Sphere formation assay protocol

Sphere formation assay was carried out as reported previously [24 (link)]. Concisely, transfected MDA-MB-231 or MCF-7 cells were inoculated in ultra-low attachment 6-well plates (Corning, NY, USA; 5 × 10³ cells per well) for 10–14 days. Cells were maintained in DMEM/F12 serum-free medium (PM150312A, Procell) added with 5 μg/mL insulin (Sigma-Aldrich), 0.4% bovine serum albumin (BSA; A1933, Sigma-Aldrich), 2% B27 (MAB1285, Sigma-Aldrich), 20 ng/mL basic fibroblast growth factor (bFGF; PHG0369, Thermo Fisher Scientific), and 20 ng/mL EGF (Peprotech, Rocky Hill, NJ, USA). Generated spheres were photographed and then counted using a light microscope (Zeiss, Oberkochen, Germany). Sphere formation efficiency is calculated as (number of spheres/number of inoculated cells) × 100%, using MShot Image Analysis System. Only spheres with diameters greater than 75 μm were counted.

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An C., Hu Z., Li Y., Zhao P., Liu R., Zhang Q., Zhu P., Li Y, & Wang Y. (2022). LINC00662 enhances cell progression and stemness in breast cancer by MiR-144-3p/SOX2 axis. Cancer Cell International, 22, 184.

Publication 2022

ultra-low attachment 6-well plates PM150312A insulin A1933 EGF

Assay Basic fibroblast growth factor Bovine serum albumin Cells Insulin Light microscope Mcf 7 cells Serum

Corresponding Organization : Second Hospital of Hebei Medical University

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Variable analysis

independent variables

Transfected cell lines (MDA-MB-231 or MCF-7)

dependent variables

Sphere formation efficiency (calculated as (number of spheres/number of inoculated cells) × 100%)

control variables

Inoculation density (5 × 10^3 cells per well)
Culture duration (10-14 days)
Culture medium (DMEM/F12 serum-free medium with 5 μg/mL insulin, 0.4% bovine serum albumin, 2% B27, 20 ng/mL basic fibroblast growth factor, and 20 ng/mL EGF)
Culture plates (ultra-low attachment 6-well plates)

controls

No explicit positive or negative controls were mentioned.

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