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

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.

1. 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 per mL 1x HBSS and sterile filter. This can be stored up to 12 months at -20 °C and thawed when needed.
2. Transfer the harvested mouse lu....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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