Essential 8 flex medium

Manufactured by Thermo Fisher Scientific

Sourced in United States

Essential 8 Flex medium is a cell culture medium formulated for the maintenance and expansion of human pluripotent stem cells. It is designed to support the growth and self-renewal of these cells while maintaining their undifferentiated state.

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52 protocols using essential 8 flex medium

iPSC Multilineage Differentiation Analysis

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iPSCs were subjected to spontaneous differentiation mediated by the formation of embryoid bodies (EBs) and subsequently analyzed for three-lineage differentiation using the ScoreCard methodology (Thermo Fisher Scientific). On the day of EB formation, 60 to 80% confluent iPSCs were washed with PBS and incubated with 0.5 mM EDTA (Invitrogen) for 1 to 3 min to dissociate the colonies. Cells were harvested in Essential 8 flex medium (Gibco) and counted before gently spinning down at 300g for 5 min. Cells were plated in a 24-well Corning Ultra-Low Attachment Surface plates (Corning) at a density of 1.5 × 10⁶ cells per well. The plates were incubated overnight at 37°C. The following day, half of the Essential 8 flex medium was replaced with Essential 6 medium (Gibco). For the spontaneous differentiation, EBs were kept for 14 days in E6 medium, which was changed every 2 days. After 14 days in culture, EBs were collected for RNA extraction with the GenElute Mammalian Total RNA Kit (Sigma-Aldrich). Complementary DNA (cDNA) synthesis was performed with SuperScript III (Thermo Fisher Scientific), and qPCR was performed according to the manufacturer’s protocol with TaqMan hPSC Scorecard assay (Thermo Fisher Scientific). Data were analyzed with the online Scorecard software (Thermo Fisher Scientific).

Fumagalli L., Young F.L., Boeynaems S., De Decker M., Mehta A.R., Swijsen A., Fazal R., Guo W., Moisse M., Beckers J., Dedeene L., Selvaraj B.T., Vandoorne T., Madan V., van Blitterswijk M., Raitcheva D., McCampbell A., Poesen K., Gitler A.D., Koch P., Berghe P.V., Thal D.R., Verfaillie C., Chandran S., Van Den Bosch L., Bullock S.L, & Van Damme P. (2021). C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility. Science Advances, 7(15), eabg3013.

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Cardiac Organoid Differentiation from hiPSCs

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Human Ventricular Cardiac Fibroblasts (NHCF-V) were purchased from Lonza (CC-2904) and cultured under manufacturer indications. Feeder-free hiPSCs (1390C1) were maintained in StemFit^® AK02N medium (Reprocell) on iMatrix-511 (nippi) coated plates. Human heart organoids were differentiated from PSCs by using a published method. Briefly, iPSCs were dissociated with Accumax and resuspended in Essential 8 Flex medium (Gibco) containing 10 μM ROCK inhibitor Y-27632. To generate EBs, 10,000 cells were seeded at a final volume of 100 μl per well in round bottom low-attachment HEMA-coated 96-well plates on day -2. Fresh Essential 8 Flex medium was added the next day. On day 0, Essential 8 Flex medium was removed, and differentiation was performed in RPMI 1640/B-27, minus insulin (Gibco) containing CHIR99021 (4 μM), BMP4 (1.25 ng/ml) and ActivinA (1 ng/ml). On day 1, the medium was replaced with fresh RPMI 1640/B-27, minus insulin. On day 2, the medium was changed to RPMI 1640/B-27, minus insulin containing Wnt-C59 (2 μM). On day 4, the medium was replaced with fresh RPMI 1640/B-27, minus insulin. On day 6, the medium was replaced with fresh RPMI 1640/B-27 (Gibco). On day 7, organoids were treated with 2 μM CHIR99021 in RPMI 1640/B-27 for 1 h. From day 7 onwards, the medium was changed every other day until day 15.

Tian Y., Tsujisaka Y., Li V.Y., Tani K., Lucena-Cacace A, & Yoshida Y. (2022). Immunosuppressants Tacrolimus and Sirolimus revert the cardiac antifibrotic properties of p38-MAPK inhibition in 3D-multicellular human iPSC-heart organoids. Frontiers in Cell and Developmental Biology, 10, 1001453.

