For long-term live cell imaging, EZRIN– or PODXL–GFP–expressing hESCs (see cloning in the Constructs and cell lines section) prepared for the apicosome formation assay or the aggregate lumen formation assay were plated on six-well plates (Nunc) in mTeSR1 medium (Stem Cell Technologies), and time-lapse images were taken at 37°C using the IncuCyte Zoom live cell imaging system (Essen Bioscience). Alternatively, cells were plated on a glass bottom culture dish (MatTek) in mTeSR1 medium (Stem Cell Technologies) and were imaged in a LiveCell chamber (Pathology Devices) configured for an A-1 or Fluoview 1000 confocal microscope at 37°C. Live ER-Tracker (E34250; Invitrogen) staining was performed as described in the manufacturer’s manual. FRAP was performed using a Fluoview 1000 confocal microscope with a SIM scanner using PODXL–GFP cells growing in mTeSR1 medium (Stem Cell Technologies) at 37°C. Fig. S3 E is plotted using normalized fluorescent recovery values (Higashi et al., 2016 (link)) obtained from 10 quantitated cells. 3D reconstruction, volume rendering, and tracking analyses were performed using Imaris 7.6 (Bitplane), and movies were generated using Imaris 7.6 and Photoshop (Adobe).
Mtesr1 medium
Manufactured by STEMCELL
Sourced in Canada, United States, France, Germany, United Kingdom, Japan, Italy
MTeSR1 is a complete, serum-free medium designed for the maintenance of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) in an undifferentiated state. It provides the necessary components to support the growth and self-renewal of pluripotent stem cells.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by Corning (135 mentions)
Matrigel, by BD (131 mentions)
Relesr, by STEMCELL (62 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (48 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (44 mentions)
Geltrex, by Thermo Fisher Scientific (43 mentions)
Dispase, by STEMCELL (34 mentions)
Accutase, by STEMCELL (33 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (32 mentions)
Dmem f12, by Thermo Fisher Scientific (27 mentions)
Glutamax, by Thermo Fisher Scientific (26 mentions)
Gentle cell dissociation reagent (gcdr), by STEMCELL (24 mentions)
L glutamine, by Thermo Fisher Scientific (24 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (24 mentions)
Accutase, by Thermo Fisher Scientific (23 mentions)
Accutase, by Merck Group (19 mentions)
Accutase, by Innovative Cell Technologies (16 mentions)
Matrigel coated plate, by BD (16 mentions)
B27 supplement, by Thermo Fisher Scientific (15 mentions)
Growth factor reduced matrigel, by Corning (14 mentions)
812 protocols using mtesr1 medium
1
Live Cell Imaging of hESCs
2
Healthy Control Donor hiPSC Generation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The generation of hiPSCs from a healthy control donor was performed as previously described [40 ]. Peripheral blood mononuclear cells from healthy control donor were collected for iPSC induction. Cells were transduced with the integration-free CytoTune-iPS Sendai Reprogramming Kit (Life Technologies, Carlsbad, CA, USA), which utilizes Sendai virus particles of the four factors (OCT4, SOX2, c-MYC, and KLF4). Transduced cells were plated on vitronectin-coated culture dishes and fed iPSC medium, which was replaced by StemPro 34 SFM (Life Technologies) from days 3 to 7. On day 7, the medium was replaced by feeder-free mTeSR1medium (STEMCELL Technologies, Vancouver, BC, Canada) until small colonies were formed. The growth of small colonies was maintained for another 3–4 weeks, and cell colonies were manually picked and mechanically dissociated for the first four passages. The hiPSCs were maintained on Matrigel-coated plates (BD Bioscience, Franklin Lakes, NJ, USA) with mTeSR1 medium (STEMCELL Technologies), and passaged every 4–5 days using 1 mg/mL dispase (Life Technologies). All experimental protocols including human stem cell use were approved by the Ethics Committee at the First Affiliated Hospital of Sun Yat-sen University.
