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B16 Cells - Fundamental Guide to B16 Melanoma Cells in Oncological Research

B16 is the skin cancer (melanoma) cell line of murine origin. This cell line is an effective in vitro model for studying human skin cancers. It is frequently used to investigate solid tumour formation and metastasis of cancer cells.

This article will assist you in understanding the basics of the B16 melanoma cell line. Specifically, it will go through the following:

1.      General characteristics and origin of the B16 cell line

This section of the article will cover the characteristic features of the B16 melanoma cell line. You will learn the answers to the following frequently asked questions. Such as What is the B16 cancer cell line? Where are B16 cells derived from? What is the size of B16 cells?

  • The B16 cell line was established in 1954. These cells were derived from C57BL/6J mice which spontaneously got a tumour in the skin at the Jackson Laboratories in Maine.
  • These are melanin-producing epithelial cells having the ability to metastasize in the spleen, liver, and lungs.
  • The melanoma B16 cells grow as monolayers and exhibit epithelial-like and spindle-shaped cell morphology.
  • The size of the B16 cell line is approximately 15.4 μm.
  • There are distinct subclones of B16 cells, including B16GMCSF, B164A5, B16FLT3, and B16F10. These sublines are different from parent B16 cells and retain some specific features. Such as, they possess a difference in morphology, cell size, and other properties. B16F10 has high lung metastatic ability, and B164A5 is the most aggressive skin cancer cell line compared to B16F10, B16-GMCSF, and B16FLT3 [1].

3D animation of a close-up of a growing skin cancer, such as malignant melanoma, which inflames the surrounding tissue.

2.      Culturing information of B16 cell line

Before maintaining or culturing a cell line, you might look for key information about doubling time, cell type, growth media, culturing conditions, etc. Here this section contains all the necessary information for culturing B16 cells.

Key Points for Culturing B16 Cells

Population Doubling Time:

The mean population doubling time for B16 cells is estimated to be 24 hours.

Adherent or in Suspension:

B16 cells are adherent and grow in monolayers.

Seeding Density:

B16 cells are recommended to seed at 1 to 2 x 104 cells/cm2 cell density. Attached B16 cells are rinsed with 1 x PBS and dissociate from the surface using Accutase solution. Cells are centrifuged, and the cell pellet is resuspended in the growth medium. Later these cells are dispensed into a new flask for growth.

Growth Medium:

B16 cells are cultured in EMEM (Eagle's Minimum Essential Medium) medium containing 10 % fetal bovine serum (FBS). The growth medium should be renewed 2-3 times a week.

Growth Conditions:

A humidified incubator with a 5 % CO2 supply and 37 °C temperature is used to grow the B16 cell line.


These cells are stored at below -150 °C temperature or in the vapour phase of liquid nitrogen to protect cell viability.

Freezing Process and Medium:

CM-1 or CM-ACF freezing medium is used to freeze B16 cells using a slow freezing process.

Thawing Process:

Frozen B16 cells are thawed at 37 °C in a water bath containing an antimicrobial agent. Thawed cells can be directly cultured by dispensing them into flasks containing growth medium. Besides, these cells can be centrifuged to remove freezing media components and then cultured in new media.

Biosafety Level:

The B16 cell line should be handled or maintained in the biosafety level one laboratory.

Semi-confluent layer of B16 melanoma cells under 10x and 20x magnification.

3.      B16 cell line: Advantages & disadvantages

Like other cell lines, B16 possesses a unique mixture of advantages and disadvantages. Some significant pros and cons of this melanoma cell line are listed in this section.


B16 is the first effective murine tool widely used in metastasis research due to the advantages it offers. Some advantages of this skin cancer cell line are:

Easy to grow

The B16 cell line is easy to culture in research laboratories. It is widely used to study cancer cell biology, signalling pathways, and more.

Fast growing

The B16 melanoma cell line exhibits a high proliferation rate, making it suitable for studying cell division and growth processes.


B16 is a tumorigenic cell line with tumour-like properties such as invasion, migration, and proliferation. It is valuable for studying tumour formation, progression, and metastasis.



The disadvantages associated with the B16 cell line are:

Lack of human relevance

Due to B16 being a mouse melanoma cell line may not accurately represent human skin cancer biology, limiting the translatability of research findings.


B16 cells are heterogeneous, displaying varied genetic and phenotypic properties within the same culture. This can impact result reliability and reproducibility.


