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 lung to the second, clean 1x HBSS well for that sample. Swirl using forceps to remove any remaining blood. Transfer clean lung to empty 3.5 cm tissue culture plate. Mince lung with scissors. Rinse plate with 2.5 mL of 1x HBSS, transfer 1x HBSS and lung pieces into a 15 mL conical tube already containing collagenase/elastase cocktail (5 mL total).
3. Incubate for 75 minutes at 4 °C. Continue mixing samples during this time, so place tubes on a rocker or rotating wheel. During this incubation step, label 50 mL centrifuge tubes and 10 cm tissue culture plates for each mouse. If doing a dilution, label enough 10 cm tissue culture plates for the dilutions.
   NOTE: Label the lid of the tissue culture plates. If labeling the plate itself, the writing will interfere with Fiji-ImageJ analysis.
4. Bring volume of each tube up to 10 mL total with 1x HBSS. Pour contents over a 70 μm cell strainer into a 50 mL conical tube for each sample. Use the plunger of a 1 mL syringe to gently grind the sample through the strainer to allow more cells to filter through.
5. Centrifuge for 5 minutes at 350 x g at room temperature (RT). Discard the supernatant and wash pellet with 10 mL of 1x HBSS. Repeat this step twice.
6. Resuspend pellet in 10 mL of 60 μM 6TG complete culture media, either RPMI or IMDM. Plate samples in 10 cm cell culture plates, using a dilution scheme if desired. Incubate at 37 °C, 5% CO₂ for 5 days.
   NOTE: 1:2, 1:10, and 1:100 are common dilutions that will need to be empirically determined based on study parameters.
   CAUTION: 6TG is toxic. Use caution when handling and follow all Environmental Health and Safety guidelines for disposal.

2. Staining plates

1. Pour culture media off plates into appropriate waste container. Fix cells by adding 5 mL of undiluted methanol per plate and incubate for 5 minutes at RT, making sure to swirl methanol so that it covers the entire plate.
   CAUTION: Methanol is hazardous if ingested, inhaled, or is on skin. Use a fume hood for this step.
2. Pour methanol off plates into appropriate waste container. Rinse plates with 5 mL of distilled water per plate and pour water into appropriate waste container. Add 5 mL of 0.03% methylene blue per plate and incubate for 5 minutes at RT, making sure to swirl methylene blue solution so that it covers the entire plate.
3. Pour methylene blue into appropriate waste container. Rinse plates again with 5 mL of distilled water per plate. Turn plates upside down and blot against a paper towel to remove excess liquid. Place plate on its lid and let air dry overnight at RT.
   NOTE: Metastatic colonies will be blue. Once plates are dried, they can be stored at RT indefinitely.

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