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 …

Divulgaciones

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).

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