. Eed^−/− mouse stem cells were gift from Dr. Terry Magnuson and grown in MEMalpha (Invitrogen) with 20% ES qualified FBS (ATCC, Manassas, VA), 1mM Sodium Pyruvate (Invitrogen), 2mM L-Glutamine (Invitrogen), 1X NEAA (Invitrogen), 100μM β -Mercaptoethanol (Invitrogen), 1000unit/ml LIF (Millipore, Billerica, MA). XEN cells were grown in MEMalpha with 20% ES qualified FBS, 1mM Sodium Pyruvate, 2mM L-Glutamine, 1X NEAA (Invitrogen), 100μM β-Mercaptoethanol. A list of antibodies with dilutions used for immunoprecipitation (IP), immunoblot, ChIP and Immuno-Fluorescence (IF) is presented in Supplemental Table 3.

Cao Q., Wang X., Zhao M., Yang R., Malik R., Qiao Y., Poliakov A., Yocum A.K., Li Y., Chen W., Cao X., Jiang X., Dahiya A., Harris C., Feng F.Y., Kalantry S., Qin Z.S., Dhanasekaran S.M, & Chinnaiyan A.M. (2014). The Central Role of EED in the Orchestration of Polycomb Group Complexes. Nature communications, 5, 3127.

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Cell lines and lentiviral transduction protocol

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Embryoid Body Formation from ESCs

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For the formation of well-defined embryoid bodies (EBs),
1.8×10⁶ cells resuspended in 5 ml EB culture medium
(Knock-out DMEM (Gibco, 10829018) supplemented with 15% ES-qualified FBS (Gibco,
10439024), 1% GlutaMAX (Gibco, 35050061), 1% MEM Non-Essential Amino Acids
(Gibco, 11140050), 0.1 mM beta- mercaptoethanol (Gibco, 21985023), 10 μM
all-trans retinoic acid (R2625–50MG, Sigma-Aldrich)) and were seeded into
a well of AggreWell^™ 800 plate (34825, STEMCELL Technologies)
following pretreatment instructions. Every two days, removed and replaced with
3.75 ml of fresh EB culture medium by slowly pipetting down the wall of the
well.

Zhu Z., Chen X., Guo A., Manzano T., Walsh P.J., Wills K.M., Halliburton R., Radko-Juettner S., Carter R.D., Partridge J.F., Green D.R., Zhang J, & Roberts C.W. (2023). Mitotic bookmarking by SWI/SNF subunits. Nature, 618(7963), 180-187.

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Mouse Embryonic Stem Cell Culture

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Mouse embryonic stem cells (mESC) lines used in this study included Dnmt1/3a/3b triple knockout [30 (link)] on a J1 (129S4/SvJae) background, as well as WT J1 and V6.5 (C57BL/6 × 129S4/SvJae) lines. Cells were cultured in Knockout-DMEM (Gibco) with 15% ES qualified FBS (Gibco), ESGRO mouse LIF 1000U/mL (Millipore), 1X PenStrep (Invitrogen), 1X Glutamax (Invitrogen), 55 µM b-mercaptoethanol (Invitrogen), 1X non-essential amino acids (Invitrogen) and 1X Primocin (Invivogen). Cells were maintained at 37 °C and 5% CO₂ on dishes coated with 0.1% gelatin (Millipore) unless otherwise stated. Experiments in which mESCs were plated on a layer of mitomycin C-treated mouse embryonic fibroblasts (MEFs) (ThermoFisher Scientific) are explicitly stated.

Azevedo Portilho N., Saini D., Hossain I., Sirois J., Moraes C, & Pastor W.A. (2021). The DNMT1 inhibitor GSK-3484862 mediates global demethylation in murine embryonic stem cells. Epigenetics & Chromatin, 14, 56.

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Lentiviral shRNA Knockdown of Sam68 in HEK293 Cells

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HEK293 and HEK293T cells were maintained in DMEM (Gibco-Invitrogen, Grand Island, NY, #11960-51 +4.5g/L D-Glucose, - l-glutamine, -sodium pyruvate) supplemented with 20mM l-glutamine (Gibco), 1kU/ml penicillin/streptomycin (HyClone, Logan UT), 1x non-essential amino acids (Gibco), and 10% ES-qualified FBS (Gibco). Doxycycline inducible lentiviruses (Thermo Fisher, Pittsburgh, PA) encoding shRNAs specific for different regions of Sam68 were produced in HEK293T cells by CaPO4 co-transfection of lentiviral backbone, packaging plasmid p8.74 and a plasmid encoding VSV-G protein⁴⁰. Lentivirus were used to infect naïve HEK293 cells and stably transduced cell lines were selected by serial single cell density plating and puromycin selection according to manufacturers recommendations, and confirmed by visual detection of RFP co-expression upon doxycycline induction. For knockdown experiments, control and knockdown samples were plated in parallel, and lentiviral shRNA expression was induced for 24 hrs by addition of 100 ng/ml doxycycline to culture medium of knockdown samples. Protein and RNA samples were simultaneously extracted using RNeasy columns (Qiagen), and proteins further purified and desalted by acetone precipitation. cDNA synthesis and qPCR for siRNA experiments were as described as above.

