PTM Based Tumor Microenvironment Monitoring: A Method for Tracking Changes in ECM Rigidity Using Tumor Spheroids
Published: April 30, 2023
Abstract
Source: Jones, D. P., et al. Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology. J. Vis. Exp. (2014).
This protocol describes a technique for monitoring extracellular matrix rigidity changes by using particle-tracking microrheology (PTM). This is done by culturing 3D pancreatic tumor spheroids into ECM with embedded fluorescent probes for video particle-tracking and analysis.
Protocol
1. Preparing 3D Tumor Spheroids Embedded in ECM Prepare a work station with needed materials inside a laminar flow hood. Prepare a diluted mixture of carboxylate-modified 1 μm diameter fluorescent tracer probes by adding 2 parts stock probes (2% solids) to 25 parts sterile water. Remove a bottle of 3.1 mg/ml bovine collagen from the refrigerator and place it on ice. Aliquot 125 μl of collagen into an empty 2 ml vial. Add 50 μl of diluted …
Açıklamalar
The authors have nothing to disclose.
Materials
| Bovine type 1 collagen | BD Biosciences, San Jose, CA | 354231 | |
| PANC-1 | American Type Cell Culture, Manassas, VA | CRL1469 | or other appropriate cell type |
| Fluorescent Microspheres | Life Technologies, Carlsbad, CA | 906906 | |
| Agarose | Fisher Bioreagents, Waltham, MA | C12H18O9 | |
| NaOH | Fisher Bioreagents, Waltham, MA | NC0480985 | |
| 96-well Imaging plates | Corning Inc., Corning, NY | 3904 | |
| DMEM | Hyclone, Waltham, MA | SH30243.01 | or appropriate cell culture media |
| Zeiss AxioObsever Microscope | Zeiss, Oberkochen, Germany | includes high-speed camera and imaging software |
Bu Makaleden Alıntı Yapın
PTM Based Tumor Microenvironment Monitoring: A Method for Tracking Changes in ECM Rigidity Using Tumor Spheroids. J. Vis. Exp. (Pending Publication), e20323, doi: (2023).