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Essential 8 flex media

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Essential 8 Flex media is a cell culture medium designed for the maintenance and expansion of human pluripotent stem cells. It provides the necessary nutrients and growth factors to support the undifferentiated growth of these cells.

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Lab products found in correlation

Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Dmem f12, by Thermo Fisher Scientific (2 mentions) Laminin 521, by BioLamina (1 mentions) Mem neaa, by Thermo Fisher Scientific (1 mentions) Xav939, by Bio-Techne (1 mentions) 2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions) Neurobasal media, by Thermo Fisher Scientific (1 mentions) Vitamin a, by Thermo Fisher Scientific (1 mentions) Rock inhibitor, by Bio-Techne (1 mentions) Accutase, by Thermo Fisher Scientific (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Insulin, by Merck Group (1 mentions) Vitronectin, by Thermo Fisher Scientific (1 mentions) Oct4a, by Cell Signaling Technology (1 mentions) Relesr, by STEMCELL (1 mentions) Nanog, by Cell Signaling Technology (1 mentions) Tra 1 60, by Thermo Fisher Scientific (1 mentions) Insect xpress protein free insect cell medium with l glutamine, by Lonza (1 mentions) Vitronectin xf, by STEMCELL (1 mentions)

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Essential 8 (61 citations) Essential 8 media (53 citations) Essential 8 Flex (6 citations)

5 protocols using essential 8 flex media

1

Generating Isogenic iPSC Lines

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GBA1 N409S heterozygous PD patient-derived iPSCs (NH50187, MUTANT) and their isogenic control cells (NH50186, ISOGENIC) were obtained from the Rutgers University Cell and DNA Repository (RUCDR). Normal karyotypes of NH50187 and NH50186 were confirmed by RUCDR. Genotypes, pluripotency, and mycoplasma infection were routinely assessed before starting new experiments (Supplementary Figure S2a,b). Undifferentiated iPSCs were grown on Laminin 521 (Biolamina, Stockholm, Sweden, LN521)-coated plates and maintained in Essential 8 Flex media (Thermo Fisher Scientific, Waltham, MA, USA, A2858501) at 37 °C, 5% CO2 until starting differentiation.
Kojima R., Paslawski W., Lyu G., Arenas E., Zhang X, & Svenningsson P. (2024). Secretome Analyses Identify FKBP4 as a GBA1-Associated Protein in CSF and iPS Cells from Parkinson’s Disease Patients with GBA1 Mutations. International Journal of Molecular Sciences, 25(1), 683.
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2

Forebrain Organoid Generation from hPSCs

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Forebrain organoids were generated from hPSCs following the protocol described in (Zhou et al., 2017 (link)). Briefly, hPSCs were dissociated into single cells using Accutase (#A11105–01, Thermo Fisher Scientific) for 30 minutes at 37°C. Afterwards, cells were aggregated using low-adhesion V-bottom plates (#MS-9096V, Wako) in Essential 8 Flex media (#A28558501, Thermo Fisher Scientific) with 10 μM ROCK inhibitor (Y-27632, #1254, Tocris) (day-1). At day0, media was changed to Essential 6 medium (E6, #A1516401, Thermo Fisher Scientific) in the presence of 500 nM LDN193189 (#04–0074, Stemgent) and 2mM XAV-939 (#3748, Tocris) until day 4. XAV-939 was removed from day 4 till day 18. Afterwards, organoids were maintained in organoid differentiation medium (50% DMEM F-12 (#11320–033, Thermo Fisher Scientific), 50% Neurobasal media (#21103–049, Thermo Fisher Scientific), 0.5xN2 supplement (#17502–048, Thermo Fisher Scientific), 0.025% insulin (#I-034, Sigma), 5 mM L-Glutamine (#25030–024, Thermo Fisher Scientific), 0.7 mM MEM-NEAA (#11140050, Thermo Fisher Scientific), 50 U/mL Penicillin-Streptomycin (#15140–122, Thermo Fisher Scientific), 55 mM 2-mercaptoethanol (#21985–023, Thermo Fisher Scientific), 1xB27 supplement - Vitamin A (#12587010, Thermo Fisher Scientific).
Padmanabhan Nair V., Liu H., Ciceri G., Jungverdorben J., Frishman G., Tchieu J., Cederquist G.Y., Rothenaigner I., Schorpp K., Klepper L., Walsh R.M., Kim T.W., Cornacchia D., Ruepp A., Mayer J., Hadian K., Frishman D., Studer L, & Vincendeau M. (2021). Activation of HERV-K(HML-2) disrupts cortical patterning and neuronal differentiation by increasing NTRK3. Cell stem cell, 28(9), 1566-1581.e8.
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3