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Generation of hiPSC-derived Holograms

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The stem cell line used in this study was a wild-type C human induced pluripotent stem cell (WTC-hiPSC) line with genetically engineered GCaMP6f (Chen et al., 2013 (link); Huebsch et al., 2015 ). hiPSCs were cultured in 6-well plates coated with growth factor reduced Matrigel (Corning) and maintained in Essential 8 Flex medium (E8, Gibco) at 37 °C in a humidified incubator supplied with 5% CO₂. After reaching 60–80% confluency, hiPSC cultures were passaged into new 6-well plates or collected to fabricate hHOs. After at least three passages post-thawing, hiPSC cultures were ready for hHOs fabrication.

Ming Y., Hao S., Wang F., Lewis-Israeli Y.R., Volmert B.D., Xu Z., Goestenkors A., Aguirre A, & Zhou C. (2022). Longitudinal morphological and functional characterization of human heart organoids using optical coherence tomography. Biosensors & bioelectronics, 207, 114136.

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Generation of hiPSC-derived Holograms

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Maintaining Human Induced Pluripotent Stem Cells

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The human induced pluripotent stem cell (hiPSC) line HPSI1213i-babk_2 was obtained from the Wellcome Trust Sanger Institute in Cambridge, UK and was used for all experiments. hiPSCs were maintained in Essential 8™ Flex Medium (Gibco) in 6-well tissue culture plates coated with 10 μg/mL Vitronectin XF (StemCell Technologies) in a 37 °C incubator containing 95% air 5% CO₂. hiPSCs were passaged at 70% confluency using ReLeSr™ (StemCell Technologies). Essential 8™ Flex Medium was supplemented with Y-27632, a specific Rho-associated, coiled-coil containing protein kinase (ROCK) Inhibitor (10 μM) (Tocris) for 24 h following passaging.

Treacy N.J., Clerkin S., Davis J.L., Kennedy C., Miller A.F., Saiani A., Wychowaniec J.K., Brougham D.F, & Crean J. (2022). Growth and differentiation of human induced pluripotent stem cell (hiPSC)-derived kidney organoids using fully synthetic peptide hydrogels. Bioactive Materials, 21, 142-156.

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Generation of Trisomic 21 iPSCs

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The trisomic 21 iPSC line (T21.2; 47XY, +21) was a gift from Stella Chou and Mitchell Weiss. It was derived as previously described (14 (link)) through reprogramming of fetal stromal cells after transduction with pMXs-based retroviral supernatant encoding human OCT4, SOX2, KLF4, or MYC. iPSCs were maintained in Essential 8 or Essential 8 Flex Medium (Gibco; Thermo Fisher Scientific) on plates coated with N-truncated human recombinant vitronectin (Gibco; Thermo Fisher Scientific). Cell passages were performed mechanically or by using the StemPro Accutase Solution (Gibco; Thermo Fisher Scientific). A mycoplasma screening was routinely performed, according to the manufacturer’s instructions (MilliporeSigma). A list of manufacturers with the catalog number of each product is provided in Supplemental Table 2.

Arkoun B., Robert E., Boudia F., Mazzi S., Dufour V., Siret A., Mammasse Y., Aid Z., Vieira M., Imanci A., Aglave M., Cambot M., Petermann R., Souquere S., Rameau P., Catelain C., Diot R., Tachdjian G., Hermine O., Droin N., Debili N., Plo I., Malinge S., Soler E., Raslova H., Mercher T, & Vainchenker W. (2022). Stepwise GATA1 and SMC3 mutations alter megakaryocyte differentiation in a Down syndrome leukemia model. The Journal of Clinical Investigation, 132(14), e156290.

