CD4+ and CD4− ILC1 were sorted from freshly isolated PBMC as described above and ~10,000 cells were added to a single well in a 96-well round bottom plate coated with irradiated mouse embryonic fibroblasts (R&D Systems) and cultured for 7 days in cRPMI/10% HS with 20 ng/mL IL-2 (Biolegend) and 20 ng/mL IL-7 (Biolegend). Cells were then stained with Fixable Viability Dye eFluor 780 (eBioscience), then stained for surface markers and analyzed by flow cytometry.
Irradiated mouse embryonic fibroblasts
Manufactured by R&D Systems
Sourced in United States
Irradiated mouse embryonic fibroblasts are primary cells derived from mouse embryos. They have been exposed to radiation to inhibit cell division. These cells can be used as feeder layers to support the growth of other cell types in culture.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (3 mentions)
Interleukin 7 (il 7), by BioLegend (1 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by BioLegend (1 mentions)
Collagenase type 4, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
β fgf, by Thermo Fisher Scientific (1 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Esgro lif, by Merck Group (1 mentions)
Knockout dmem with 4.5 g l d glucose, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol bme, by Thermo Fisher Scientific (1 mentions)
Knockout serum replacement ksr, by Thermo Fisher Scientific (1 mentions)
Mem nonessential amino acids mem neaa, by Thermo Fisher Scientific (1 mentions)
C57bl 6j, by Jackson ImmunoResearch (1 mentions)
3 protocols using irradiated mouse embryonic fibroblasts
1
Expansion of CD4+ and CD4- ILC1 from PBMC
2
Maintenance and Passage of iPSCs
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iPSCs were maintained on irradiated mouse embryonic fibroblasts (R&D Systems) in iPSC medium containing KnockOut-DMEM/F12 supplemented with 20% KnockOut serum replacement (ThermoFisher), 1× GlutaMax (ThermoFisher), 100 μM non-essential amino acids (ThermoFisher), 100 μM β-mercaptoethanol (Sigma), and 10 ng/ml fibroblast growth factor (FGF)-β (ThermoFisher). Cultures were passaged with 1 mg/ml collagenase type IV (ThermoFisher) every week.
3
Generating Mouse Induced Pluripotent Stem Cells
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We utilized mouse induced pluripotent stem cell lines derived at the UMN Stem Cell Institute as previously described (Terzic et al. 2016 (link)) from primary cultures of C57BL/6J and CAST/EiJ E13.5 mouse embryonic fibroblasts obtained from Jackson Laboratories (Bar Harbor, ME, USA), using octamer-binding transcription factor-4 (Oct4), sex-determining region y-box 2 (Sox2), and Kruppel-like factor-4 (Klf4) as reprogramming factors, introduced using pMXs retroviral vectors.
Induced pluripotent stem cells were cultured on irradiated mouse embryonic fibroblasts (R and D Systems, Minneapolis, MN, USA) in miPSC medium: knockout DMEM with 4.5 g/ L d-glucose (Gibco, Grand Island, NY, USA), 10% knockout serum replacement (KSR) (Gibco), 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA), 1x MEM nonessential amino acids (MEM NEAA) (Gibco), 1x GlutaMAX (Gibco), 0.1 mM 2-mercaptoethanol (BME) (Life Technologies, Grand Island, NY, USA), and 0.02% ESGRO-LIF (Millipore, Billerica, MA, USA). Cells were incubated at 37°C in 5% CO2.
Induced pluripotent stem cells were cultured on irradiated mouse embryonic fibroblasts (R and D Systems, Minneapolis, MN, USA) in miPSC medium: knockout DMEM with 4.5 g/ L d-glucose (Gibco, Grand Island, NY, USA), 10% knockout serum replacement (KSR) (Gibco), 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA), 1x MEM nonessential amino acids (MEM NEAA) (Gibco), 1x GlutaMAX (Gibco), 0.1 mM 2-mercaptoethanol (BME) (Life Technologies, Grand Island, NY, USA), and 0.02% ESGRO-LIF (Millipore, Billerica, MA, USA). Cells were incubated at 37°C in 5% CO2.
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