Cultivation and Characterization of iPSCs

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IPSCs and WA09 (H9) cells were cultured on feeder-free substrate vitronectin (Thermofisher) with Essential 8 Flex media (ThermoFisher) or mTeSR and Matrigel. Cells were passaged 1:18-1:24 every 5–7 days using ReLeSR (Stem Cell Technologies). Pluripotency was confirmed via immunostaining for TRA-1-60 (1:100, ThermoFisher), NANOG (1:200, Cell Signaling Technologies), and OCT4A (1:400, Cell Signaling Technologies). Cells were utilized between passage 18 and 50, and outgrowth analysis confirmed the conservation of neuronal outgrowth phenotypes between early and late passages (Supplementary Fig. 4B).
Catlett T.S., Onesto M.M., McCann A.J., Rempel S.K., Glass J., Franz D.N, & Gómez T.M. (2021). RHOA signaling defects result in impaired axon guidance in iPSC-derived neurons from patients with tuberous sclerosis complex. Nature Communications, 12, 2589.
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4

hPSC Maintenance and Authentication

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Human pluripotent stem cells (hPSCs) (H9 derivatives (H9-CRISPRa, H9-CRISPRi, H9-CRISPRa-control, H9-CRISPRa-HERV-K(HML-2), H9-CRISPRi-control, H9-CRISPRi-HERV-K(HML-2), H9-TRE-CRISPRa-control, H9-TRE-CRISPRa-HERV-K(HML-2), H9-CRISPRa-CLSTN2, H9-CRISPRa-CHRDL1, H9-CRISPRa-EPHA4 and H9-CRISPRa-NTRK3)) were maintained with Essential 8 Flex media (#A28558501, Thermo Fisher Scientific) in feeder-free conditions on vitronectin (VTN-N) substrate (#A14700, Thermo Fisher Scientific). hPSCs were passaged as clumps with 0.5M EDTA (0.5M EDTA+ 5M NaCl+ 1X PBS). The pluripotency levels of the cells in culture was routinely authenticated for markers Nanog and Oct-4 and checked for mycoplasma contamination.
Padmanabhan Nair V., Liu H., Ciceri G., Jungverdorben J., Frishman G., Tchieu J., Cederquist G.Y., Rothenaigner I., Schorpp K., Klepper L., Walsh R.M., Kim T.W., Cornacchia D., Ruepp A., Mayer J., Hadian K., Frishman D., Studer L, & Vincendeau M. (2021). Activation of HERV-K(HML-2) disrupts cortical patterning and neuronal differentiation by increasing NTRK3. Cell stem cell, 28(9), 1566-1581.e8.
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5

Fluctuation Analysis of Bu-1 Loss

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DT40 cell culture, cell survival assays, fluctuation analysis for generation of Bu‐1 loss variants (Fig EV1), and genetic manipulation of the BU‐1 locus were performed as previously described (Simpson & Sale, 2003, 2006; Schiavone et al, 2014). Drosophila S2 cells were grown in Insect‐XPRESS Protein‐Free Insect Cell Medium with l‐glutamine (Lonza), supplemented with 1% penicillin–streptomycin at 27°C, ambient CO2 with 105 rpm agitation. BOBSC human induced pluripotent stem (hiPS) cells (Yusa et al, 2011) were cultured feeder‐free on dishes coated with Vitronectin XF (07180; Stem Cell Technologies) in Essential 8 Flex media (A2858501; Thermo Fisher Scientific) at 37°C and 5% CO2. Cells were split 1:10–1:15 every 3–4 days depending on confluence. All cells tested negative for mycoplasma.
Šviković S., Crisp A., Tan‐Wong S.M., Guilliam T.A., Doherty A.J., Proudfoot N.J., Guilbaud G, & Sale J.E. (2018). R‐loop formation during S phase is restricted by PrimPol‐mediated repriming. The EMBO Journal, 38(3), e99793.
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