3
Feeder-free Human ESC Maintenance and Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human ESC lines were maintained on human foreskin fibroblast feeders or in feeder-free culture conditions on Matrigel (BD Biosciences) in mTeSR1 medium (STEMCELL Technologies) as previously described (Narva et al., 2012 (link), Konki et al., 2016 (link)). In feeder-free culture conditions the cells were maintained on Matrigel (BD Biosciences) in mTeSR1 medium (STEMCELL). Differentiation of hESCs was performed as described by Narva et al. (2012) (link). In brief, for spontaneous embryonic body differentiation the cells were plated without feeders and were grown in suspension in standard hESC medium without fibroblast growth factor 2. For retinoic acid-induced differentiation, cells were plated in feeder-free conditions and medium was supplemented with 13.7 μM retinoic acid (Sigma). The karyotypes of the lines were routinely monitored with G-banding and/or KaryoLite BoBs assay (Lund et al., 2012 (link)).
4
hPSC Culturing and Maintenance
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human embryonic stem cells (H9) (Thomson et al. 1998 ) was obtained from WiCell Research Institute. Human induced pluripotent stem cells (DC60-3, DC87-3) were generated from human peripheral blood mononuclear cells (PBMNC) in our lab previously (Tao et al. 2020 (link)). Undifferentiated hPSCs were cultured in mTeSR1™ medium (STEMCELL Technologies) on Matrigel (Corning, hESCs-qualified)-coated cell culture plates. mTeSR1™ medium was changed every day and hPSCs clones were disassociated into small clumps with ReLeSR (STEMCELL Technologies) as required and passaged every 3-4 days.
5
Differentiation of hiPSCs into Islet-like Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The procedure has been described elsewhere [5 (link),19 ]. In brief, the undifferentiated hiPSC line NCRM-1 (Lonza/NIH, Walkersville, MD, USA) was cultured in mTeSR1 medium (Stem Cell Technologies, Vancouver, BC, Canada) on Growth Factor Reduced Matrigel (Corning, Corning, NY, USA) for 3–4 days at 37 °C in a humidified 5% CO2 incubator. When cells became almost confluent, they were subjected to single-cell dispersion, seeded at 5 × 105 cells/mL into Matrigel-coated Transwell culture plates (Corning) containing mTeSR1 medium (Stem Cell Technologies) and cultured for 24 h. Then, their differentiation was induced sequentially under six different medium conditions. At the end of the six-stage induction, cells were removed from the Transwell culture plates, resized and placed into Ultra Low Attachment flasks (Corning) in suspension as stage seven for eight days. Upon completion of stage seven, the resulting hiPSC-islets were obtained for in vitro quality testing and intraocular transplantation [5 (link),19 ]. All reagents were purchased from R&D Systems (Minneapolis, MN, USA) unless otherwise stated.
6
Generation of Pluripotent Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MEFs: Primary Pou5f1-GFP MEFs were prepared from E12 embryos (http://jaxmice.jax.org/strain/008214.html ) and reprogrammed by transduction with OKS using four HCT116-transducing units (HC-TU) per cell as previously described (Pasi et al. 2011 (link)). CD34+ cells from human cord blood were obtained and prepared as previously described (Barde et al. 2013 (link)), before transducing 250,000 cells with OKS using 100 HC-TU per cell. After 5 d, cells were switched to mTeSR1 medium (Stemcell Technologies no. 05859) and grown on a mouse fibroblast feeder layer until reprogrammed colonies emerged (∼21 d). Individual human iPSC clones were then picked and expanded. Primary human hepatocytes were isolated from liver biopsies as previously described (Birraux et al. 2009 (link)) and plated on Matrigel or collagen before being transduced with OKS or OKSM using 20 HC-TU per cell. After 5 d, cells were switched to mTeSR1 medium (Stemcell Technologies no. 05859) and grown until reprogrammed colonies emerged (∼25 d).
