Human hepatocyte suspensions (see Isolation of EpCAM+ cell from primary human liver) were washed as described, spun down and resuspended in 35 ml Advanced DMEM/F12 (GIBCO) + 13.5 ml Percoll (GE healthcare, density 1.130 g/ml) + 1.5 ml 10x HBSS (GIBCO). Cells were pelleted at 100 g for 10 min and washed 3 times in Advanced DMEM/F12 (GIBCO). Viable cells were counted with Trypan blue and 10.000 viable cells per 50 ul drop were seeded into matrigel. Remaining cells were stained for EpCAM as described above (see Isolation of EpCAM+ cells from primary human liver) or seeded onto collagen coated tissue culture plates for subsequent determination of cytochrome 3A4 activity. To measure Cyp3a4 in primary hepatocytes, the seeded cells were cultured in Williams E medium (GIBCO) containing Hepatocyte plating supplement pack (GIBCO) for 4 days with daily medium changes. On day 0 and day 4 the cells were incubated with Luciferin-PFBE substrate (50 μM) and Cytochrome P450 activity was measured using the P450-Glo Assay Kit (Promega) according to manufacturer’s instructions and normalized to the number of cells in the plate. HepG2 cells cultured in the same medium served as controls.
Advanced dmem f12
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Japan, Germany, France, Netherlands
Advanced DMEM/F12 is a cell culture medium formulated for the growth and maintenance of a variety of cell types, including stem cells and other sensitive cell lines. It is a modified version of the standard DMEM/F12 medium, providing a more optimized nutrient and growth factor composition. The core function of Advanced DMEM/F12 is to support the in vitro culture of cells while maintaining their viability and proliferative capacity.
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Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (205 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (125 mentions)
Hepes, by Thermo Fisher Scientific (102 mentions)
B27 supplement, by Thermo Fisher Scientific (102 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (83 mentions)
Matrigel, by Corning (72 mentions)
N acetylcysteine, by Merck Group (72 mentions)
N2 supplement, by Thermo Fisher Scientific (57 mentions)
Noggin, by Thermo Fisher Scientific (53 mentions)
Matrigel, by BD (52 mentions)
Nicotinamide, by Merck Group (49 mentions)
L glutamine, by Thermo Fisher Scientific (44 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (43 mentions)
A83 01, by Bio-Techne (36 mentions)
Primocin, by InvivoGen (25 mentions)
Matrigel, by Thermo Fisher Scientific (25 mentions)
Penicillin, by Thermo Fisher Scientific (25 mentions)
Neurobasal medium, by Thermo Fisher Scientific (24 mentions)
Streptomycin, by Thermo Fisher Scientific (23 mentions)
Epidermal growth factor (egf), by R&D Systems (22 mentions)
505 protocols using advanced dmem f12
1
Hepatocyte Isolation, Culture, and Cytochrome P450 Assay
2
Tonsil Organoid Culture Protocol
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The tonsil samples were chopped and washed with Dulbecco's phosphate-buffered saline (D-PBS; Welgene, Daegu, Korea) and then enzymatically digested with 1 mg/mL collagenase II (Gibco) in advanced DMEM/F12 (Gibco) for 2 h at 37 °C. After digestion, isolated cells were embedded in Matrigel (Corning Inc., Corning, NY, USA) in a 48-well plate (SPL Inc., Seongnam, Korea) and incubated at 37 °C for 10 min to polymerize the matrices. Tonsil organoids were cultured in advanced DMEM/F12 supplemented with Antibiotic–Antimycotic, Glutamax (Thermo Fisher Scientific), B27 (Invitrogen, Carlsbad, CA, USA), 10% conditioned media from Cultrex HA-R-spondin1-Fc 293T cells (R&D Systems, Minneapolis, MN, USA), and the following growth factors: 50 ng/mL recombinant murine HGF (Peprotech, Rocky Hill, NJ, USA), 100 ng/mL noggin (ProSpec, St. Paul, MN, USA), 20 nM A83-01 (Sigma), 50 ng/mL human FGF10 (ATGen, Seongnam, Korea), 20 ng/mL human bFGF (Peprotech), 10 μM prostaglandin E2 (BioGems, Westlake Village, CA, USA), and 10 mM nicotinamide (Sigma). For passage, organoids were dissociated by incubation in 0.25% trypsin-EDTA (Invitrogen, Waltham, Massachusetts, USA) every 7–10 days depending on the number and size of organoids. For the first 2 days at every passage, 10 μM Y-27632 (Tocris Biosciences, Bristol, UK) was added to the culture medium.