4.      Applications of B16 cells

The B16 cell line is extensively used in research studies. A few promising applications of this cell line are:

  • Tumour biology: This murine skin cancer cell line is tumorigenic and widely used to understand tumour biology. Several studies have been conducted to explore the cellular mechanisms behind tumour cell growth, proliferation, and metastasis using B16 cells. A research study conducted in 2020 utilized B16 cells to investigate the role of long non-coding RNA, LncRNA MEG3, in melanoma formation, growth, and metastasis. This research found that the non-coding RNA modulates the miRNA-21/E-Cadherin axis to stimulate these cellular events [2]. Like this, research was carried out to investigate the potential role of Notch1 signalling in tumour-induced immunosuppression using B16 cells [3].
  • Drug discovery: B16 cells are used to validate and test the potential therapeutic effects of drug candidates. A study evaluated the anti-tumour effect of neogambogic acid, a natural compound, using a B16 cell line. The study outcomes revealed that this compound modulates PI3K/Akt/mTOR signalling pathway to cause cancer cell death [4]. Another study investigated the anti-melanoma effect of Ginsenoside Rg3, a saponin, using the B16 cell line. The research proposed that this natural compound caused anti-tumour activity by downregulating ERK and Akt pathways [5].

5.      Research Publications Featuring B16 Cells

Here are some significant research publications featuring the B16 melanoma cell line.

LncRNA MEG3 promotes melanoma growth, metastasis and formation through modulating miR-21/E-cadherin axis

This publication in the Cancer Cell International journal (2020), proposed that long non-coding RNA MEG3 enhances the formation, growth, and metastasis of B16 melanoma cells by modulating the miRNA-21/E-Cadherin axis.

A novel psoralen derivative-MPFC enhances melanogenesis via activation of p38 MAPK and PKA signaling pathways in B16 cells

This paper was published in the International Journal of Molecular Medicine in 2018. This study investigated the melanogenic effect and mechanisms of a psoralen derivative- 4-methyl-6-phenyl-2H-furo[3,2-g] chromen-2-one (MPFC) in B16 cells. The study proposed that this derivative promotes melanogenesis by stimulating PKA and p38 MAPK cell signalling.

Notch1 signaling in melanoma cells promoted tumor-induced immunosuppression via upregulation of TGF-β1

This research was published in 2018 in the Journal of Experimental & Clinical Cancer Research. The study findings suggest that activation of Notch1 signalling in B16 cells may prevent antitumor immunity by upsurging the expression of the TGF-β1 gene.

Neogambogic acid induces apoptosis of melanoma B16 cells via the PI3K/Akt/mTOR signaling pathway

This study was conducted by Chunlan Wu and his colleagues in 2020 and published in the Acta Biochimica Polonica journal. This research states that neogambogic acid, a natural compound, can cause B16 melanoma cell death by modulating PI3K/Akt/mTOR signalling cascade.

An iridium (III) complex as potent anticancer agent induces apoptosis and autophagy in B16 cells through inhibition of the AKT/mTOR pathway

This research paper was published in the European Journal of Medicinal Chemistry in 2018. In this study, researchers investigated the anti-cancer activity of a compound, iridium (III) complex, using B16 melanoma cells.

Ailanthone induces cell cycle arrest and apoptosis in melanoma B16 and A375 cells

This study proposed that a plant bioactive, Ailanthone possesses anticancer potential as it can induce apoptosis and cell cycle arrest in B16 and A375 melanoma cells. This paper was published in Biomolecules in 2019.

6.      Resources for B16 Cell line: Protocols, Videos, and More

There are limited resources on the B16 cell line explaining its culturing and transfection protocols.

The following are some cell culture protocols for B16 cells. 

  • Culturing B16 cells: This website contains all the necessary information for culturing B16 cells, including growth media, subculturing, thawing, and freezing cells.


  1. Danciu, C., et al., Behaviour of four different B 16 murine melanoma cell sublines: C57 BL/6J skin. International journal of experimental pathology, 2015. 96(2): p. 73-80.
  2. Wu, L., et al., LncRNA MEG3 promotes melanoma growth, metastasis and formation through modulating miR-21/E-cadherin axis. Cancer cell international, 2020. 20: p. 1-14.
  3. Yang, Z., et al., Notch1 signaling in melanoma cells promoted tumor-induced immunosuppression via upregulation of TGF-β1. Journal of Experimental & Clinical Cancer Research, 2018. 37(1): p. 1-13.
  4. Wu, C., et al., Neogambogic acid induces apoptosis of melanoma B16 cells via the PI3K/Akt/mTOR signaling pathway. Acta Biochimica Polonica, 2020. 67(2): p. 197-202.
  5. Meng, L., et al., Antitumor activity of ginsenoside Rg3 in melanoma through downregulation of the ERK and Akt pathways. International Journal of Oncology, 2019. 54(6): p. 2069-2079.


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