Methylene Blue Staining to Assess Metastatic Colony Formation In Vitro: A Staining Procedure to Assess Metastasis by Quantifying Harvested Cancer Cells From Cancer Mouse Model
Published: April 30, 2023
Abstract
Source: Nagai-Singer, M. A., et al. Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model. J. Vis. Exp. (2020)
In this video, we describe the methylene blue staining procedure to quantify the metastatic burden by counting stained colonies obtained by culturing cancer cells harvested from distant organs of a mouse model following injection.
Protocol
All animal model procedures have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Processing Tissues NOTE: All steps in this section should be done using sterile technique. Label 1 15 mL conical tube per mouse and add 2.5 mL of type IV collagenase mixture and 30 units of elastase to each tube. To make type IV collagenase mixture, dissolve 2 mg of type IV collagenase …
Declarações
The authors have nothing to disclose.
Materials
| Methanol | Fisher Scientific | A412SK-4 | |
| Methylene blue | Sigma-Aldrich | 03978-250ML | |
| RMPI-1640 Medium | ATCC | 30-2001 | |
| Rocker or rotating wheel | Any | Any | |
| Sterile disposable filter with PES membrane | ThermoFisher Scientific | 568-0010 | |
| Type IV collagenase | Sigma-Aldrich | C5138 | |
| 3.5 cm tissue culture plates | Nunclon | 153066 | |
| 10 cm tissue culture plates | Fisher Scientific | 08-772-22 | |
| 15 mL centrifuge tube | Fisher Scientific | 14-959-70C | |
| 1X Hank’s Balanced Saline Solution (HBSS) | Thermo Scientific | SH3026802 | |
| 50 mL centrifuge tube | Fisher Scientific | 14-959-49A | |
| 70 μM cell strainer | Fisher Scientific | 22-363-548 | |
| 70% ethanol | Sigma Aldrich | E7023 | Dilute to 70% with DI water |
| Incubator | Needs to be capable of 5% CO2 and 37 °C | ||
| Forceps | Roboz | RS-8100 | |
| Centrifuge | Any | Any | Needs to be capable of 125 x g and 300 x g |
| Distilled water | Any | Any | |
| Elastase | MP Biomedicals | 100617 |
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Methylene Blue Staining to Assess Metastatic Colony Formation In Vitro: A Staining Procedure to Assess Metastasis by Quantifying Harvested Cancer Cells From Cancer Mouse Model. J. Vis. Exp. (Pending Publication), e21037, doi: (2023).