Exactly 14 days after sorting, CD73+ cells were cultured for 2 days in EZSPHERE® (AGC Techno Glass, Shizuoka, Japan), in order to generate spheroids. Spheroids were collected and suspended in PBS. Cells (1.0 × 106 cells in 200 μL of PBS) were transplanted via enteral administration 0 and 5 days after DSS treatment. Trans-anal transplantation was performed as previously described, with slight modifications [22 (link)]. The cell suspension was infused into the murine colonic lumen by using a thin, flexible catheter (diameter: 2.1 mm) or a stainless steel needle (diameter: 1.9 mm). After transplantation, the mice were housed as usual, and colon tissues were collected and analyzed by flow cytometry. Control mice received an equal volume of PBS, or a solution of 0.01 mg/mL 5-aminosalicylic acid (5-ASA; Zeria Pharmaceutical Co., Ltd., Tokyo, Japan). The concentration of 5-ASA (100 mg/kg) was based on a previous paper, and 5-ASA was administered enterally in the same manner as the spheroids [23 (link)].
Ezsphere
Manufactured by AGC Techno Glass
Sourced in Japan
The EZSPHERE is a lab equipment product designed for spherical glass processing. It is capable of producing spherical glass components with high precision and consistency.
Automatically generated - may contain errors
Lab products found in correlation
Activin a, by R&D Systems (2 mentions)
Vascular endothelial growth factor (vegf), by R&D Systems (2 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions)
Egm 2, by Lonza (1 mentions)
Stemfit ak03n, by Ajinomoto (1 mentions)
Y27632, by Ajinomoto (1 mentions)
Dorsomorphine, by Merck Group (1 mentions)
Basic fibroblast growth factor (bfgf), by R&D Systems (1 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (1 mentions)
Basic fibroblast growth factor (bfgf), by ReproCELL (1 mentions)
Skov3, by Sumitomo Pharma (1 mentions)
Hepg2, by Sumitomo Pharma (1 mentions)
Penicillin streptomycin mixture, by Merck Group (1 mentions)
Y 27632, by Fujifilm (1 mentions)
24 well culture plate, by Corning (1 mentions)
Fluo 8, by AAT Bioquest (1 mentions)
Orca r2 ccd camera, by Hamamatsu Photonics (1 mentions)
Phenol red free dmem, by Nacalai Tesque (1 mentions)
Penicillin streptomycin, by Nacalai Tesque (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
6 protocols using ezsphere
1
Enteral Transplantation of CD73+ Spheroids for Murine Colitis
2
Differentiation of hiPSCs into CD31+ Endothelial Cells
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CD31+ cells were prepared from differentiated hiPSCs (Ff-I14). To generate embryoid bodies (Ebs), hiPSCs were seeded onto EZSPHERE (AGC TECHNO GLASS Co., Ltd., Shizuoka, Japan). Approximately 3 × 105 hiPSC cells/mL were cultured in the StemFit AK03N medium containing 2 ng/mL BMP4 and 10 µM Y27632 in the absence of component C (Ajinomoto, Co., Ltd., Tokyo, Japan) (day 0). On day 1, 9 ng/mL BMP4, 10 ng/mL bFGF, and 6 ng/mL Activin A (R&D Systems, Inc., Minneapolis, MN, USA) were added to the medium. From days 2 to 7, Ebs were cultured in a single-use bioreactor and magnetic stirrer (ABLE Corporation & Biott Corporation, Tokyo, Japan). On day 2, the medium was supplemented with 9 ng/mL BMP4, 10 ng/mL bFGF, and 6 ng/mL Activin A (R&D Systems, Inc., Minneapolis, MN, USA), and removed on day 4. On day 4, the medium was supplemented with 25 ng/mL vascular endothelial growth factor (VEGF) (R&D Systems, Inc., Minneapolis, MN, USA) and 8 ng/mL bFGF, and removed on day 7. On day 7, Ebs were enzymatically dissociated and subjected to MACS (Miltenyi Biotec Inc., Tokyo, Japan) to separate CD31+ cells. CD31+ cells were maintained in EGM2 (Lonza, Inc., Basel, Switzerland) on collagen-type IV-coated tissue culture dishes. They were passaged every two days until being harvested for cryopreservation on day 13.
