ARIAIII Cell sorter (BD biosciences, USA) was used to precisely transfer 1 single viable cell (i.e. propidium iodide-negative cell) into each well of an ultra-low adherence 96-well plate (Corning) containing 100 µL of CSC medium. Fresh EGF, FGF-b and heparin were added every 3 days. This experiment was performed in order to assess the tumorsphere formation ability of cells originating from adherent cultures or from previously formed primary or secondary tumorspheres. After 10 days of culture, the number of wells containing a tumorsphere larger than 50 µm was determined using a phase contrast microscope.
Ultra low adherence 96 well plate
Manufactured by Corning
Sourced in United States
The Ultra-low adherence 96-well plates are designed to minimize cell attachment and promote the growth of suspension cultures. The plates feature a specialized surface treatment that reduces the binding of cells, proteins, and other biomolecules, allowing for the cultivation of spheroids, organoids, and other 3D cell models.
Automatically generated - may contain errors
Lab products found in correlation
Thermo arrayscan, by Thermo Fisher Scientific (2 mentions)
Growth factor reduced matrigel, by Corning (2 mentions)
Aria 3 cell sorter, by BD (1 mentions)
Growth factor reduced matrigel, by BD (1 mentions)
Alamarblue reagent, by Thermo Fisher Scientific (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 csu, by Leica (1 mentions)
9 protocols using ultra low adherence 96 well plate
1
Clonal Expansion of Single Cells
2
Mammosphere Formation Assay Protocol
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Mammosphere formation assays were conducted as previously described39 (link),62 (link). Briefly, cells were cultured at concentrations of 2, 5, 10, 25, 50 and 100 cells per well in 8 wells of an ultra-low adherence 96-well plate (Corning). After 10 days, each well was assessed for the presence of mammospheres (defined as cell structures greater than 40 μM in diameter39 (link),63 (link)) and results were put into the Extreme Limiting Dilution Analysis (ELDA) software (https://bioinf.wehi.edu.au/software/elda/ ) to calculate mammosphere forming cell frequency.
3
Spheroid Formation and Co-culture Assay
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Spheroids were first generated by plating TS/A-pc cells at 2000 cells/well into ultra low adherence -96-well plates (Corning, Tewksbury, USA) [6 (link)]. These plates stimulate spontaneous formation of a single spheroid of cells, within 24 hours of incubation at 37°C, 5% CO2. Three day later, medium or 1000 NIH-3T3 cells or 800 NIH-3T3 and 800 EA. hy926 cells were added to wells. Next day (D0 on Figure 7 ) medium or C5M (final concentration of 1 μM) were added. The final volume is the same for all the wells (200 μL/well). Spheroids culture is performed in RPMI 1640 media supplemented with 1% glutamine and 10% foetal bovine serum.
Spheroids were imaged, each day, with a 10-X objective and then the area of the larger section was measured for each spheroid by Image J software.
Spheroids were imaged, each day, with a 10-X objective and then the area of the larger section was measured for each spheroid by Image J software.
4
Evaluating APE1Ref-1 Inhibitor Efficacy
5
3D Tumor Spheroid Co-Culture Assay
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Pa03C and Pa02C cells express TdTomato and CAF19 express EGFP to enable us to track their growth over time. Cells were grown in co−culture as 3D tumor spheroids as previously described (6 (link)). The total intensity of the red or green signal from the spheroids over time was quantitated as described in our previous studies (6 (link), 22 (link)). Briefly, PDAC and CAF (Cancer Associated Fibroblasts) cells (500:2,000 cell/well, 1:4 ratio) were seeded in ultralow adherence 96−well plates (Corning Inc.) in media containing 5% FBS and 3% reduced growth factor Matrigel (Corning Inc.). Spheroids were fed or treated on days 4, 8, and 11 with fluorescent intensity measured on days 4, 8, 11, and 14 following plating using the Thermo ArrayScan (Thermo Fisher Scientific) as previously described. Fold change was calculated to assess the effect of drug treatment on spheroid growth and was calculated compared to media control total intensity on Day 14 (23 (link)).
6
Sphere Formation Assay Protocol
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A total of 4 × 103 cells were washed with PBS and resuspended in culture medium in ultra-low adherence 96-well plates (Corning). Spheres, >75 μM diameter, were counted after 7 days by light microscopy.
7
Pancreatic Cancer Spheroid Co-culture Assay
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PDAC cells were grown in co‐culture as 3‐dimensional tumour spheroids as described previously using cancer‐associated fibroblasts (EGFP‐positive) to mimic stroma and pancreatic cancer cells (TdTomato (red)‐positive). The intensity of the red or green signal from the spheroids over time was quantitated as described in our previous studies.8 , 43 , 48 Briefly, PDAC and CAF cells (500:2000 cell/well, 1:4 ratio) were seeded in ultralow adherence 96‐well plates (Corning Inc) in media containing 5% FBS and 3% reduced growth factor Matrigel (Corning Inc). Spheroids were fed or treated on days 4, 8 and 12 with red and green fluorescence intensity measured on days 4, 8, 12 and 14 following plating using the Thermo ArrayScan (Thermo Fisher Scientific). Fold change was calculated to assess the effect of drug treatment on spheroid growth and was calculated compared to media control.
8
Sphere Formation Assay for Cell Culture
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The cells were detached from culture flasks with 0.25% trypsin and suspended in sphere formation medium (50 ml DMEM/F12 containing 100 mg/ml EGF, 100 mg/ml bFGF and 1 ml B-27® Supplement; Gibco; Thermo Fisher Scientific, Inc.). The cells were then filtered into a single-cell suspension and seeded. Cells (200 cells/well) were seeded in ultra-low adherence 96-well plates (Corning, Inc.) and were cultured in NBS-free medium for 14 days and the spheroids were observed under a light microscope.
9
Metabolic Labeling of 3D Spheroids
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Spheroids were generated by plating BT-549 cells at 5000 cells per well into ultralow adherence-96-well plates (Corning). Spheroids grew in complete medium as in 2D-cultures in the final volume of 200 μL. After 72 h, spheroids were treated with 50 μM Ac4ManNAz and 10 μM DBCO-ABMs in the same manner as in 2D-cultures. Spheroids were imaged using confocal microscope (TCS SP8 CSU, Leica, laser excitation at λ = 448 nm and fluorescence emission collected between λ = 495 and 545 nm) equipped with a 40× objective and analysis was performed with the ImageJ software.
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