Essential 6

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Essential 6 is a compact, multi-functional laboratory instrument designed for essential analytical tasks. It provides accurate and reliable results for a range of common laboratory applications.

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Lab products found in correlation

Essential 8, by Thermo Fisher Scientific (4 mentions) Xav939, by Bio-Techne (3 mentions) Sb431542, by Fujifilm (2 mentions) Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (2 mentions) Dmem f12, by Thermo Fisher Scientific (2 mentions) Mem neaa, by Thermo Fisher Scientific (2 mentions) Essential 8 medium, by Thermo Fisher Scientific (2 mentions) Sb431542, by STEMCELL (2 mentions) Chir99021, by Bio-Techne (2 mentions) Accutase, by R&D Systems (2 mentions) Accutase, by Innovative Cell Technologies (2 mentions) Y 27632, by R&D Systems (2 mentions) Bone morphogenetic protein 4 (bmp4), by R&D Systems (1 mentions) Vascular endothelial growth factor (vegf), by R&D Systems (1 mentions) Stem cell factor (scf), by R&D Systems (1 mentions) Flt3 ligand, by R&D Systems (1 mentions) Stemline 2 medium, by Merck Group (1 mentions) Chir99021, by Fujifilm (1 mentions) Sb431542, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Essential 6 medium (48 citations) Essential 6 media (6 citations) Essential 6TM medium (5 citations) Essential 6 (E6, (3 citations)

13 protocols using essential 6

TGF-β Removal Enhances Reprogramming

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Reprogramming was performed as described for protocol#1, but without TGF‐β from day 8 until colony isolation. On day 8 after the transduction medium was switched from DMEM + 10% FCS to Essential 6 (Thermo Fisher Scientific) + 100 ng/ml FGF2, colonies were switched back to E8 following isolation. The removal of TGF‐β prevented ACL cells from rolling up into a single sheet and gave developing PSC colonies space to grow.
Reprogramming efficiency was calculated by dividing the number of identified pluripotent colonies by the number of cells transduced. Not all identified pluripotent colonies were taken forward into established lines, but all were still taken into account when calculating efficiency.

Woods S., Bates N., Dunn S.L., Serracino‐Inglott F., Hardingham T.E, & Kimber S.J. (2019). Generation of Human‐Induced Pluripotent Stem Cells From Anterior Cruciate Ligament. Journal of Orthopaedic Research, 38(1), 92-104.

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Hematopoietic Cell Induction from hPSCs

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We modified our previously reported protocol^{23 (link)} to induce hematopoietic cells from hPSCs. The medium of PSC colonies that grew to a diameter of 750 to 1000 μm was replaced with Essential 8 (catalog #A1517001; Thermo Fisher Scientific, Waltham, MA) containing 2 μM of GSK-3 inhibitor CHIR-99021 (catalog #038-23101; Wako, Osaka, Japan), 80 ng/mL of BMP4 (catalog #314-BP-010; R&D Systems, Minneapolis, MN), and 80 ng/mL of vascular endothelial growth factor (catalog #293-VE-010; R&D Systems) on days 0 to 2 to induce cells equivalent to those of the primitive streak. Next, the medium was replaced with Essential 6 (catalog #A1516401; Thermo Fisher Scientific) containing 2 μM of ALK5 inhibitor SB431542 (catalog #031-24291; Wako), 50 ng/mL of stem cell factor (catalog #255-SC-010; R&D Systems), and 80 ng/mL of vascular endothelial growth factor on days 2 to 4 to induce hemangioblast-like bipotent progenitor cells. To induce hematopoietic progenitor cells,^{24 (link)} the medium was replaced with Stemline-II medium (catalog #S0192; Sigma-Aldrich, St Louis, MO) containing 50 ng/mL of stem cell factor, 10 μM of thrombopoietin, and 50 ng/mL of Flt-3 ligand (catalog #308-FK-005; R&D Systems) on days 4 to 15.