Rivers E.R., Horton A.J., Hawk A.F., Favre E.G., Senf K.M., Nietert P.J., Chang E.Y., Foley A.C., Robinson C.J, & Lee K.H. (2014). Placental Nkx2.5 and Target Gene Expression in Early-Onset and Severe Preeclampsia. Hypertension in pregnancy, 33(4), 412-426.

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Culturing Mouse Embryonic Stem Cells

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ESCs constitutively expressing histone H2B-GFP were provided by Dr Kat Hadjantonakis [37 (link)]. Mitomycin-inactivated mouse embryonic fibroblasts, which served as the feeder layer for ESCs, were plated on dishes coated with 1% gelatine (Sigma-Aldrich) in DMEM supplemented with 10% FBS and 1% AB. Twenty-four hours later ESCs were seeded onto the inactivated mouse embryonic fibroblasts and cultured in knockout DMEM (Life Technologies) supplemented with 15% ES-qualified FBS (Life Technologies), 0.1 mM nonessential amino acids (Sigma-Aldrich), 200 mM l-glutamine (Life Technologies), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), 1% AB, and 500 U/ml leukaemia inhibitory factor (Chemicon, Billerica, MA, United States). Prior to transfection, ESCs were separated from mouse embryonic fibroblasts by the preplating technique and cultured on dishes coated with Matrigel Matrix Growth Factor Reduced (1 mg/ml DMEM; BD Biosciences, Becton-Dickinson, San Jose, CA, United States). The morphology of cells was analysed using a Nikon Eclipse TE200 microscope with Hoffman contrast. The cells were subjected either to Sdf-1 treatment, transfection with siRNA complementary to mRNA encoding CXCR4 or CXCR7, quantitative RT-PCR, immunolocalisation, or western blotting or used for co-culture experiments.

Brzoska E., Kowalski K., Markowska-Zagrajek A., Kowalewska M., Archacki R., Plaskota I., Stremińska W., Jańczyk-Ilach K, & Ciemerych M.A. (2015). Sdf-1 (CXCL12) induces CD9 expression in stem cells engaged in muscle regeneration. Stem Cell Research & Therapy, 6(1), 46.

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Fibroblast Reprogramming into iPSCs

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One control and one GRN (GRN #3) fibroblast line were reprogrammed into iPSCs using the Cytotune 2.0 Kit (Life Technologies) per the manufacturer’s protocol. In brief, early passage fibroblast (P < 10) were grown to approximately 50–80 % confluency in fibroblast medium consisting of 10 % ES-qualified FBS (Life Technologies, 0.1 mM NEAA, 55 μM β-mercaptoethanol, high glucose DMEM (Life Technologies). On Day 0, fibroblasts were transduced with three Sendai viruses encoding Klf4–Oct3/4–Sox2 (KOS), hc-Myc, and hKlf4, each at an MOI of 5). Cells were fed with fibroblast medium every other day for 7 days. On day 7, cells were passaged onto vitronectin (Life Technologies) coated dishes at a density of 2.5 × 10⁵ - 5.0 × 10⁵ cells/well. Beginning on day 8, cells were fed every day in Essential 8 medium (Life Technologies). Resulting iPS colonies were manually picked and transferred to a dish coated with either vitronectin or Matrigel (BD). iPSCs were maintained on Matrigel coated dishes and mTesR1 medium (Stem Cell Technologies) and passaged every 5–7 days.

Holler C.J., Taylor G., McEachin Z.T., Deng Q., Watkins W.J., Hudson K., Easley C.A., Hu W.T., Hales C.M., Rossoll W., Bassell G.J, & Kukar T. (2016). Trehalose upregulates progranulin expression in human and mouse models of GRN haploinsufficiency: a novel therapeutic lead to treat frontotemporal dementia. Molecular Neurodegeneration, 11, 46.

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Isolation and Culture of Chick Primordial Germ Cells

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Circulating PGCs were collected from male WL embryonic blood at Hamburger and Hamilton’s developmental stage
14 (HH14) [25 (link)]. Embryos were sexed by PCR using W chromosome-specific
primers [26 (link)], and whole blood cells containing PGCs were grown on a BRL
feeder layer. The PGC culture medium was prepared as described in previous studies with some modifications
[1 (link), 5 ]. The PGC culture medium
contained KnockOut DMEM (KO-DMEM; Life Technologies), 50% BRL-conditioned KO-DMEM, 7.5% ES qualified FBS (Life
Technologies), 2.5% chicken serum (Biowest, Nuaillé, France), 2 mM GlutaMAX (Life Technologies), 1 mM sodium
pyruvate (Life Technologies), 1 × nonessential amino acids (Life Technologies), 1 × nucleosides (Millipore,
Billerica, MA, USA), 0.5 mM monothioglycerol (Wako Pure Chemical Industries, Osaka, Japan) and 5 ng/ml human
FGF2 (Wako Pure Chemical Industries). Half the volume of the culture medium was replaced every 2 days during
the culture period for PGCs, and the cultured PGCs were passaged until 90% confluence. Several male PGCs were
proliferated to large numbers, and could be maintained for more than 100 days. The established PGC lines were
used for subsequent assays.

MIYAHARA D., OISHI I., MAKINO R., KURUMISAWA N., NAKAYA R., ONO T., KAGAMI H, & TAGAMI T. (2015). Chicken stem cell factor enhances primordial germ cell proliferation cooperatively with fibroblast growth factor 2. The Journal of Reproduction and Development, 62(2), 143-149.

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