Matrigel was gently disrupted by pipetting and transferred together with the media to a 35 mm dish (121 V, Thermofisher Scientific). Single organoids were picked under a light microscope, collected in Eppendorf tubes and then mechanically disrupted and seeded in Matrigel in a single well of a 48-well plate (CLS3548-100EA, Qiagen). Selection media was overlaid in the well once the Matrigel had solidified.
35 mm dish
Manufactured by Thermo Fisher Scientific
Sourced in United States, Denmark
The 35 mm dish is a laboratory cell culture vessel commonly used in cell biology research. It provides a circular growth area with a surface area of approximately 8 square centimeters for the cultivation of cells. The dish is made of optically clear plastic and features a flat bottom design.
Automatically generated - may contain errors
Lab products found in correlation
Basic fibroblast growth factor (bfgf), by Fujifilm (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Methylcellulose media, by R&D Systems (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Ibright fl1500, by Thermo Fisher Scientific (1 mentions)
Insulin like growth factor igf 1, by Fujifilm (1 mentions)
Phosphate buffered saline (pbs), by Welgene (1 mentions)
Epidermal growth factor (egf), by Fujifilm (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Vascular endothelial growth factor (vegf), by R&D Systems (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Heparin, by Ajinomoto (1 mentions)
H4236, by STEMCELL (1 mentions)
Pluronic acid, by Merck Group (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Bovine collagen 1, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Fluo 3 am, by Biotium (1 mentions)
Fv300, by Olympus (1 mentions)
Phenylalanine, by Merck Group (1 mentions)
14 protocols using 35 mm dish
1
Organoid Isolation and Expansion
2
Bone Marrow and Spleen Colony Assay
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A total of 5 × 105 cells from the bone marrow or spleen were added to 5 mL of methylcellulose media (R&D System), and 1 mL was poured into a 35 mm dish (Thermo Fisher Scientific). These were incubated in a humid atmosphere at 37°C with 5% CO2, and the number of CFU-G, CFU-M, and CFU-GM was enumerated on day 14.
3
Copper-catalyzed Azide-Alkyne Cycloaddition
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SCCVII cells (3.0 × 104 cells) were seeded on a 35 mm dish (#150460, Thermo Scientific), allowed to adhere overnight, and incubated for probe labeling as described above. After 2 h incubation in a probe-free medium, the cells were washed with DPBS(–) twice and lysed by adding cell lysis buffer (#9803, Cell Signaling Technology) and scraping. The lysate was centrifuged, and the supernatant was collected. To 86 μL of lysate, a pre-mixed cocktail of 5.5 μL of DMSO, 2 μL of 100 mM BTTAA in DMSO, and 2.5 μL of 1 mM Cy5-PEG4-azide or CF647 azide in DMSO was added. After adding 3 μL of 100 mM CuSO4 in H2O and 1 μL of 500 mM NaAsc in H2O, the lysate was shaken in the dark at 700 rpm and 25 °C for 1 h. After that, 2 μL of 0.5 M EDTA (pH 8.0) was added to quench the reaction, and proteins in the lysate were precipitated by acetone, and the pellet was washed with acetone to remove the CuAAC reagents. After resolubilization by cell lysis buffer, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with SuperSep Ace Mini, 10–20%, 17well (FUJIFILM Wako). The in-gel fluorescence was imaged by a gel imager (iBright FL1500, Invitrogen), followed by Coomassie Brilliant Blue (CBB) staining.
4
Satellite Cell Colony Formation Assay
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Freshly isolated satellite cells were seeded in a 35 mm dish (Cat# 150460, Thermo Fisher Scientific) to form colonies, which were counted 5 days after culture under a microscope.
5
Isolation and Characterization of EPCs from HUCB
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Human CD34+, CD34−, or both CD34+ and CD34− cells isolated from HUCB were cultured in methylcellulose-containing medium, H4236 (StemCell Technologies, Vancouver, Canada), supplemented with 20 ng/mL stem cell-derived factor (Kirin, Tokyo, Japan), 50 ng/mL vascular endothelial growth factor (VEGF; R&D Systems, Minneapolis, MN, USA), 20 ng/mL interleukin (IL)-3 (Kirin), 50 ng/mL basic fibroblast growth factor (bFGF; Wako, Osaka, Japan), 50 ng/mL epidermal growth factor (EGF; Wako), 50 ng/mL insulin-like growth factor (IGF)-1 (Wako), 2 U/mL heparin (Ajinomoto, Tokyo, Japan), and 10% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA) on a 35-mm dish (Thermo SCIENTIFIC, Rockford, IL) for 8 d. The cell density of each sample was 5×102 cells per dish or was adjusted depending on the assay. The EPCs were identified as small EPC-CFUs or large EPC-CFUs by visual inspection using a light microscope (OLYMPUS, Tokyo, Japan) under 40x magnification. Small EPC-CFUs were composed of round adhesive cells, and large EPC-CFUs were composed of spindle-shaped cells. Nonattached cells were isolated as small EPCs by washing with PBS (WELGENE, Daegu, Korea), while attached cells were harvested as large EPCs by treatment with 5 mM EDTA (Sigma-Aldrich, St. Louis, MO) in PBS (5 mmol/L) for 5 min at 37°C.
