Sphere Formation Assay: An In Vitro Method to Identify Pancreatic Cancer Stem Cells and Screen Therapies

Abstract

Source: Lonardo, E. et al. Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies. J. Vis. Exp.(2015).

This video demonstrates the protocol for developing pancreatic cancer stem cell spheroids in vitro. The assay can serve as a screening tool to identify compounds that potentially target cancer stem cells.

Protocol

1.Sphere Forming Assay and Analysis

1. Sphere Formation Assay and Metformin Treatment
   1. Take the required number of cells and add the appropriate volume of CSCs medium to prepare a cell concentration of 2,000 cells/ml. Do not keep the cell suspension on ice for no longer than 1h and mixed well prior to plating.
   2. Add 500 µl of 1X PBS to the first and last row of a 24-well plate for humidification in order to help minimize medium evaporation. Seed the cells into ultra-low attachment cells plates at a density of 2,000 cells per well in 1 ml of tumor sphere medium (2,000 cells/ml).
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Results

Figure 1. Metformin Treatment Inhibits Oxygen Consumption and Induces ROS Production. (A) Metformin addition inhibits oxygen consumption (OCR) of sphere-derived cells. Two different PDAC secondary spheres were treated with.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24-well Ultra-low Attachment Plates	Corning	3474
Sterile 1x Dulbecco’s Phosphate Buffered Saline	Sigma	D8537
Metformin	Sigma	D150959-5G
Trypsin solution, 0.05%	Life technologies	25300054
Incubator with CO<sub>2</sub> input
CASY Cell Counter and Analyzer for proliferation and viability measurement	Roche Innovatis	AG CASY Model TTC 45,60,150 &mu;m
Counting Chamber/ Hemocytometer	Hausser Scientific Co	3200
50 ml centrifuge tubes	Corning	430828
15-ml polypropylene conical tube	BD Falcon	352097
50-ml polypropylene conical tube	BD Falcon	352070