Cells (103/well) were seeded on 24-well Ultra Low Attachment culture plates (Corning Costar, Cambridge, MA) in complete culture medium containing 0.5% methyl cellulose (Sigma-Aldrich, St. Louis, MO), allowed to grow for 14 days and spheroids scored by image acquisition and spheroid area quantification with ImageJ.
Ultra low attachment culture plate
Manufactured by Corning
Sourced in United States
Ultra-low attachment culture plates are designed for the cultivation of cells in a three-dimensional (3D) environment. These plates feature a specially treated surface that minimizes cell attachment, promoting the formation of spheroids, organoids, and other 3D cellular structures. The ultra-low attachment design helps maintain the undifferentiated state and natural characteristics of the cultured cells.
Automatically generated - may contain errors
Lab products found in correlation
Basic fibroblast growth factor (bfgf), by R&D Systems (4 mentions)
Epidermal growth factor (egf), by R&D Systems (4 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Insulin transferring selenium, by Thermo Fisher Scientific (2 mentions)
Accutase, by Merck Group (2 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions)
Methylcellulose, by Merck Group (1 mentions)
Heparin, by Merck Group (1 mentions)
Dmem f12 culture medium, by Thermo Fisher Scientific (1 mentions)
Nunclon sphera, by Thermo Fisher Scientific (1 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
Insulin transferrin selenium a, by Thermo Fisher Scientific (1 mentions)
24 protocols using ultra low attachment culture plate
1
Tumor Spheroid Formation Assay
2
Prostatosphere Formation Assay
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Single-cells derived from monolayer cultures were seeded (3 × 103 cells/well) in 24-well Ultra Low Attachment culture plates (Corning Costar, Cambridge, MA) in triplicates in 1 ml of DMEM-F12 culture medium (Invitrogen) containing 1% methyl cellulose, 60 μg/ml glucose, 1 μM hydrocortisone, 1 μg/ml putrescine, 10 μg/ml transferrin, 3 nM sodium selenite, 2.5 ug/ml insulin, 10 ng/ml β-FGF, 20 ng/ul, EGF5 U.I./ml heparin (Sigma-Aldrich) and 0.4% B27 (Invitrogen). Prostatospheres were fed every two days and allowed to grow for 14 days. The number of spheres (> 50 cells/sphere) were then counted and expressed as ratio of spheres/1000 plated cells (Sphere Forming Efficiency, SFE).
3
Differentiation of YY1-knockout mESCs
4
Generating Neural Spheres from hDPC
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hDPC cultures were detached with the corresponding Trypsin EDTA treatment (as described above) and resuspended in sphere medium: DFNB, 1× P/S (Sigma), 20 ng/mL bFGF (R&D Systems), 20 ng/mL EGF (R&D Systems) at 10 000 cells per 100 μL in ultra‐low attachment culture plates (Costar, UK). Neurospheres were fed by adding 50 μL of the medium at day 3 and 6. The number of individual spheres and their diameter was measured on day 4. Neurospheres were harvested or used in downstream applications from days 6 to 8. Where indicated, 10 ng/mL BMP4 (R&D Systems) was supplemented to the sphere medium.
5
Sphere Formation Assay for Progenitor Cells
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Cells were trypsinized and resuspended at a density of 1.0 × 103 cells/well in D/F12 (Invitrogen Gibco) medium supplemented with 10 ng/mL epidermal growth factor (R&D Systems Inc., Minneapolis, MN, USA), 10 ng/mL basic fibroblast growth factor (R&D Systems Inc.), 1× insulin-transferring selenium (Invitrogen, Carlsbad, CA, USA), 0.5% bovine serum albumin (Invitrogen), and 0.5% FBS in ultra-low attachment culture plates (Corning Inc., Corning, NY, USA). Adherent cultured cells, as a control, were seeded in culture dishes (Nalgene Nunc Intl, Rochester, NY, USA) with sphere-formation medium. After seven days, cells were collected and dissociated with Accutase (Sigma-Aldrich, St. Louis, MO, USA).
6
Induction of Neural Lineage from BMSCs
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The induction method 18 (link) is briefly described below. P3 generation BMSCs were collected, and the cell concentration was adjusted to 1×105 cells/ml with mixed induction medium containing 1:1 (v/v) Dulbecco's Modified Eagle's Medium (DMEM)/F12 and Neurobasal medium (Invitrogen) supplemented with basic fibroblast growth factor (bFGF; 40 ng/ml; Peprotech), epidermal growth factor (20 ng/ml) (Peprotech), and B27 (2%, v/v, Invitrogen). Then, the cells were seeded onto ultralow attachment culture plates (Corning). Three weeks later, neurospheres were collected and seeded into poly-lysine-coated flasks. After the cells had attached to the wall, the medium was changed to a mixed medium composed of α-MEM (Gibco) supplemented with 10% FBS, forskolin (5 mM), platelet-derived growth factor (PDGF)-AA (5 ng/ml), bFGF (10 ng/ml), and β-heregulin (200 ng/ml). Cells were cultured for two additional weeks before the detection of the S-100, O4, and GFAP proteins. These B-dSCs were further cultured in basal medium (DMEM/F12 supplemented with 10% FBS).
7
Characterizing Cancer Stem Cell Proportion
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The HCC cells were seeded in 96-well ultralow attachment culture plates (Corning Incorporated Life Sciences) (2, 4, 8, 16, 32, and 64 cells per well (n = 8)) and cultured in DMEM/F12 (Gibco) supplemented with 1% FBS, 20 ng/mL bFGF, and 20 ng/mL EGF for 7 days. The proportion of CSCs was assessed using ELDA software (http://bioinf.wehi.edu.au/software/elda/index.html ) [20 (link)]. The results were repeated three times.
8
Spheroid Formation and Attachment Assay
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JCRB1033 cells (shCtrl and shDsg2) were seeded (1 × 106 cells/well) in 2 ml of DMEM in six-well Ultra-Low Attachment culture plates (Corning) and cultured for 72 h. Subsequently, the cells were transferred back to adhesive tissue culture plates and photographed at 18 h later (Fig. 2e ). The experiments were repeated three times.
9
Cancer Stem Cell Quantification
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The PC cells were cultured in DMEM/F12 (Gibco), supplemented with 20 ng/mL EGF, 20 ng/mL bFGF, and 1% FBS for 1 week after being seeded in 96-well ultra-low attachment culture plates (Corning Incorporated Life Sciences). Cell seeding densities included 2, 4, 8, 16, 32, and 64 cells per well. The ELDA software was utilized to calculate the percentage of CSCs [22 (link)]. The experimental process was repeated three times.
10
Spheroid Formation Assay for PC Cells
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The PC cells were cultured in DMEM/F12 (Gibco) supplemented with 20 ng/mL EGF, 20 ng/mL bFGF, and 1%FBS for one week after being seeded on 96-well ultra-low attachment culture plates (Corning Incorporated Life Sciences) at a density of 300 cells/well. Subsequently, a spheroid count was performed, and representative images of the samples were captured. The experiment was repeated three times.
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