Yoshida M., Nakabayashi K., Yang W., Sato-Otsubo A., Tsujimoto S.I., Ogata-Kawata H., Kawai T., Ishiwata K., Sakamoto M., Okamura K., Yoshida K., Shirai R., Osumi T., Moriyama T., Nishii R., Takahashi H., Kiyotani C., Shioda Y., Terashima K., Ishimaru S., Yuza Y., Takagi M., Arakawa Y., Kinoshita A., Hino M., Imamura T., Hasegawa D., Nakazawa Y., Okuya M., Kakuda H., Takasugi N., Inoue A., Ohki K., Yoshioka T., Ito S., Tomizawa D., Koh K., Matsumoto K., Sanada M., Kiyokawa N., Ohara A., Ogawa S., Manabe A., Niwa A., Hata K., Yang J.J, & Kato M. (2021). NUDT15 variants confer high incidence of second malignancies in children with acute lymphoblastic leukemia. Blood Advances, 5(23), 5420-5428.

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Reprogramming Protocols for iPS Cell Differentiation

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The two reprogramming protocols used for iPS cell differentiation are depicted in Figure 2. FGF2 was purchased from PeproTech (Rocky Hill, NJ, USA). SB431542 was purchased from FUJIFILM Wako Pure Chemical Corporation. In protocol 1, cell culture was performed using TGF-β-containing Essential 8 (Thermo Fisher Scientific^®, Waltham, MA, USA) immediately after gene transfection. Penicillin-streptomycin (100 IU/mL penicillin and 100 µg/mL streptomycin) was added from day 2 post-transfection. From then onward, the medium was changed every other day. In protocol 2, from day 1 to day 5 post-transfection, a culture medium in which FGF2 (10 ng/mL) and SB431542 (5 µM) was added to Essential 6 (Thermo Fisher Scientific^®) without TGF-β was used. Penicillin-streptomycin (100 IU/mL penicillin and 100 µg/mL streptomycin) was added from day 2. After day 6, the culture medium was switched to Essential 8.

Tanaka N., Kato H., Tsuda H., Sato Y., Muramatsu T., Iguchi A., Nakajima H., Yoshitake A, & Senbonmatsu T. (2020). Development of a High-Efficacy Reprogramming Method for Generating Human Induced Pluripotent Stem (iPS) Cells from Pathologic and Senescent Somatic Cells. International Journal of Molecular Sciences, 21(18), 6764.

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Forebrain Organoid Differentiation from H9 hESCs

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To induce forebrain organoid differentiation, H9 hESC line (WA-09, WiCell) hPSCs were dissociated to single cells using EDTA, and 9000 cells were reaggregated using low-adhesion V-bottom 96 well plates (Wako) in Essential8 Medium (Fisher Scientific) with 10 μM Y-27632 (Tocris Biosciences). After 24 h (day 0), medium was changed to Essential6 (Fischer Scientific) supplemented with 10 μM SB431542 (Tocris Biosciences), 500 nM LDN193189 (Stem Cell Technologies), and 2 μM XAV939 (Tocris Biosciences) until day 4. From day 4 to day 18, XAV939 was removed. Medium was changed every other day. From Day 18, organoids were maintained in organoid differentiation medium (50% DMEM F-12 (Fisher Scientific), 50% mL Neurobasal (Fisher Scientific), 0.5xN2 supplement (Stem Cell Technologies), 0.025% insulin (Sigma), 5 mM L-Glutamine (1x, Fischer Scientific), 0.7 mM MEM-NEAA (1x, Fischer Scientific), 50 U/mL Penicillin-Streptomycin (1x, Fischer Scientific), 55 μM 2-mercaptoethanol (1x, Fischer Scientific), 1xB27 supplement without Vitamin A (Fischer Scientific).

Zhou T., Tan L., Cederquist G.Y., Fan Y., Hartley B.J., Mukherjee S., Tomishima M., Brennand K.J., Zhang Q., Schwartz R.E., Evans T., Studer L, & Chen S. (2017). High Content Screening in hESC-Neural Progenitors Identifies Drug Candidates that Inhibit Zika Virus Infection in Fetal-like Organoids and Adult Brain. Cell stem cell, 21(2), 274-283.e5.