7
Differentiation of iPSCs into Cardiomyocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human induced pluripotent stem cells (iPSC) were acquired from the Coriell Institute (GM23338), as characterized previously102 (link). iPSC colonies were maintained on growth-factor reduced Matrigel (Corning)-coated tissue culture plates in mTeSR1 medium (Stem Cell Technologies). iPSCs were differentiated into iPSC-derived cardiomyocytes (iPSC-CMs) following a sequential protocol of manipulating the WNT pathway103 (link), using 12.5 μM CHIR99021 (Stem Cell Technologies) for 24 hours and 5 μM IWP4 (Tocris Bioscience) for 48 hours on day 3 after the induction of differentiation. Subsequently, the differentiating cultures were maintained in RPMI + B2-insulin until day 11 when spontaneous beating was observed. Media was then changed to RPMI + B27 + insulin for 2 days, followed by 4 days of metabolic selection using DMEM without glucose and 4 mM lactate104 (link). iPSC-CMs were kept in RPMI + B27 + insulin until day 21 after the start of differentiation when they were dissociated and re-plated on a 12-well plate coated with 50 μg/ml fibronectin (bovine, Sigma-Aldrich)105 (link). iPSC-CMs were cultured for a subsequent 2 days in RPMI + B27 + insulin before experiments were performed.
8
Feeder-free hiPSC Culture using mTeSR1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In this work, the hiPSC cell line iPS-DF6-9-9T.B, purchased from WiCell Bank, was used. This cell line is vector free and was derived from foreskin fibroblasts with a karyotype 46, XY. The hiPSC culture was performed using mTeSR1 medium (STEMCELL Technologies, Vancouver, BC, Canada) in 6-well tissue culture plates coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) diluted 1:30 in DMEM/F12. The medium was changed daily. Enzyme-free passaging was performed using an EDTA (Thermo Fisher Scientific, Waltham, MA, USA) solution diluted in PBS at a concentration of 0.5 mM. The cells were incubated for 5 min with EDTA at room temperature and flushed with culture medium. Splits from 1:3 to 1:8 were usually performed. For cell counting, a sample of 100 µL was incubated in 400 µL of Accutase (Thermo Fisher Scientific) for 7 min at room temperature and the samples were diluted in trypan blue. Phase contrast images were obtained using a Leica DMI 3000B microscope and a Nikon DXM 1200 digital camera.
9
Cultivation of Primate Induced Pluripotent Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chimpanzee and bonobo iPSCs were cultured on plates pre-coated with Matrigel (BD Biosciences) in commercial mTeSR1™ medium (Stemcell technology, 85850). Cells were passaged every 5-6 days with TrypLE (Life Technologies) at a split ratio of 1:10. ROCK inhibitor Y27632 (10 µM) was added at least one hour prior to and 24 hours after passaging. Marmoset iPSCs were cultured on MEFs in CDF12 media containing DMEM/F12 (Life Technologies, 11330-032), 20% Knockout Serum Replacement (KSR, Life Technologies, 10828), 2 mM Glutamax (Life Technolgies, 35050-061), 0,1 mM NEAA (Life Technologies, 11140-050), 0.1 mM β-mercaptoethanol (Gibco, 21985) and 10 ng/ml FGF2 (Peprotech). Marmoset iPSCs were passaged every 6-7 days either using Collagenase IV (Life Technologies) at or TrypLE (Life Technologies) at 1:5 ratio. When passaged using TrypLE ROCK inhibitor Y27632 (10 µM) was added at least one hour prior to and 24 hours after passaging. Tests for mycoplasma contamination were routinely performed for all the cell lines using PCR-based approach or MycoAlert mycoplasma detection kit (Lonza) following manufacturer’s recommendation every 10 passages.
10
Cell Culture Protocols for Research
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa, SH-SY5Y, 293T, and U2OS cells were all purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China). They were cultured in DMEM-high glucose (Gibco, Carlsbad, CA, USA), which was supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin at 37 °C in 5% CO2. In addition, human embryonic stem cells (ESCs) H9 were obtained from Dr. Hongkui Deng, Peking University Stem Cell Research Center (Beijing, China) [17 (link)]. Coat plates with 1% matrigel (354277, Corning, New York, NY, USA) were heated at 37 °C for 1 h, cultured in mTeSR™1 medium (85850, STEMCELL, Vancouver, Canada), and supplemented with 1% penicillin/streptomycin and Y27632 (10 μM).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!