LCL were cultured in LCL medium (RPMI 1640 with GlutaMAX [Thermo Fisher Scientific]) supplemented with 15% fetal bovine serum (FBS) (Life Technologies) and 1 μM sodium pyruvate. Human embryonic kidney cells (HEK293, ATCC [American Type Culture Collection] line CRL-1573) and human fibroblasts were cultured in fibroblast medium (Dulbecco’s Modified Eagle Medium [DMEM, Sigma-Aldrich, D6546]) supplemented with 10% FBS, 2 mM GlutaMAX (GIBCO), and 100 μL/mL penicillin-streptomycin (Sigma-Aldrich). iPSCs were cultured in plates coated with Matrigel (Corning) in E8 (GIBCO) or E8 Flex (GIBCO) medium. The medium was changed every other day. All of the cells were kept in an incubator at 37°C and 5% CO2 and were routinely tested for mycoplasma contamination.
E8 flex
Manufactured by Thermo Fisher Scientific
Sourced in United States
The E8 Flex is a high-performance liquid chromatography (HPLC) system designed for a wide range of analytical applications. It features a modular design, allowing for customization to meet specific laboratory needs. The E8 Flex offers precise control over flow rate, pressure, and temperature to ensure accurate and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by Corning (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Stempro accutase, by Thermo Fisher Scientific (1 mentions)
Revitacell supplement, by Thermo Fisher Scientific (1 mentions)
Matrigel hesc qualified matrix, by Corning (1 mentions)
Mtesr medium, by STEMCELL (1 mentions)
Essential 8 medium, by Thermo Fisher Scientific (1 mentions)
Geltrex, by Thermo Fisher Scientific (1 mentions)
Vitronectin, by Thermo Fisher Scientific (1 mentions)
Essential 8 flex medium, by Thermo Fisher Scientific (1 mentions)
Mtesr1, by STEMCELL (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Polybrene, by Thermo Fisher Scientific (1 mentions)
Growth factor reduced basement membrane matrix, by Corning (1 mentions)
Laminin 521, by Thermo Fisher Scientific (1 mentions)
Chir99021, by Bio-Techne (1 mentions)
Imr90 4, by WiCell (1 mentions)
6 protocols using e8 flex
1
Culturing Various Cell Lines
2
Differentiation of hiPSCs into Hepatocytes and Endothelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The hiPSC line, Sigma 0028, was purchased from Sigma–Aldrich and maintained on coated plates with Corning Matrigel hESC‐Qualified Matrix (Corning, 734–1440) in Essential 8 Flex Medium (E8 Flex; Gibco, A2858501). The cells were passaged at 65% confluency using 0.1% Ethylenediaminetetraacetic acid (EDTA, Life Technologies, 15575020) in PBS. The hiPSCs were genetically engineered to induce the overexpression of 3 transcription factors (TFs), namely HNF1A, FOXA3, and PROX1 (named HC3X‐PSC) as described earlier to generate hepatocyte‐like progeny; or one TF, namely ETV2 (named iETV2‐PSC) to generate endothelial cells.[33 (link),
69 (link)
] When the cells reached 65% confluency, cells were dissociated into single cells using StemPro Accutase (Gibco, A2644501) cell dissociation reagent. Cells were plated on Matrigel Based Membrane Growth Factor Reduced coated plates (Corning, 734–0270) at a density of ±8.75 × 104 cells cm−2 or 3 × 105 cells mL−1 in mTeSR medium (Stem Cell Technologies, 85850) supplemented with 1:100 RevitaCell Supplement (Gibco, A2644501). When a cellular confluency of 70–80% was obtained, cells were differentiated toward either the hepatocyte or endothelial lineage. The use of hiPSCs for research was approved by the “Commissie Medische Ethiek,” UZ KU Leuven/Onderzoek U.Z. Gasthuisberg, Herestraat 49, B 3000 Leuven, under file no. S52426.
69 (link)
] When the cells reached 65% confluency, cells were dissociated into single cells using StemPro Accutase (Gibco, A2644501) cell dissociation reagent. Cells were plated on Matrigel Based Membrane Growth Factor Reduced coated plates (Corning, 734–0270) at a density of ±8.75 × 104 cells cm−2 or 3 × 105 cells mL−1 in mTeSR medium (Stem Cell Technologies, 85850) supplemented with 1:100 RevitaCell Supplement (Gibco, A2644501). When a cellular confluency of 70–80% was obtained, cells were differentiated toward either the hepatocyte or endothelial lineage. The use of hiPSCs for research was approved by the “Commissie Medische Ethiek,” UZ KU Leuven/Onderzoek U.Z. Gasthuisberg, Herestraat 49, B 3000 Leuven, under file no. S52426.