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Quantification of Residual bFGF by ELISA

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Residual bFGF in the bFGF solutions with the P10 sheet after a specific adsorption period was quantified by ELISA. After various adsorption periods of up to 48 h, 10 μL aliquots were sampled from the bFGF solutions and frozen at −80 °C before ELISA. The as-prepared bFGF solutions were also frozen at −80 °C before ELISA. The sampled bFGF solutions were diluted with a serum-free acellular culture medium (Essential 6; Thermo Fisher Scientific, USA) and assayed for bFGF using a human FGF basic Quantikine® ELISA kit (R&D Systems, Inc., USA). The Essential 6 medium is a chemically defined basic medium commonly used for stem cell culture (components based on Chen's “E8” medium^{28 (link)}), and will be used in the assays in Sections 2.6, 2.7, and 2.8. Standard bFGF solutions were prepared by diluting the bFGF source with the same medium. The bFGF residual rate was determined as a percentage of the residual bFGF concentration (C) in the bFGF solutions for a specific adsorption period among the initial bFGF concentrations (C₀) in the as-prepared bFGF solutions as follows:
As a control, the same bFGF solution without the P10 sheet was assayed in the same manner as described above. The amount of bFGF adsorbed onto each sheet was estimated by subtracting the residual bFGF in the bFGF solution from that in the control solution without sheets.

Oyane A., Araki H., Nakamura M., Aiki Y., Higuchi K., Pyatenko A., Adachi M, & Ito Y. (2019). Controlled release of basic fibroblast growth factor from a water-floatable polyethylene nonwoven fabric sheet for maintenance culture of iPSCs. RSC Advances, 10(1), 95-104.

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Neural Induction of iPSCs using CRISPR

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Neural induction of human iPSCs was performed using the dual SMAD inhibition paradigm.⁶ (link)^,²¹ (link) Engineered CRISPRi-iPSCs⁶² (link) were grown in Essential 8 (Thermo Fisher Scientific) media on Matrigel (Corning) to 80% confluency. Cells were rinsed with DPBS and dissociated with Accutase (StemPro). After centrifugation at 300 x g for 3 min and resuspension in Essential 8 media with 10 μM Y-27632 ROCK inhibitor (Selleckchem), cells were replated at a density of 250,000 cells/cm² overnight at 37°C. The next day (D0), cells were rinsed with DPBS and changed to neural induction media, which consisted of Essential 6 media (Thermo Fisher Scientific) with freshly-added SMAD inhibitors 500 nM LDN193189 (Selleckchem) and 10 μM SB431542 (Selleckchem). Media was replaced every 2 days until the endpoint of interest, as described.²¹ (link)

Wu D., Poddar A., Ninou E., Hwang E., Cole M.A., Liu S.J., Horlbeck M.A., Chen J., Replogle J.M., Carosso G.A., Eng N.W., Chang J., Shen Y., Weissman J.S, & Lim D.A. (2022). Dual genome-wide coding and lncRNA screens in neural induction of induced pluripotent stem cells. Cell Genomics, 2(11), 100177.

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Dermal Fibroblast iPSC-Derived Retinal Differentiation

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A skin biopsy was obtained from a 12‐year‐old female patient carrying a compound heterozygous variant c.2548G>A p.(Gly850Ser) and c.4006‐10A>G in the CRB1 gene and a healthy control (WT). Human dermal fibroblasts were reprogrammed using integration‐free episomal vectors: pCXLE‐hOCT3/4‐shp53‐F (Addgene, Watertown, MA, USA; Catalogue No. 27077), pCXLE‐Hsk (Addgene; Catalogue No. 27078), pCXLE‐Hul (Addgene; Catalogue No. 27080), and pCXWB‐EBNA1 (Addgene; Catalogue No. 37624) as previously described [48 (link)]. hiPSC were differentiated towards a retinal lineage [49 (link)]; briefly, hiPSCs were grown to 80–90% confluency (day 0), mTesR™ Plus was replaced by Essential 6™ (Thermo Fisher Scientific; Catalogue No. A1516401). From day 2 to day 28, differentiating cells were subjected to a neural induction medium Essential 6™, 1% N2 supplement (Stemcell Technologies, Cambridge, UK; Catalogue No. 17502048) and 0.1% Pen/Strep. By 3–4 weeks, neural retina‐like structures emerged from the cell layer and were manually dissected at day 28 using a 21G needle and further cultured until day 35 in maturation media [DMEM/F12 (Thermo Fisher Scientific; Catalogue No. 21331020), 1% MEM NEAA (Thermo Fisher Scientific; Catalogue No. 10370021), 2% B27 supplement (Thermo Fisher Scientific; Catalogue No. 12587010), 10 ng/ml FGF2 (Thermo Fisher Scientific; Catalogue No. 100‐18B), and 0.1% Pen/Strep].

