5‐ALA (Sigma‐Aldrich, St. Louis, MO, USA) was reconstituted to 2.9 M in sterile 0.22 μm filtered 1X phosphate buffered saline (PBS; ThermoFisher Scientific, Waltham, MA, USA) and stored at −20°C. On day 0, all three cell lines were plated at a density of 1 million cells on 100 mm diameter plates (Nunc EasYDish; Thermo Fisher Scientific, Waltham, MA) in 10 ml of media. On day 1, the media was replaced with EV‐free media: DMEM, containing 3% EV‐depleted foetal bovine serum (EV‐free FBS; Life Technologies Corporation, Carlsbad, CA, USA) and 1% Pen/Strep. The cells were also dosed with a final concentration of 0.8 mM 5‐ALA or mock‐dosed with 1X PBS of the same volume, according to a previously identified optimal in vitro dosage (Jones et al., 2019 (link)). On day 2, conditioned media was collected for analysis and processed as described below. All the experiments were performed in a dark room, with the plates and conical tubes always covered with aluminium foil to minimize PpIX photobleaching as previously established (Jones et al., 2019 (link)). Cell viability, maintained at 80% and above, was assessed using the Countess II FL Automated Cell Counter (ThermoFisher Scientific, Waltham, MA, USA).
Nunc easydish
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nunc EasYDish is a disposable cell culture dish designed for routine cell culture applications. It features a flat bottom and a slanted wall design to facilitate easy handling and observation of cells under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Countess 2 fl automated cell counter, by Thermo Fisher Scientific (1 mentions)
5 ala, by Merck Group (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
70 μm cell strainer, by Greiner (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Collagenase, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Fujifilm (1 mentions)
Crystal violet, by Fujifilm (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Scepter 2.0 cell counter, by Merck Group (1 mentions)
6 protocols using nunc easydish
1
5-ALA Dosing and Conditioned Media Collection
2
Isolation and Cryopreservation of Human Synovial MSCs
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This study was approved by the Medical Research Ethics Committee of Tokyo Medical and Dental University, and informed consent was obtained from all study subjects. Human synovium was harvested from the knees of four donors during total knee arthroplasty operations performed to treat osteoarthritis. The synovium was minced and digested in a solution of 3 mg/mL collagenase (Sigma-Aldrich Japan, Tokyo, Japan) at 37 °C for 3 h, and the digested cells were filtered through a 70 μm cell strainer (Greiner Bio-One GmbH, Frick-enhausen, Germany). The obtained nucleated cells were cultured in a growth medium consisting of α-MEM (Thermo Fisher Scientific, Rockford, IL, USA), 1% antibiotic-antimycotic (Thermo Fisher Scientific), and 10% fetal bovine serum (FBS, Thermo Fisher Scientific) under 5% CO2 at 37 °C. After 14 days, the human synovial MSCs were detached with 0.25% trypsin and 1 mM EDTA, harvested, and cryopreserved at − 80 °C for future use at a concentration of 106 cells/mL in a freezing vessel (BICELL, Japan Freezer, Tokyo, Japan). The cryopreservation medium consisted of 95% growth medium plus 5% dimethyl sulfoxide (DMSO, Fujifilm Wako Pure Chemical, Osaka, Japan). For colony formation assays, 100 cells, at 1.67 cells/cm2 in a 60 cm2 dish (Nunc EasYDish, Thermo Fisher Scientific) were cultured for 14 days and then stained with crystal violet (Fujifilm Wako Pure Chemical).
3
Caco2 Cell IFNγ Treatment Kinetics
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In a 150 mm Petri-dish (Nunc EasYDish, Thermo Scientific), Caco2 cells (seeding density: 8000 cells/cm3) were treated with 10 ng/ml IFNγ (PeproTech, Rocky Hill, NJ, USA) for 3, 7, and 11 days. The cells were washed with DPBS three times and stored at −80 °C until use.
4
Murine Osteoblast Mechanical Stimulation
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Murine MC3T3-E1 osteoblasts (3 × 105 cell/dish) were incubated in cell culture dishes (3.5-cm in diameter; Thermo Scientific™ Nunc™ EasYDish™; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with DMEM and 10% fetal bovine serum till 80% confluence. A PMVS Ceramic piezoelectric transducer was attached to the bottom of each cell culture dish using adhesive tape. Sixty Hz, 120 Hz, or 180 Hz PMVS was applied to each dish for 1 h or 3 h. Cell cultures incubated in dishes without PMVS were designated in controls. All protocols were performed in a 5% O2, 37 °C humidified incubator.
5
Isolation and Culture of Fish Leukocytes
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Cells from DI and HK were harvested and grown at 12°C in Leibovitz’s L-15 Medium (L-15; Sigma, Oslo, Norway) as described previously by Park, Zhang (8 (link)). Briefly, the isolated DI or HK leukocytes were allowed to adhere on a cell culture dish (Nunc EasYDish, Thermo Fisher Scientific, Oslo, Norway) with 2 mL L-15+ (L-15 medium with 50 U/mL penicillin, 50 μg/mL streptomycin, 2% fetal bovine serum and 10 U/mL heparin) for 2 days at 12°C. Thereafter the medium was removed and the adherent cells on the culture dish were detached by washing three times with 1.5 mL ice-cold PBS (Sigma) supplemented with 5 mM EDTA (Sigma). The cells were centrifuged (500 × g, 5 min, 4°C) and re-suspended with 2 mL L-15+. Then, the adherent intestinal cells or adherent head kidney cells were counted using a portable cell counter (Scepter™ 2.0 cell counter, EMD Millipore, Darmstadt, Germany) for further analysis.
6
Ultrasound-Mediated Endosomal Escape Visualization
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To observe endosomal escape, the OCSCs were seeded in the 35 mm cell culture plate (Thermo Scientific™ Nunc™ EasYDish™, Waltham, MA), and then incubated with 10 μL PSP@MB and 10 μg FITC-labeled pDNA for 1 min at 37 °C. The OCSCs were exposed by ultrasound with parameters (0.8 W/cm2, 20%, 1 min, and 1 MHz). There were four groups: (A) Control group, (B) PSP@MB group, (C) US group, and (D) US + PSP@MB group. After ultrasound exposure, the OCSCs were incubated with rhodamine-labeled Lyso Tracker with final concentration of 1:15,000 for 8 h at 37 °C. The nucleus was stained with 1 mg/mL DAPI for 5 min. The fluorescent images were observed with CLSM.
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