Around 100 freshly isolated crypts were counted and embedded in 50 μl of undiluted Matrigel (356231; Cultek) and culture DMEM/F12 media (D8437; Sigma-Aldrich) supplemented with Pen/Strept, 1× (2 mM) Glutamax (35050038; Gibco or Life Technologies/Thermo Fisher Scientific), 10 mM Hepes (15630049; Gibco or Life Technologies/Thermo Fisher Scientific), 2 mM N-acetyl cysteine (A8199-10G; Sigma-Aldrich), 1× B27 supplement (17504-044; Life Technologies), 10 mM Nicotinamide (N0636-100G; Sigma-Aldrich), 50 ng/ml of recombinant mEGF (PMG8044; Gibco), 100 ng/ml of recombinant Nogging (250-38; PeproTech), 3.5 μM CHIR99021 (GSK3 inhibitor; 2520691; PeproTech), 1 μg/ml murine R-spondin 1 (120-38; Peprotech), 10 μM Y-27632 inhibitor (1293823; PeproTech), and 1% Normocyn (ant-nr-1; InvivoGen; Barriga et al., 2017 (link)). Notably, CHIR99021 (GSK3 inhibitor; 2520691; PeproTech) was kept in culture for 12 h to establish the culture, and the medium was replaced without this inhibitor. The media was changed every 3 d. Depending on the experiment, the crypt culture media was supplemented with 1.5 μg/ml doxycycline (D9891; Sigma-Aldrich) or 1 μM of 4-OHT (H6278; Sigma-Aldrich). Organoids were paraffin-embedded and processed for IF and IHC following the protocols described below.
D8437
Manufactured by Merck Group
Sourced in United States
The D8437 is a laboratory equipment product offered by Merck Group. It is designed to perform a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f12, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Doxycycline, by Merck Group (1 mentions)
Murine r spondin 1, by Thermo Fisher Scientific (1 mentions)
Normocyn, by InvivoGen (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
4 oht, by Merck Group (1 mentions)
Chir99021, by Thermo Fisher Scientific (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
Nicotinamide, by Merck Group (1 mentions)
1321n1 cell line, by Merck Group (1 mentions)
Sh sy5y cell line, by American Type Culture Collection (1 mentions)
Ls174t, by Merck Group (1 mentions)
Ccd841 con, by Merck Group (1 mentions)
Sw1116, by American Type Culture Collection (1 mentions)
Hca 2, by American Type Culture Collection (1 mentions)
Ls174t, by American Type Culture Collection (1 mentions)
P4083, by Merck Group (1 mentions)
6 protocols using d8437
1
Murine Small Intestinal Organoid Culture
2
Cell Culture of Common Cell Lines
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The human embryonic kidney HEK293 cell line (85120602, Sigma-Aldrich) and human astrocytoma 1321N1 cell line (86030402, Sigma-Aldrich) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; 41966029, ThermoFisher scientific) containing 10% fetal bovine serum (FBS; 10270106, ThermoFisher scientific), and incubated at 37°C in a humidified atmosphere of 5% CO2. The human neuroblastoma SH-SY5Y cell line (CRL-2266, ATCC) was cultured in a 1:1 mixture of DMEM and DMEM/F12 (D8437, Sigma-Aldrich) supplemented with 10% FBS, and incubated at 37°C in a humidified atmosphere of 5% CO2.
3
Colorectal Cancer Cell Lines Cultivation
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Three colorectal cancer cell lines were used in the experiments: HCA-2 (Duke C), LS 174T (Duke B), and SW1116 (Duke A), together with a normal cell line, CCD 841 CoN. All cell lines were supplied by ATCC (American Type Culture Collection ATCC®, Old Town Manassas, VA, USA). To ensure optimal conditions for cell growth, appropriate culture media were used: Eagle’s minimum essential medium (EMEM) (ATCC 30-2003) for CCD 841CoN and LS 174T cell lines, and Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 Ham (DMEM), Sigma-Aldrich D8437). For SW1116 and HCA-2 cell lines, both media were supplemented with 10% foetal bovine serum ((FBS), ATCC 30-2020) and 1% penicillin–streptomycin–neomycin stabilised solution (Sigma-Aldrich P4083).
4
Cultivation of A549 Lung Epithelial Cells
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Exponentially growing type II lung epithelial cells (A549 ATCC® CCL-185) were cultivated at 37 °C under 5% CO2, in 1:1 DMEM:F-12 medium (Sigma-Aldrich D8437, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS, Gibco, 10270-106, Thermo Fisher Scientific, Waltham, MA, USA). Cells were passaged approximately every 3–4 days, to prevent cells from reaching confluence. Subculture was carried out by first washing the cell monolayers with phosphate buffered saline (PBS) followed by incubation with TrypLE Trypsin-EDTA (0.05%, Gibco, 25300-062, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C as above. Cell number and viability were determined by staining with trypan blue and counting with a hemocytometer.
5
Isolation and Culture of Mouse Organ of Corti
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After the skin was cleaned with 75% alcohol, the postnatal day 3 C57BL/6 mice were euthanized by cold CO2 inhalation and decapitated painlessly. The skin was peeled off the scalp, and the skull was cut into two pieces posterior to the eye. The cochleae were removed and placed in Leibovitz's L-15 (Procell, PM151012, Wuhan, China). The organ of Corti was separated away from the stria vascularis and then adhered to separate culture plates. Each plate contained 1 ml/well of culture medium, which comprised Dulbecco's modified Eagle's medium/Ham's F-12 (DMEM/F-12; 1 : 1 Mix) (Sigma-Aldrich, D8437, St. Louis, MO, USA) with 10% bovine serum albumin (Sigma-Aldrich, A1595, St. Louis, MO, USA) and 30 U/mL penicillin G (Sigma-Aldrich, P3032, St. Louis, MO, USA). The organ of Corti explants was cultured in a humidified atmosphere with 5% CO2 at 37°C.
6
Assessing Decellularized Nucleus Pulposus Cytotoxicity
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Decellularized NP samples were eluted for 72 hours at 37°C in 10 mL of medium containing 89% DMEM:F12 (D8437, Sigma), 10% fetal bovine serum (16000044, Life Technologies, Carlsbad, CA, USA), and 1% penicillin-streptomycin (51606, EMD Millipore, Billerica, MA, USA). A total of 16 dNPs were eluted from four distinct spines (n=3-6 per spine). After 72 hours, eluted medium from each sample was removed and 250 _L was added to a 48-well plate containing ~7,500 human neonatal dermal fibroblasts (PCS-201-010, ATCC, Manassas, VA, USA) in triplicate per sample. Control wells containing fresh medium were also cultured (n=3). After 48 hours, a metabolic assay (DAL1100, Thermo Fisher) was performed to assess the metabolic activity of eluted dNP samples medium versus control medium and determine if cytotoxic residuals were present.
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