Sphere Formation Assay: An In Vitro Method to Identify Pancreatic Cancer Stem Cells and Screen Therapies
Published: April 30, 2023
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 Sphere Formation Assay and Metformin Treatment 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. 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 evaporati…
Representative 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 metformin and OCR changes were measured by extracellular flux analysis. Rotenone injection was used as a control for total mitochondrial OCR …
Divulgaciones
The authors have nothing to disclose.
Materials
| 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 CO2 input | |||
| CASY Cell Counter and Analyzer for proliferation and viability measurement | Roche Innovatis | AG CASY Model TTC 45,60,150 μm | |
| 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 |
Citar este artículo
Sphere Formation Assay: An In Vitro Method to Identify Pancreatic Cancer Stem Cells and Screen Therapies. J. Vis. Exp. (Pending Publication), e20320, doi: (2023).