WTC-10 human induced pluripotent stem cells (hiPSCs) were obtained from the lab of Bruce Conklin and genome edited using TALENs to endogenously express AP2-tagRFP-T at one allele at an internal loop of the µ2 subunit (Hong et al., 2015 (link)). We grew these cells on matrigel (hESC-Qualified Matrix, Corning) (80 µg/mL, 1 mL/well) in StemFlex (Thermo Fisher) with Penicillin/Streptomycin (Thermo Fisher), and passaged with Gentle Cell Dissociation reagent (EDTA-based clump passaging; StemCell Technologies). Parental and genome-edited cells were tested for mycoplasma and authenticated by STR profiling. For single-cell applications (genome editing, flow cytometry, transfections) we trypsinized the cells with the recombinant trypsin TrypLE Select (Thermo Fisher) and grew the cells in StemFlex supplemented with the specific rho kinase inhibitor RevitaCell (Thermo Fisher).
Hesc qualified matrix
Manufactured by Corning
The HESC-Qualified Matrix is a specialized lab equipment product developed by Corning. It is designed to meet the requirements of HESC (Highly Engineered Sterile Containers) qualification standards. The core function of the HESC-Qualified Matrix is to provide a reliable and controlled environment for a variety of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Revitacell, by Thermo Fisher Scientific (1 mentions)
Tryple select, by Thermo Fisher Scientific (1 mentions)
Stemflex, by Thermo Fisher Scientific (1 mentions)
Gentle cell dissociation reagent (gcdr), by STEMCELL (1 mentions)
Antibiotic antimyotic, by Thermo Fisher Scientific (1 mentions)
Intesticult, by STEMCELL (1 mentions)
Accutase, by Merck Group (1 mentions)
Hescs h9, by WiCell (1 mentions)
Mtesr1, by STEMCELL (1 mentions)
Knockout serum, by Thermo Fisher Scientific (1 mentions)
Fibroblast growth factor 2 fgf 2, by R&D Systems (1 mentions)
Collagenase type 4, by Thermo Fisher Scientific (1 mentions)
Gentle cell reagent for passaging, by STEMCELL (1 mentions)
Primocin, by InvivoGen (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Mtesr1 medium, by STEMCELL (1 mentions)
Mtesr plus kit, by STEMCELL (1 mentions)
6 protocols using hesc qualified matrix
1
Genome-edited hiPSC line expressing AP2-tagRFP-T
2
Isolation and Culture of Human Intestinal Organoids
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Fresh tissue from human jejunum
was stripped of muscle and connective tissue using scissors to isolate
the mucosa. Small biopsies were then taken and treated with PBS +
antibiotic-antimyotic (1:100) (Thermo Scientific) for 4 × 2 min
and then PBS + DTT (10 mM) for 3 × 2 min. The tissue was then
incubated in PBS + EDTA (2 mM), 4 °C, on rotation for 1 h and
then violently shaken to isolate the crypts. The crypts were then
seeded into matrigel (hESC-Qualified Matrix, Corning) and cultured
in Intesticult (Stem Cell) to keep the cells in a stem cell state.
If growing well and forming spheres after three passages, the cultures
were considered to be stable enteroid lines and were used for further
expansion and experiments.
was stripped of muscle and connective tissue using scissors to isolate
the mucosa. Small biopsies were then taken and treated with PBS +
antibiotic-antimyotic (1:100) (Thermo Scientific) for 4 × 2 min
and then PBS + DTT (10 mM) for 3 × 2 min. The tissue was then
incubated in PBS + EDTA (2 mM), 4 °C, on rotation for 1 h and
then violently shaken to isolate the crypts. The crypts were then
seeded into matrigel (hESC-Qualified Matrix, Corning) and cultured
in Intesticult (Stem Cell) to keep the cells in a stem cell state.
If growing well and forming spheres after three passages, the cultures
were considered to be stable enteroid lines and were used for further
expansion and experiments.
3
hESC Culture and CLEVER-seq Library Prep
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The hESCs (H9) were purchased from WiCell Institute. hESCs were cultured on the hESC-Qualified Matrix (Corning) in a feeder-free condition. And the complete E8 culture medium were used in cell culture. With regular passaging, we collected the colonies of hESCs and digested them into single-cell suspension using Accutase (Sigma) at 37°C for 1 h. Then the single cells of hESCs were used for CLEVER-seq library construction.
4
Culturing hESCs in mTeSR1 Medium
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H1 hESCs (XY) and modified cell lines were cultured in mTeSR1 (STEMCELL Technologies) using hESC-Qualified Matrix (Corning). Cells were incubated at 37°C with 5% CO2 and passaged every 3 to 4 days. Cells were routinely screened for mycoplasma.
5
Feeder-free iPSC Maintenance and Targeting
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iPSCs were maintained in feeder-free conditions in mTeSR1 medium (STEMCELL Technologies, 05850) on hESC-Qualified Matrix (Corning, 8774552), using Gentle Cell reagent for passaging (STEMCELL Technologies, 07174). For targeting, iPSCs were replated on mitotic inactivated mouse embryonic fibroblast (MEF) feeder cells in human iPS medium [20% knockout serum (Life Technologies), GlutaMAX (Life Technologies), nonessential amino acids (Life Technologies), fibroblast growth factor 2 (FGF2) (10 ng/ml; R&D Systems), 100 μM β-mercaptoethanol (Life Technologies), and Primocin (InvivoGen) in Dulbecco’s modified Eagle’s medium/D12 (Life Technologies)]. iPSCs on feeders were passaged with collagenase type 4 (1 mg/ml; Gibco). All iPSCs displayed a normal karyotype when analyzed by G-banding (Cell Line Genetics). Additional details pertaining to iPSC derivation, characterization, and culture are available for free download at https://crem.bu.edu/cores-protocols/ . iPSCs used here are available upon request from the CReM iPSC repository at https://stemcellbank.bu.edu .
6
Differentiating iPSCs into Microglia
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IPSCs were differentiated into microglia following an established protocol (McQuade et al., 2018 (link)). In brief, iPSCs were maintained in mTeSR Plus Kit (Stemcell Technologies # 05825) in 10 cm dishes. At DIV−1 of HPCs differentiation, 5- to 7-day-old iPSC cultures were then used to generate a cluster of differentiating 43 positive (CD43+) hematopoietic progenitor cells (HPCs) following a 12-day commercially available kit (STEMdiff Hematopoietic Kit; Stemcell Technologies # 05310). IPSCs were cleaned and 1/3 dish was gently dissociated with dispase (Stemcell Technologies # 07923) for 12–15 min at 37°C. IPSCs were then collected and spun down at 500 rpm for 1–2 min and resuspended in 2 mL of mTeSR Plus media with 20 μM ROCK inhibitor Y-27632 (Stemcell Technologies # 72304). Matrigel, hESC-Qualified Matrix (Corning # 354277), coated six-well plates were used to start the HPC differentiation. Using a 5-mL serological pipette, one drop or two drops of iPSCs were seeded per well into matrigel. Then, the next day, on DIV 0 of HPCs differentiation, wells with 80 small colonies per well were selected to start HPCs differentiation. IPSCs were fed following the manufacturer's instructions. On DIV 12 of HPCs differentiation, only the non-adherent HPCs were transferred to a new six-well plate to start microglia differentiation (DIV 12/0).
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