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Feeder-free hiPSC Expansion and Banking

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The WTSli020 hiPSC line from fibroblasts of dermis of a healthy female that was provided by EBiSC (European Bank for induced pluripotent Stem Cells) was cultured in feeder-free conditions using Vitronectin-coated culture vessels (VTN-N; Thermo Fisher Scientific, Waltham, MA, USA) and Essential 8 Flex medium (Gibco, Grand-Island, NY, USA) supplemented with Penicillin/Streptomycin (1:1000 PenStrep; Gibco). Another hiPSC line derived from a healthy female was also used as previously described [38 (link)]. Briefly, cells were thawed and manually expanded over five supplementary passages. For manual passaging, StemPro EZPassage tool (Thermo Fisher Scientific) was used. The automated cell culture system CompacT SelecT (Sartorius, Gottingen, Germany) was then used to generate a working cell bank using 0.25 mM EDTA (Thermo Fisher Scientific) in Phosphate-Buffered Saline (PBS; Gibco) without calcium or magnesium for cell passaging. Finally, cells were dispensed into cryovials using the automated system Fill-It (Sartorius) and frozen using CryoMed Controlled-Rate Freezer (Thermo Fisher Scientific). Quality controls (mycoplasma detection, pluripotency marker expression, genomic integrity) were performed before and after amplification.

de Lamotte J.D., Polentes J., Roussange F., Lesueur L., Feurgard P., Perrier A., Nicoleau C, & Martinat C. (2021). Optogenetically controlled human functional motor endplate for testing botulinum neurotoxins. Stem Cell Research & Therapy, 12, 599.

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Culturing and Maintaining Cell Lines

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HEK293FT cells, U2OS, and U2OS.eGFP-PEST cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen), and Jurkat cells were cultured in RPMI-1640 medium (RPMI; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 1x penicillin-streptomycin (Penstrep; Gibco) and 1x sodium pyruvate (Gibco) at 37°C in a 5% CO₂ atmosphere. The stable iPSCs expressing the CRISPR repressor were cultured in a xeno-free and feeder–cell-free state in Essential 8^™ Flex Medium (Gibco) along with the addition of Essential 8^™ Flex Supplement (50X) supplied by manufacturer. iPSC cells were subcultured according to the protocol mentioned for Gibco A18945. Cells matched their expected cell-type morphology and were routinely maintained at <90% confluency.

Sreekanth V., Jan M., Zhao K.T., Lim D., Davis J.R., McConkey M., Kovalcik V., Barkal S., Law B.K., Fife J., Tian R., Vinyard M.E., Becerra B., Kampmann M., Sherwood R.I., Pinello L., Liu D.R., Ebert B.L, & Choudhary A. (2023). A molecular glue approach to control the half-life of CRISPR-based technologies. bioRxiv.

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Cardiac Organoid Fabrication from iPSCs, Fibroblasts, and Stem Cells

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Human iPSCs were purchased from Thermo Fisher Scientific (cat. #A18945, Waltham, MA, USA). They were grown on Matrigel (Corning #356231, NY, USA) coated 6-well plates and maintained in Essential 8 Flex Medium (Gibco, Thermo Fisher Scientific) at 37°C with 95% air and 5% CO₂. Cells were passaged at 70%–80% confluence using TrypLE Express Enzyme (Gibco).
Human cardiac ventricular fibroblasts (HCVFBs) were purchased from Lonza (#CC-2904, Bend, OR, USA) and cultured in FGM-2 medium (CC-3132, Lonza). Human adipose-derived stem cells (hADSCs) were purchased from Lonza (#PT5006) and cultured in low-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin, 1% glutamine, and 1% antimycin (Gibco). Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (cat. #C2519A) and cultured in EGM-3 medium (Lonza). HCVFBs, hADSCs, and HUVECs were used at passages of 3–5 after purchasing for cardiac organoid fabrication.

Wang P., Li H., Zhu M., Han R.Y., Guo S, & Han R. (2022). Correction of DMD in human iPSC-derived cardiomyocytes by base-editing-induced exon skipping. Molecular Therapy. Methods & Clinical Development, 28, 40-50.

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Human iPSC Derivation on Vitronectin

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Human iPSC were purchased from ThermoFisher and cultured on vitronectin (ThermoFisher)-coated 6 well plates in Essential 8™ Flex Medium (Gibco). Cells were adapted to single cell passaging for 4–5 passages before starting the differentiation protocol.

Alvarez-Palomo B., Sanchez-Lopez L.I., Moodley Y., Edel M.J, & Serrano-Mollar A. (2020). Induced pluripotent stem cell-derived lung alveolar epithelial type II cells reduce damage in bleomycin-induced lung fibrosis. Stem Cell Research & Therapy, 11, 213.

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