Human intestinal organoids were generated and maintained as previously described (Munera and Wells, 2017 (link); Watson et al., 2014 (link)). Human embryonic stem cells and induced pluripotent stem cells were grown in feeder-free conditions in six-well Nunclon surface plates (Nunc) coated with Matrigel (BD Biosciences) and maintained in mTESR1 media (Stem Cell Technologies). For induction of definitive endoderm (DE), human ES or iPS cells were passaged with Accutase (Invitrogen) and plated at a density of 100,000 cells per well in a Matrigel-coated, Nunclon surface 24-well plate. For Accutase split cells, 10 μM Y27632 compound (Sigma) was added to the media for the first day. After the first day, media was changed to mTESR1 and cells were grown for an additional 24 hours. Cells were then treated with 100 ng/mL of Activin A for 3 days as previously described (Spence et al., 2011 (link)). DE was then treated with hindgut induction medium (RPMI 1640, 2 mM L-glutamine, 2% decomplemented FBS, penicillin-streptomycin and 100 ng/mL Activin A) for 4 d with 500 ng/mL FGF4 (R&D) and 3 μM Chiron 99021 (Tocris) to induce formation of mid-hindgut spheroids.
Nunclon surface plates
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States, Canada
Nunclon surface plates are a type of cell culture vessel used in laboratories. They feature a proprietary surface treatment that promotes cell attachment and growth. The plates are available in a range of sizes and well configurations to accommodate different experimental needs.
Automatically generated - may contain errors
Lab products found in correlation
Mtesr1 media, by STEMCELL (6 mentions)
Matrigel, by BD (5 mentions)
Chiron 99021, by Bio-Techne (3 mentions)
Matrigel, by Thermo Fisher Scientific (3 mentions)
Line h1 embryonic stem cells, by WiCell (2 mentions)
Epidermal growth factor (egf), by R&D Systems (2 mentions)
Accutase, by STEMCELL (2 mentions)
Dispase, by Thermo Fisher Scientific (2 mentions)
Advanced dmem f12, by Thermo Fisher Scientific (2 mentions)
Y27632 compound, by Merck Group (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
Accutase, by Merck Group (1 mentions)
Nrk 52e, by Merck Group (1 mentions)
Matrigel, by R&D Systems (1 mentions)
Dispase solution, by Thermo Fisher Scientific (1 mentions)
Geltrex, by Thermo Fisher Scientific (1 mentions)
Mtesr1 medium, by STEMCELL (1 mentions)
Tesr e8 media, by STEMCELL (1 mentions)
Gentle cell dissociation reagent (gcdr), by STEMCELL (1 mentions)
8 protocols using nunclon surface plates
1
Generation of Human Intestinal Organoids
2
Derivation and Maintenance of Human Intestinal Organoids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HIOs were generated and maintained as previously described2 (link)–4 . Briefly, line H1 embryonic stem cells (WiCell Research Institute, Inc.) were grown in feeder-free conditions in Matrigel (BD Biosciences) coated six-well Nunclon surface plates (Nunc) and maintained in mTESR1 media (Stem Cell Technologies). For induction of definitive endoderm (DE), cells were passaged with Accutase (Stem Cell Technologies) and plated at a density of 65,000 cells per well in 24-well Nunc plates. Cells were allowed to grow in mTESR1 media for two days before treatment with 100 ng/ml of Activin A for three days as previously described. DE was then treated with hindgut induction medium (RPMI 1640, 100x NEAA, 2% dFCS,) for four days with 100 ng/ml FGF4 (R&D) and 3 µM Chiron 99021 (Tocris) to induce formation of mid-hindgut spheroids. Spheroids were then plated in Growth Factor Reduced (GFR) Matrigel and maintained in intestinal growth medium (Advanced DMEM/F-12, N2 supplement, B27 supplement, 15 mM HEPES, 2 mM L-glutamine, penicillin-streptomycin) supplemented with 100 ng/ml EGF (R&D) to generate human intestinal organoids (HIOs). Media was changed twice weekly thereafter. HIOs were replated in fresh Matrigel every 14 days. HIOs were utilized for surgical transplantation between days 28 and 36.
