Cell Culture and Differentiation Protocols

The human embryonal carcinoma NTERA-2 (NT2) cell line was obtained from ATCC. NT2, 293T, and HeLa cells were cultured in DMEM high glucose with GlutaMAX (Invitrogen) supplemented with 10% fetal bovine serum (FBS; HyClone, Piscataway, NJ). Large scale culture of NT2 cells were described (Fong et al., 2011 (link)). Mouse ES cell line D3 was purchased from ATCC (Manassas, VA) and adapted to feeder-free condition as described (Fong et al., 2011 (link)). Differentiation of D3 cells was induced by maintaining cells in LIF-free ES cell medium containing 2–5 mM all-trans retinoic acid (Sigma Aldrich) for up to 7 days. Human ES cell line H9 (WiCell, Madison, WI) was maintained in feeder-independent conditions, using Synthemax SC-II Substrate (Corning) and grown in TeSR-E8 (Stemcell Technologies, Canada). Media was changed daily and cell cultures were passaged using Dispase (Stemcell Technologies), according to the manufacturer's protocol.

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Fong Y.W., Ho J.J., Inouye C, & Tjian R. (2014). The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells. eLife, 3, e03573.

Publication 2014

GlutaMAX Synthemax SC-II Substrate TeSR-E8 Dispase

All trans retinoic acid Cell cultures Cell line Cells Cells differentiation Dispase Embryonal carcinoma Es cell Glucose Hela cells Human Human es cell Mouse Stemcell

Corresponding Organization :

Other organizations : Howard Hughes Medical Institute, University of California, Berkeley

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Variable analysis

independent variables

Cell lines used (NTERA-2 (NT2), 293T, HeLa, D3 mouse ES cells, H9 human ES cells)

dependent variables

Cell culture conditions (media, supplements, feeder-free/feeder-dependent)

control variables

Passage number/cell culture conditions for each cell line
Media composition (DMEM high glucose with GlutaMAX, 10% FBS)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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