3
Embryo Culture and Differentiation
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E3.5 embryos were recovered by flushing uteri with M2 medium. Zona pellucida were removed by brief exposure to acidic Tyrode’s solution (Sigma). Zona-freed blastocysts were seeded on ibiTreat microscopy plastic μ plates (Ibidi), filled with prewarmed IVC1 medium (Advanced DMEM/F12 (GIBCO) containing 20% FCS (Biosera) and supplemented with 2 mM L-glutamine (GIBCO), 1 mM sodium pyruvate (GIBCO), penicillin (25 units/ml)/streptomycin (25 μg/ml) (GIBCO,), 1 × ITS-X (Invitrogen), 8 nM β-estradiol (Sigma), 200 ng/ml progesterone (Sigma), and 25 μM N-acetyl-L-cysteine (Sigma). In the following 36–48 hr, embryos attached to the surface of the plate as TE differentiated into giant cells. The medium was then exchanged and emerging egg cylinders cultured in serum-free, chemically defined IVC2 medium (Advanced DMEM/F12 (GIBCO) containing 30% KSR (KnockOut Serum Replacement, GIBCO) and supplemented with 2 mM L-glutamine (GIBCO), 1 mM sodium pyruvate (GIBCO), penicillin (25 units/ml)/streptomycin (25 μg/ml) (GIBCO), 1 × ITS-X (Invitrogen), 8 nM β-estradiol (Sigma), 200 ng/ml progesterone (Sigma), and 25 μM N-acetyl-L-cysteine (Sigma). Embryo culture was performed at 37°C in 5% CO2. Procedures used for imaging living or fixed preparations of cultured embryos are given in the Extended Experimental Procedures .
4
Establishing Cancer Organoid Cultures
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Cancer organoid cultures were established and propagated as previously described (Sato et al., 2011 (link); Schütte et al., 2017 (link)). Briefly, resected tumor samples were enzymatically digested with Collagenase IV (C9407, Sigma-Aldrich), DNaseI (A3778,0050, AppliChem) and Dispase (07913, Stem Cell Technologies) at 37°C for 60 min. Suspensions were washed, filtered, and depleted of red blood cells using Red Blood Cell Lysis Solution (00-4333-57, Invitrogen). Cells were mixed with phenol-red free growth factor-reduced Matrigel (356231, Corning) and seeded into 24-well plates. Solidified droplets were overlaid with culture medium consisting of Advanced DMEM/F12 (12634-010, Gibco) supplemented with 1% penicillin/streptomycin, 1% HEPES buffer (1064859, Fisher Scientific), 1% Glutamax, 1x N2 (#17502-048, Invitrogen), 1x B27 (17504-044, Invitrogen), 50 ng/mL EGF (E9644, Sigma), and 1mM N-acetylcysteine (A9165-5G, Sigma) and maintained at 37°C. Organoids were released from Matrigel and passaged by adding 5 mL Advanced DMEM/F12 followed by centrifugation and digestion of pellets with TrypLE Express (12604-013, Gibco) (Regan, 2022 (link)).
5
Organoid Generation from Primary Hepatocytes
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Three lots of PHHs (lots DOO; Celsis, HC10-10; XENOTECH, HC4-24; XENOTECH) were used to generate organoids (Table S1 ). PHHs were washed with cold Advanced DMEM/F12 (Thermo Fisher Scientific) and spun at 400 g for 5 min. The cell pellet was mixed with Matrigel (growth factor reduced, Corning) and 1 × 104 cells were seeded per well in a 24-well plate. After the Matrigel had solidified, 500 μl of organoid expansion medium was added to each well. The organoid expansion medium was prepared as described in a previous report [13 (link)]. Briefly, Advanced DMEM/F12 was supplemented with 1% Antibiotic Antimycotic Solution and 1 × GlutaMAX (GIBCO), 10 mM HEPES (Nacalai Tesque), 2% B27 supplement (GIBCO), 1.25 mM N-Acetylcysteine (Sigma), 10 mM Nicotinamide (Sigma), 10 nM recombinant gastrin (Merk), 50 ng/ml EGF (R&D), 10% R-Spondin1 conditioned medium (homemade), 100 ng/ml recombinant human FGF10 (peprotech), 25 ng/ml recombinant human HGF (R&D), 5 μM A83-01 (Wako), and 10 μM Forskolin (Wako). During cultivation, the medium was refreshed every 3 days. For the establishment of the organoids, the medium was supplemented with 25 ng/ml Recombinant Noggin (R&D), 7.5 ng/mL Recombinant Wnt3a (R&D) and 10 μM Y27632 (Wako) for the first 3–4 days. Passage was performed in a 1:3 split ratio once every 10–14 days. Organoids passaged 5–10 times were used in all experiments.
