Name: 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
Uploaded: 2023-04-30T14:57:59
Description: 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. ...

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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.
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	<li>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 ...

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

Source: Nagai-Singer, M. A., et al. <a href="https://www.jove.com/v/61805">Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model.</a> 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.

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

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. ...

During cancer metastasis, tumor cells from a primary tumor travel through the circulatory system, extravasate to distant organs, and establish secondary tumors.
To evaluate lung metastasis in a breast cancer mouse model that has been injected with 4T1 cells, highly metastatic breast cancer cells, prepare a cell suspension, including 4T1 cells, isolated from the harvested lungs. Centrifuge the suspension and resuspend the cells in media containing 6-thioguanine, 6-TG - a guanine analog. Seed the cell suspension in a culture plate.
6TG enters normal cells where specific transferase enzyme converts it to 6-TG nucleotide, a toxic nucleotide. These cytotoxic nucleotides get incorporated into DNA, causing DNA damage, and selectively eliminate cells from culture. 4T1 cells have decreased ability to form toxic nucleotides, facilitating their survival and adherence to the plate. Depending on the proliferative ability of cells, they form colonies.
Treat the cells with methanol. Methanol permeabilizes the cells and denatures the cellular proteins. Incubate the cells with methylene blue.
The positively-charged dye enters the cell, interacts with the negatively-charged DNA, intercalating into spaces between the DNA strands&#8217; adjacent base pairs. It also binds to RNA in the cytoplasm, but with lower affinity. The nuclei stain dark blue due to high DNA concentration than the cytoplasm owing to the low RNA concentration. Metastatic colonies appear as blue dots.
Image the plate. Use software to determine the metastatic colonies, quantifying the metastatic burden in the mouse lungs.

methylene-blue-staining-to-assess-metastatic-colony-formation-vitro

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Staining Techniques

Cell staining

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the formation of a colored precipitate. It works by soaking the acrylamide or agarose DNA gel in a solution of 1x (equivalent to 2.0 &#181;M) SYBR Green I (SG I) and 0.20 mM nitro blue tetrazolium that produces a purple precipitate of formazan when exposed to sunlight or specifically blue light. Also, DNA recovery tests were performed using an ampicillin resistant plasmid in an agarose gel stained with our method. A larger number of colonies was obtained with our method than with traditional staining using SG I with ultraviolet illumination. The described method is fast, specific, and non-toxic for DNA detection, allowing visualization of biomolecules to the "naked eye" without a transilluminator, and is inexpensive and appropriate for field use. For these reasons, our new DNA staining method has potential benefits to both research and industry.

This method allows selective staining and quantification of DNA in gels by soaking the gel in a SYBR Green I/Nitro Blue Tetrazolium solution and then exposing it to sunlight or a blue light source. This produces a visible precipitate and requires almost no equipment, making it ideal for field use.

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the ...

JoVE Journal

Biochemistry

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...

Medicine

Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection

Urinary tract junction obstruction defects are congenital anomalies inducing hydronephrosis and hydroureter. Murine urinary tract junction obstruction defects can be assessed by tracking methylene blue dye flow within the urinary system. Methylene blue dye is injected into the renal pelvis of perinatal embryonic kidneys and dye flow is monitored from the renal pelvis of the kidney through the ureter and into the bladder lumen after applying hydrostatic pressure. Dye accumulation will be evident in the bladder lumen of the normal perinatal urinary tract, but will be constrained between the renal pelvis and the end point of an abnormal ureter, if urinary tract obstructions occur. This method facilitates the confirmation of urinary tract junction obstructions and visualization of hydronephrosis and hydroureter. This manuscript describes a protocol for methylene blue dye injection into the renal pelvis to confirm urinary tract junction obstructions.

Methylene blue dye injection into the renal pelvis facilitates the assessment of urinary tract junction obstruction defects during mouse embryonic urinary tract development. Here, a protocol for methylene blue dye injection into the renal pelvis is described.

Urinary tract junction obstruction defects are congenital anomalies inducing hydronephrosis and hydroureter. Murine urinary tract junction obstruction ...

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

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