6
Micropatterned Substrates for Adherent Cell Culture
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hCECs were seeded onto commercial micropatterned glass substrates (CYTOO, Grenoble, France) which have been customized to contain an array of circular micropatterns (65 and 80 µm diameter) on which cells can adhere. The area surrounding the circular micropatterns was selectively passivated to attenuate cell and protein adhesion. Before cell seeding, the micropatterned substrates were coated with a mixture consisting of Fibronectin (Sigma-Aldrich, Burlington, MO, USA F1141 0.03 mg/mL), Bovine Collagen I (5 mg/mL Gibco, Agawam, MA, USA, A1064401, 0.01 mg/mL) and Bovine Serum Albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA, A331, USA, 0.01 mg/mL) in 1× HBSS, and incubated at 37 °C for 24 h. Coated micropattern substrates were transferred to a 35 mm dish (Thermo Scientific, China, 150460), which was passivated with 10% Pluronic acid (Sigma-Aldrich, St. Louis, MO, USA, P2443) in 1× HBSS overnight. Cells were seeded onto the micropatterned substrates at a density of 300,000 cells/mL. Unattached cells were removed after 2 h by washing with 1× HBSS 5 times, followed by passivation with 0.2% Pluronic acid for 10 min, and finally replaced with 2 mL of complete medium. The cells were cultured on the micropattern substrates for 24–48 h based on the assay being performed.
7
Bone Marrow Cell Culture in MC Medium
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Using a micropipette, 1 μl of the medium containing 20000 BM cells was injected into a 35-mm dish (Thermo scientific, 153066) of MC medium and then cultured in a CO2 incubator at 37 °C in 5% CO2 (Fig. 1 a).![]()
MC medium method. Schematic model of the experiments. Dispersed-state BM cells (including blood cells) were injected into the 3% MC medium (a). Liquid culture medium absorbed to the surrounding MC medium. Remaining BM cells were aggregate within approximately 30 min (b–e). After the culture, organized BM cells (BM-like tissue) could be removed from the MC medium within 24 h. BM-like tissues were embedded in alginate gel, and paraffin sections were made. Scale bars = 100 μm.
8
Measuring Calcium Levels in Coronary Artery SMCs
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Ca2+i in human coronary artery SMCs was determined with a confocal laser scanning microscope (Olympus FV300, Tokyo, Japan) as described previously [53 (link)]. Briefly, the cells were cultured in a 35 mm dish (Thermo Fisher Scientific, MA, USA) for 48 h and then loaded with 5 μmol/L fluo-3 AM (Biotium, Hayward, CA, USA) for 30 min at 37°C, incubated in serum-free bath solution for 40 min in the dark, and then washed with bath solution. The bath solution contains (in mmol/L) NaCl 140, KCl 5.0, MgCl2 1.0, CaCl2 1.8, HEPES 10, glucose 10 (pH adjusted to 7.3 using NaOH), and 5% FBS. Fluorescence was excited by an argon laser at 488 nm and emission was detected at 525 nm. The Ca2+i level was expressed as ratio of basal fluorescence intensity.
9
Real-time Bioluminescence Recording of Circadian Rhythms
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Real-time bioluminescence recording was conducted according to a previously described method with minor modification (Lee et al., 2016 (link)). A day before the real-time recording, MEF cells were seeded in 35 mm dish (Fisher Scientific, USA) at 50% confluence. After 24 h, culture media was changed to synchronization media with 200 nM dexamethasone (Sigma-Aldrich, USA) to synchronize circadian rhythms of cell population (So et al., 2009 (link)) for 2 h. To record the real-time bioluminescence, synchronized cells were cultured in recording media with 200 μM D-luciferin (Promega, USA) and culture dishes were sealed with parafilm (Sigma-Aldrich). Next, the dishes were placed in Kronos, a real-time bioluminescence recording device (ATTO, Japan), at 37°C and 5% CO2, and luciferase (Luc) activity in each dish was measured for 1 min every 10 min for 3 to 5 days. For the dose-response effect of 4-OH-PPA and PPA, the cells were treated with four different doses, 0.125 mM, 0.5 mM, 1 mM, and 2 mM of 4-OH-PPA (Sigma-Aldrich) and PPA (Sigma-Aldrich) using two different administration modes, either immediately after the nadir or the peak of the oscillation (Fig. 1B ). To verify the effect of 4-OH-PPA and PPA, their precursors, tyrosine (Sigma-Aldrich) and phenylalanine (Sigma-Aldrich), were examined initially as the control experiments.
10
Isolation and Culture of Primary Corneal Epithelial Cells
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VDR WT and VDR KO mice were obtained and bred from the Jackson Labs (Strain: B6.129S4-Vdrtm1Mbd; Bar Harbor, ME, USA). All animal studies were approved by the University Institutional Animal Care and Use Committee, and animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Primary corneal epithelial cell cultures were established using a modification of the established explant culture method.38 (link),39 (link) Briefly, eyes were enucleated, and the cornea was washed with Ca++-free PBS (pH 7.2). Each cornea was cut in half and placed in a 35-mm dish (Fisher Scientific) with the epithelial side up. One and half milliliters of DMEM with 10% serum containing 40 μg/mL gentamicin, 1% ITS, and 100 ng/mL cholera toxin (LIST Biological Laboratories, Inc., Campbell, CA, USA) was added and the tissue was cultured in a humidified incubator at 37°C with 5% CO2. Culture medium was replaced every 2 days. Cells were passged using 0.25% trypsin (Fisher Scientific), and subculturing in DMEM with 3% serum containing 40 μg/mL gentamicin, 1% ITS, and 100 ng/mL cholera toxin.
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