3
Directed Differentiation of Human iPSCs
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Human iPSC (Ff-I14) cultured on iMatrix511 were dissociated with TrypLE Select and a single cell suspension was cultured on EZSPHERE (10 cm diameter) (AGC Techno Glass, Shizuoka, Japan) for 4 days in StemFit AK03N medium not containing component C with Y27632 (10 μM) at 37 °C in humid air with 5% CO2 and 1% O2. At day 4, cell aggregates were collected from EZSPHERE dishes and resuspended in StemFit AK03N medium not containing component C and cultured in a 100 mL stirred bioreactor system (ABLE) until day 13 or 14. The following growth factors and small molecules were used at the corresponding days: days 0–1, 2 ng/mL BMP4 (R&D systems); days 1–4, 10 ng/mL BMP4, 6 ng/mL Activin A (R&D systems), 10 ng/mL bFGF (ReproCell); days 4–8, 1 μM IWP-3 (Stemgent, Lexington, MA, USA), 0.6 μM Dorsomorphine (Sigma–Aldrich, St. Louis, MO, USA), 5.4 μM SB432542 (Sigma Aldrich); after day 8, 5 ng/mL VEGF (R&D Systems) and 10 ng/mL bFGF. At days 6, 8, 10, and 12, the culture medium was exchanged.
4
Culturing and Spheroid Formation of Human Cancer Cell Lines
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Human cancer cell lines were obtained from DS Pharma Biomedical (Osaka, Japan; A549, SKOV3, and HepG2). A549 and HepG2 cells were cultured in DMEM with 10% FBS. SKOV3 cells were cultured in McCoy's 5A medium with 15% FBS. These media were supplemented with a 1% penicillin–streptomycin mixture (Sigma‐Aldrich, St. Louis, MO, USA). Cells were cultured at 37°C in a humidified atmosphere flushed with 5% CO2 in air.
HepG2 cell spheroids were made for the dispersion test by using EZSPHERE (AGC Techno Glass, Tokyo, Japan). After 6–10 days of culture on the plate (initial cell number, 250 000 cells/100‐mm dish), spheroids of 100–200 μm in diameter were collected by centrifugation at 420g for 10 min.
HepG2 cell spheroids were made for the dispersion test by using EZSPHERE (AGC Techno Glass, Tokyo, Japan). After 6–10 days of culture on the plate (initial cell number, 250 000 cells/100‐mm dish), spheroids of 100–200 μm in diameter were collected by centrifugation at 420
5
Cardiac Differentiation and Co-Culture of iPSCs
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Following cardiac differentiation of human iPS cells, cells were cultured for 2 days in 24-well culture plates (Corning) at a density of 2.1 × 105 cells/cm2 in DMEM supplemented with 10% FBS at 37 °C in humid air with 5% CO2. One day before starting the co-culture experiment, iPS cells cultured on iMatrix511 were dissociated with TrypLE Select and a single cell suspension was cultured on EZ Sphere (AGC Techno Glass, Shizuoka, Japan) for 1 day in StemFit AK03 medium with Y27632 (10 μM) (Wako) to form cell aggregates. The following day, 50 cell aggregates of iPS cells were co-cultured with iPS-derived cardiac cells in 24-well culture plates in StemFit AK03 for 1 day. These preparations were then cultured for 2 days according to the following conditions: StemFit AK03 at 37 °C or 42 °C, 10% FBS DMEM at 37 °C or 42 °C.
6
Hypoxic Culture of hiPSC-Derived Cardiomyocytes
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hiPSC-CMs at day 23 were seeded onto EZ Sphere (AGC Techno Glass, Shizuoka, Japan) at 5 × 104 cells/cm2 to form a spheroid. The following day (day 24), spontaneous beating was confirmed, and the spheroids were moved to a normoxic (20% O2) or hypoxic (1% O2) incubator for 4 or 12 days to be cultured in DMEM supplemented with 10% FBS and penicillin–streptomycin (Sigma–Aldrich). The culture medium was changed every 4 days. To investigate intracellular Ca2+ transients, spheroids were transferred to 6-well plates and loaded with 5 μM Fluo-8 (AAT Bioquest, Sunnyvale, CA, USA) in DMEM supplemented with 10% FBS for 1 h in a humidified incubator with 5% CO2 at 37 °C. Ten minutes before observation, each well was immersed in phenol red-free DMEM (Nacalai Tesque, Kyoto, Japan) containing 10% FBS and penicillin–streptomycin. Changes in fluorescence levels were monitored at a rate of 100 ms with an ORCA-R2 CCD camera (Hamamatsu Photonics K.K., Shizuoka, Japan) and processed with Aquacosmos image processing software (Hamamatsu Photonics K.K.) for baseline correction and colored visualization. The Ca2+ transient was recorded during electrical stimulation at 1 Hz (C-Pace EP, IonOptix, Westwood, MA, USA).
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