All hESC and iPSC lines (H1, H9, HUES53, LiPSC-GR1.1) were adapted to and maintained for at least 3 passages in each media tested (E8, StemFlex, (Thermo Fisher Scientific), mTeSR (STEMCELL Technologies), MEF conditioned media (MEF-CM; R&D Systems) or StemFit (AMSBIO). 384-well white flat bottom plates (Corning) were coated with VN, LN521, or Matrigel at 37 °C for 1 h. Coating media was removed and each well immediately received 30 μl of the appropriate media supplemented with DMSO (2X), CEPT (2X), Y-27632 (20 μM), CloneR (2X; STEMCELLTechnologies), RevitaCell (2X; Thermo Fisher Scientific) or SMC3/4 (2X; BioVision). Once 384-well plates were ready, cells were dissociated with Accutase, re-suspended in the appropriate medium without supplements and dispensed into the 384-well plates at 2000 cells/well (30 μl per well, resulting in a final assay volume of 60 μl in each well and a final concentration of 1X for each supplement). After 24 h, 30 μl CTG was dispensed into each well and luminescence signals were read using the ViewLux μHTS microplate imager (Perkin-Elmer). All plates were prepared in six replicates to compensate for cell dispensing variation (see Supplementary Fig. 3 ).
Stemfit
Manufactured by AMS Biotechnology
StemFit is a cell culture medium designed for the maintenance and expansion of human pluripotent stem cells. It provides a serum-free, animal component-free formulation that supports the self-renewal and undifferentiated growth of human embryonic stem cells and induced pluripotent stem cells.
Automatically generated - may contain errors
Lab products found in correlation
White flat bottom plates, by Corning (2 mentions)
Cloner, by STEMCELL (2 mentions)
Revitacell, by Thermo Fisher Scientific (2 mentions)
Mef cm, by R&D Systems (2 mentions)
Smc3 4, by Abcam (2 mentions)
Viewlux μhts microplate imager, by PerkinElmer (2 mentions)
Stemflex, by Thermo Fisher Scientific (2 mentions)
Coated sixwell plates, by Corning (1 mentions)
Geltrex, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Merck Group (1 mentions)
Human fgf2, by R&D Systems (1 mentions)
Mtesr1, by Merck Group (1 mentions)
Matrigel, by Corning (1 mentions)
Accutase, by STEMCELL (1 mentions)
Mtesr1, by STEMCELL (1 mentions)
Y 27632, by Merck Group (1 mentions)
3 protocols using stemfit
1
Optimizing hESC and iPSC Culture Conditions
2
Optimizing hESC and iPSC Culture Conditions
3
Maintenance of human iPSC and H9 hESC lines
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Human iPSC line WTC11 (a kind gift from Bruce Conklin, Gladstone Institute of Cardiovascular Disease) was maintained in mTeSR1 (Stemcell Technologies) on Matrigel (Corning)-coated six-well plates (Corning) in a 37 °C incubator with 5% CO2. At 70–80% confluence, cells were dissociated with Accutase (Stemcell Technologies) and plated on Matrigel-coated six-well plates with mTeSR™1 containing 10 μM Rho kinase inhibitor, Y27632 (EMD Millipore). Y27632 was removed after the first 48 h and thereafter media was replaced with fresh mTeSR1 every day. Human H9 (purchased from Wicell) or H9-FP ubiquitously expressing miRFP703 fused to histone H2B (a kind gift from Dr. Andrew McMahon, University of Southern California) were maintained in StemFit (amsbio) with 100 ng/ml human FGF2 (R&D) on Geltrex (Thermo Fisher Scientific)-coated six-well plates. At 70–80% confluence, cells were dissociated with Accutase and plated on Geltrex-coated six-well plates with StemFit containing 100 ng/ml human FGF2 and 10 μM Y27632. After 48 h of culture, medium was replaced with fresh StemFit containing 50 ng/ml human FGF2. After the next 48 h, medium was replaced with fresh StemFit containing 25 ng/ml human FGF2.
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