3
Feeder-Free Human iPSC Maintenance Protocol
4
Lentiviral Knockdown of OXTR in hiPSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bacteria carrying the plasmid for shRNA-mediated knockdown of OXTR and a scrambled plasmid (both designed with VectorBuilder) were grown on LB agar plates and isolated colonies were expanded in LB broth, both containing ampicillin. The plasmids carried an ampicillin resistance cassette, allowing for their growth. Plasmid DNA was isolated from the bacteria by midiprep (Zymo Research), and purified DNA was transfected into 40% confluent HEK293T cells using Lipofectamine (Invitrogen). Lentiviral packaging plasmids (pVSVg, psPAX2) were also transfected at this time, thereby allowing the generation of a functional lentivirus containing the shRNA molecules of interest. Viral supernatant was collected and concentrated after 48 h and transduced directly into hiPSCs at low to mid-confluency along with 8 μg/ml polybrene (Fisher Scientific). Because all plasmids contained puromycin-resistance cassettes, the stem cell media was changed the next day to E8 Flex containing 0.5 μg/ml puromycin (Thermo Fisher Scientific) to facilitate colony selection. hiPSCs were maintained in puromycin for 5 days and surviving monoclonal colonies were re-plated and expanded to generate new hiPSC lines.
5
Directed Differentiation of hiPSCs into BMECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The hiPS cell line iMR90-4 (WiCell) 18) (link) was cultured in mTeSR1 medium (Stem Cell Technologies, Vancouver, Canada), and dissociated into clumps using 0.5 mM ethylenediamine tetraacetic acid (EDTA)/phosphate-buffered saline (PBS) and plated onto growth factor-reduced basement membrane matrix (Corning, Corning, NY, U.S.A.). The hiPS cell line 201B7 19) (link) was maintained in E8-Flex (Thermo Fisher Scientific, Waltham, MA, U.S.A.), which was changed daily, and dissociated into clumps using 0.5 mM EDTA/PBS and plated onto Laminin-521 (Thermo Fisher Scientific). The differentiation protocol for the induction of BMECs from hiPS cells was described previously. 11) (link) Chir99021 (Lot: 5B/198191) and BIO (Lot: 3A/178335) were purchased from TOCRIS. Chir99021 was reconstituted in dimethyl sulfoxide (DMSO) and included at concentrations of 3-6 µM depending on the experiments. BIO was reconstituted in DMSO and included at concentrations of 2-3 µM depending on the experiments. They were added into the culture medium from day 6 to day 8.
6
Generation and Characterization of Human iPSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In this study we used in total 3 control hIPSC lines, Control #1, Control #2 and Control #3. All hiPS cells used in this study were obtained from reprogrammed fibroblasts. Control line 1 was obtained from a healthy 30-year-old female, and reprogrammed via episomal reprogramming 85 .
Control line 2 was derived from a healthy 51-year-old male and reprogrammed via a nonintegrating Sendai virus by KULSTEM (Leuven, Belgium). Control line 3 was obtained from a healthy male, and reprogrammed via retroviral vectors expressing four transcription factors: Oct4, Sox2, Klf4, and cMyc. Generated clones (at least two per patient line) were selected and tested for pluripotency and genomic integrity based on single nucleotide polymorphism (SNP) arrays 75 . HiPSCs were cultured on Matrigel (Corning, #356237) in E8 flex (Thermo Fisher Scientific) supplemented with primocin (0.1 µg/ml, Invivogen) and low puromycin (0.5 µg/ml, to select for rtTA positive cells) and G418 concentrations (50 µg/ml, to select for Ngn2 or Ascl1 positive cells) at 37˚C/5% CO2. Medium was refreshed every 2-3 days and cells were passaged twice a week using an enzyme-free dissociation reagent (ReLeSR, Stem Cell Technologies).
Control line 2 was derived from a healthy 51-year-old male and reprogrammed via a nonintegrating Sendai virus by KULSTEM (Leuven, Belgium). Control line 3 was obtained from a healthy male, and reprogrammed via retroviral vectors expressing four transcription factors: Oct4, Sox2, Klf4, and cMyc. Generated clones (at least two per patient line) were selected and tested for pluripotency and genomic integrity based on single nucleotide polymorphism (SNP) arrays 75 . HiPSCs were cultured on Matrigel (Corning, #356237) in E8 flex (Thermo Fisher Scientific) supplemented with primocin (0.1 µg/ml, Invivogen) and low puromycin (0.5 µg/ml, to select for rtTA positive cells) and G418 concentrations (50 µg/ml, to select for Ngn2 or Ascl1 positive cells) at 37˚C/5% CO2. Medium was refreshed every 2-3 days and cells were passaged twice a week using an enzyme-free dissociation reagent (ReLeSR, Stem Cell Technologies).
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!