To generate the hEM15A cell-derived spheroids, hEM15A cells were seeded at 2000 cells/well in 96-well Clear Flat Bottom Ultra-Low Attachment Microplate (3474, Corning). Adding 2.5% Matrigel in the culture system (10%FBS DMEM/F-12 medium) was indispensable to form hEM15A-spheroids. α-NETA (0, 25, 50 μm) was added to the plates for 48 h, and the diameter of spheroids was recorded. Live and Dead™ Viability/Cytotoxicity Assay Kit for Animal Cells (Calcein AM, EthD-1) was purchased from Yu Heng Bio (L6023). hEM15A-spheroids were stained with Calcein AM and EthD-1 according to the manufacturer’s experimental procedure and observed under an inverted fluorescence microscope. Three independent experiments were performed.
Clear flat bottom ultra low attachment microplate
Manufactured by Corning
Sourced in United States
The Clear Flat Bottom Ultra-Low Attachment Microplate is a laboratory equipment designed to provide a non-adhesive surface for cell culture applications. It is made of clear material and has a flat bottom configuration.
Automatically generated - may contain errors
Lab products found in correlation
Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions)
Fibroblast growth factor basic, by Thermo Fisher Scientific (2 mentions)
Bz 9000 fluorescence microscope, by Keyence (1 mentions)
Bx51 epi fluorescent microscopy, by Olympus (1 mentions)
Cytation 3, by Agilent Technologies (1 mentions)
Imark microplate reader, by Bio-Rad (1 mentions)
6 protocols using clear flat bottom ultra low attachment microplate
1
hEM15A Spheroids formation and α-NETA effect
2
Breast Cancer Spheroid Formation Assay
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Breast cancer cells were harvested and re-suspended in sphere formation medium, which was composed of RPMI-1640 (for EO771FL) or DMEM (for 4T1, MDA-MB-231, and T-47D) supplemented with 1 × B27, 20 ng/mL fibroblast growth factor-basic and 20 ng/mL epidermal growth factor (Thermo Fisher Scientific, Waltham, MA, USA). Cells were plated in a 48-well Clear Flat Bottom Ultra-Low Attachment Microplate (CORNING, Corning, NY, USA) at a density of 2000, 1000, 500, 250, or 100 cells/well. After 7 days (for EO771FL cells and 4T1 cells) or 10 days (for MDA-MB-231 and T-47D cells), the spheres (defined as >20 cell/spheroid) were recorded using Olympus BX51 Epifluorescent microscopy (Olympus, Tokyo, Japan).
3
Spheroid Formation Assay Protocol
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Spheroid formation was performed by placing 1×104 cells in a 96-well Clear Flat Bottom Ultra-Low Attachment Microplate (Corning, Inc.) in DMEM/F12 medium containing 10% FBS, as described previously (17 (link)). After three days of plating, spheroid formation was monitored using a BZ-9000 fluorescence microscope (Keyence). Spheroid formation assays were performed in duplicate.
4
Sphere Formation Assay for Breast Cancer
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Breast cancer cells were harvested and re-suspended in sphere formation medium, containing RPMI-1640 or DMEM supplemented with 20 ng/mL fibroblast growth factor-basic and 20 ng/mL epidermal growth factor (Thermo Fisher Scientific, Waltham, MA, USA). Cells were plated in a 48-well Clear Flat Bottom Ultra-Low Attachment Microplate (CORNING, Corning, NY, USA) at a density of 2000, 1000, 500, 250, or 100 cells/well. After 7 days (for MCF-7 cells and MDA-MB-453 cells) or 10 days (for MDA-MB-231 and T-47D cells), the spheres (defined as >20 cell/spheroid) were recorded using Olympus BX51 Epifluorescent microscopy (Olympus, Tokyo, Japan).
5
Tumor Sphere Culture Assay for NSCLC Lines
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The tumor sphere culture medium was prepared as previously described [30 (link)]. A549, H358, and HCC827 cells were seeded at a density of 500 cells/well in a 96-well Clear Flat Bottom Ultra-Low Attachment Microplate (Corning Incorporated, Corning, NY, USA) with tumor sphere culture medium and 25 μL of the fresh medium was added twice a week. After two weeks, images of each well were analyzed using Cytation 3 (BioTek, Winooski, VT, USA). Tumor spheres in each cell line were counted when they reached the following diameter: A549, ≥150 μm; H358, ≥80 μm; HCC827, ≥100 μm. The experiments were independently replicated three times, with triplicate in each case.
6
Loliolide Cytotoxicity Evaluation
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The Water-Soluble Tetrazolium-1 (WST-1) assay (EZ-Cytox Cell Viability Assay Kit; ITSbio, Korea) was used as described in the manufacturer's protocol to determine cell viability. HDP cells (1 × 10 4 cells/well) were seeded on a 96-well, clear, flat-bottom ultra-low attachment microplate (Corning Inc., USA) to obtain spherical structures, and the cells were treated with the indicated doses of loliolide (1, 5, 10, 20, 50 , and 100 μg/ml) for 48 h. Subsequently, the WST-1 solution was added to each well, and cell viability was examined by measuring absorbance at 450 nm using an iMark microplate reader (Bio-Rad Laboratories, USA).
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