3
Derivation and Maintenance of Human Intestinal Organoids
4
Culturing Rat Kidney Cells with Babesia microti
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In the in vitro studies, rats’ kidney epithelial cells (NRK-52E; ECACC 87,012,902; Sigma-Aldrich, St. Louis, MO, USA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2 mM L-glutamine, 5% fetal bovine serum and 1% non-essential amino acids. Cultures were grown in sterile 12-well Nunclon surface plates (Nunc, Wiesbaden, Germany)—10 wells were used for B. microti infection (2 biological replicates from second passage rats and 5 technical replicates from each of 2 rats), 2 wells were used as controls. After reaching confluence, 0.5 ml of rats blood with the confirmed presence of the B. microti was added to the culture (approximately 20% parasitemia, the blood of 2 rats from second passage). The same blood was used in in vivo studies. Control cells were NRK-52E line grown in the above media with the addition of control rats’ blood. Cultures were performed at 37 °C for 48 h in a 5% CO2 atmosphere. After this time, the cultures were rinsed with DMEM and fixed in 2.5% paraformaldehyde in phosphate buffer.
5
hESC H9 Feeder-Free Maintenance
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The hESC line H9 (WiCell, USA) was grown in feeder-free conditions in six-well Nunclon surface plates (Nunc, USA) coated with Matrigel (R&D systems, USA) and maintained in mTESR1 media (Stem Cell Technologies, USA). Cells were passaged at a 1:3~4 ratio using dispase (Invitrogen, USA). All Matrigel plates were coated with a 1:80 dilution in Advanced DMEM-F12 (Gibco, USA) and incubated at room temperature for at least 1 h before use.
6
Characterization of hPSC Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two hPSC lines were used in this study: (1) WA01-H1 human embryonic stem cells purchased from WiCell (NIH approval number NIHhESC-10-0043 and NIHhESC-10-0062) and (2) human iPSC72_3 generated by the CCHMC Pluripotent Stem Cell Facility. Both cell lines have been authenticated as follows: (i) cell identity: via STR profiling by Genetica DNA Laboratory (a LabCorp brand; Burlington, NC), (ii) genetic stability: by standard metaphase spread and G-banded karyotype analysis in CCHMC Cytogenetics Laboratory, and (iii) functional pluripotency: cells were subjected to analysis of functional pluripotency by teratoma assay demonstrating ability to differentiate into each of the three germ layer. Both cell lines routinely tested negative for mycoplasma contamination. hPSC lines were maintained on feeder-free conditions in mTeSR1 medium (Stem Cell technologies, Vancouver, Canada) on six-well Nunclon surface plates (Nunc) coated with Geltrex (ThermoFisher Scientific) and maintained in mTESR1 media (Stem Cell Technologies) at 37 °C with 5% CO2. Cells were checked daily and differentiated cells were manually removed. Cells were passaged every 4 days using Dispase solution (ThermoFisher Scientific).
7
Feeder-free hESC Culture Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human embryonic stem cell (hESCs) lines, H9 and H1, were grown in feeder-free conditions in six-well Nunclon surface plates (Nunc) coated with Matrigel (BD Biosciences) and maintained in mTESR1 media (Stem Cell Technologies). Cells were passaged at a 1:3~4 ratio using dispase (Invitrogen). All Matrigel plates were coated with a 1:80 dilution in Advanced DMEM-F12 (Life Technologies) and incubated at room temperature for at least 1 hr before use.
8
Derivation and Characterization of iPSC72.3 Line
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The iPSC72.3 line was derived from primary human foreskin fibroblasts (HFFs) cultured from neonatal human foreskin tissue. Tissues were obtained through the Department of Dermatology, University of Cincinnati. iPSC72.3 was generated by the CCHMC Pluripotent Stem Cell Facility, approved by the CCHMC institutional review board and previously characterized [72 (link), 73 (link)]. iPSC72.3 had a normal male karyotype and differentiated into endoderm, mesoderm, and ectoderm lineages in an in vivo teratoma assay. Induced pluripotent stem cells were grown in feeder-free conditions in six-well Nunclon surface plates (Nunc) coated with Matrigel (BD Biosciences) and maintained in TeSR-E8 media (Stem Cell Technologies) at 37 °C with 5% CO2. Cells were checked daily for differentiation and were passaged every 4 days using Gentle Cell Dissociation Reagent (Stem Cell Technologies). iSPC72.3 were checked for karyotype and routinely checked for mycoplasma.
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!