6
Isolation and Culture of Human Intestinal Organoids
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Intestinal crypts were isolated from intestinal biopsies as previously described15 (link),28 (link), resuspended in Matrigel (Corning) and polymerized at 37 °C. Human intestinal organoids (HIO) were grown in Human intestinal stem cell (HISC) medium15 (link),28 (link), passaged at a ratio of 1:10 to 1:12 every 10–14 days and used between passages 4 and 15. The final composition of the HISC medium is shown at Table 2 . HISC medium was produced in Advanced DMEM/F-12 (Gibco, #12634) supplemented with 1%v/v Glutamax (Gibco, #35050), 1%v/v HEPES (Gibco, #15630) and 1%v/v Pen/Strep (Gibco, #15140). 2X-concentrated HISC medium was diluted in a 1:1 ratio in Wnt3a conditioned medium. To produce Wnt3a conditioned medium, L Wnt3a cells (CRL-2647) were grown in Advanced DMEM/F-12 (Gibco, #12634) supplemented with 10% FBS (Bovogen, #SFBS), 1%v/v Glutamax (Gibco, #35050), 1%v/v HEPES (Gibco, #15630) and 1%v/v Pen/Strep (Gibco, #15140) in presence of 125 µg/mL Zeocin (Thermo Fisher, #R25001).
7
Neuronal Differentiation of LUHMES Cells
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Lund human mesencephalic (LUHMES) cells were a kind gift from M. Leist and D. Scholz, University of Konstanz, Germany. LUHMES cells are conditionally immortalized cells with neuronal features that can be differentiated by shutting-down the myc transgene leading to uniformly post-mitotic neurons within 5 d84 (link). LUHMES cells were plated in 50 μg/ml poly-L-ornithine (Sigma-Aldrich) and 1 μg/ml fibronectin (Sigma-Aldrich) coated flasks/plates and cultured in proliferation medium containing advanced DMEM/F12 (Gibco), 2 mM L-glutamine (Gibco), 1% N-2 supplement (Invitrogen) and 40 ng/ml recombinant basic fibroblast growth factor (bFGF, R&D Systems) at 37 °C and 5% CO2. For differentiation, 2 × 106 cells were seeded in T75 flasks in proliferation medium. After 48 h proliferation medium was replaced by differentiation medium containing advanced DMEM/F12 (Gibco), 2 mM L-glutamine (Gibco), 1% N-2 supplement (Invitrogen), 1 μg/ml tetracycline (Sigma-Aldrich), 1 mM dibutyryl cAMP (Sigma-Aldrich) and 2 ng/ml glial cell line-derived neurotrophic factor (GDNF, R&D Systems). After additional 48 h, pre-differentiated LUHMES cells were re-seeded at a density of 1.3 × 105 cells/cm2 and kept for another 72 h in differentiation medium (5 d differentiation) prior to treatment.
8
Organoid-based Evaluation of Wnt Signaling
9
Neuronal Spheroid Formation from hBM-MSCs
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To obtain neuronal spheroids, hBM-MSCs were cultured in a low-attachment dish (Corning) with serum-free sphere-forming medium comprising advanced DMEM/F-12 (1:1) (Gibco) supplemented with 0.5 mM l-glutamine, N2 supplement (Gibco), 20 ng/mL recombinant human EGF (Wako), 20 ng/mL human FGF-2 (Wako), and 2% B27 (Gibco). Following the formation of spheroids, neuronal spheroids obtained using low-attachment dishes or MSC spheroids obtained under shaking-culture conditions were seeded on fibronectin (Wako)-coated 6-well chambers (Matsunami Glass Ind. Inc., Kishiwada, Osaka, Japan) using induction medium comprising advanced DMEM/F12 (1:1) (Gibco) supplemented with 10% FBS (Hyclone, GE Healthcare), N2 supplement (Gibco), 1% P/S (Wako), and 10 mM HEPES (Dojindo Molecular Technologies, Inc.) (Morikawa et al., 2009b (link); Choi et al., 2017 (link)). The medium was changed every 3–4 days until day 10.
10
Generation of Inducible Microglia-like Cells
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Microglia were differentiated as previously described. Briefly, hiPSCs expressing six inducible transcription factors (MAFB, CEBPα, IRF, PU1, CEBPβ, IRF5) were grown in Essential 8™ Basal Medium (Gibco/Thermo Fisher Scientific, Cat. No. A15169–01), 10 μM ROCK inhibitor, and 2 μg/ml Doxycycline on Matrigel and 10 cm Poly-D-Lysine coated plates at a density of 1.5 million cells per dish. After 2 days, cells were grown in a differentiation media consisting of Advanced DMEM/F12 (Gibco/Thermo Fisher Scientific, Cat. No. 35050–061), 1X GlutaMAX, 2ug/ml doxycycline, 100 ng/mL Human IL34 (Peprotech; Cat. No. 200–34) and 10 ng/mL Human GM-CSF (Peprotech; Cat. No. 300–03). Two days later, media was exchanged for iTF-Microglia media consisting of Advanced DMEM/F12, 1X GlutaMAX, 2 μg/mL doxycycline, 100 ng/mL Human IL-34, 10 ng/mL Human GM-CSF, 50 ng/mL Human M-CSF (Peprotech; Cat. No. 300–25) and 50 ng/mL Human TGFB1 (Peprotech; Cat. No. 100–21C). On day 8, cells were dissociated with TypLE express (Gibco/Thermo Fisher Scientific, Cat. No. 12605–028) and seeded into iAssembloids.
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