Owen N., Toms M., Tian Y., Toualbi L., Richardson R., Young R., Tracey‐White D., Dhami P., Beck S, & Moosajee M. (2023). Loss of the crumbs cell polarity complex disrupts epigenetic transcriptional control and cell cycle progression in the developing retina. The Journal of Pathology, 259(4), 441-454.

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Characterizing bFGF Release from Cryopreserved Membranes

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The F2, F4, F8, and F12 membranes, after lyophilization and subsequent cryopreservation (F4 only), were subjected to a preliminary bFGF-release test using a chemically defined serum-free acellular medium (Essential 6, Thermo Fisher Scientific, Waltham, MA, USA). For comparison, the as-prepared wet F12 membrane (before lyophilization) was also tested.
Each membrane was placed (plasma-treated surface down) over 2 mL of medium in a 24-well culture plate. As a control, medium supplemented with 10 ng/mL bFGF (in place of the membrane) was also tested. After incubation at 37 °C in a humidified 5% CO₂ atmosphere for various periods up to 72 h, 150-µL aliquots were sampled from the medium and frozen at −80 °C before use in ELISA. The resulting medium loss was compensated by adding identical amounts of the medium. In ELISA, the sampled solutions were diluted with medium and assayed for bFGF using a human FGF basic Quantikine^® ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions. Standard bFGF solutions were prepared by diluting the bFGF source with the same medium.

Oyane A., Araki H., Nakamura M., Aiki Y, & Ito Y. (2021). Storable bFGF-Releasing Membrane Allowing Continuous Human iPSC Culture. Materials, 14(3), 651.

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Directed Differentiation of iPSCs

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iPSC were cultured on matrigel and Essential 8 (Gibco) until they reached 80% confluence. Then, cells were detached using 0.5 mM EDTA in PBS and the colonies were disassociated into small clusters of cells. The cell clusters were transferred into ultralow attachment plates (Corning, 3471) and cultured on Essential 6 (Gibco). Media was supplemented with 10 μM ROCK inhibitor (Y-27632 2HCl, Seleckchem, #S1049) only for the first two days, and then changed regularly every two days. After two weeks, the spheroids were plated on gelatin-coated (0.1%, Sigma, G1890) dishes with cover glasses, and cultured for an additional week. Then, samples were immunocytochemically stained as described below.

Kenvin S., Torregrosa-Muñumer R., Reidelbach M., Pennonen J., Turkia J.J., Rannila E., Kvist J., Sainio M.T., Huber N., Herukka S.K., Haapasalo A., Auranen M., Trokovic R., Sharma V., Ylikallio E, & Tyynismaa H. (2021). Threshold of heteroplasmic truncating MT-ATP6 mutation in reprogramming, Notch hyperactivation and motor neuron metabolism. Human Molecular Genetics, 31(6), 958-974.

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Directed Differentiation of iPSCs

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EB formation was performed as described previously, with minor modifications (22 (link)). Dissociated iPSCs were seeded onto a StemFIT 3D culture dish (MicroFIT) and cultured in Essential 6 (Gibco) with 100 U/ml penicillin, 100 mg/ml streptomycin (GE Life Science), and 10 μM fasudil (Adooq) for two days. Aggregated cells were transferred to Petri dishes and cultured for six days using the same medium.

Lee Y., Han J., Hwang S.B., Kang S.S., Son H.B., Jin C., Kim J.E., Lee B.H, & Kang E. (2023). Selection of iPSCs without mtDNA deletion for autologous cell therapy in a patient with Pearson syndrome. BMB Reports, 56(8), 463-468.

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