Wilms8 Cells
General information
Description | The Wilms8 cell line was derived from a primary Wilms tumor in a pediatric patient with a germline WT1 mutation. This cell line is characterized by a homozygous nonsense mutation in the WT1 gene (c.1168 C>T, p.R390X), leading to a complete loss of WT1 function. WT1 is crucial for normal kidney development, and its inactivation is a common feature in certain aggressive subtypes of Wilms tumor, particularly those that exhibit mesenchymal differentiation. Wilms8, therefore, provides a valuable model for studying the effects of WT1 loss on tumorigenesis, especially in the context of Wilms tumors that arise with a pronounced stromal component. In addition to the WT1 mutation, Wilms8 cells harbor a mutation in the CTNNB1 gene (p.S45A), which encodes β-Catenin, a key regulator of the Wnt signaling pathway. The mutation at serine 45 disrupts the normal phosphorylation process that leads to β-Catenin degradation, causing its stabilization and accumulation in the nucleus. This results in the constitutive activation of Wnt signaling, which drives cell proliferation and contributes to the oncogenic properties of the Wilms8 cell line. The interplay between WT1 loss and aberrant Wnt signaling in Wilms8 makes it a crucial model for understanding the molecular mechanisms underlying these pathways in Wilms tumor biology. Wilms8 cells display a mesenchymal phenotype, characterized by the expression of vimentin and the absence of epithelial markers such as cytokeratin. This aligns with the stromal differentiation observed in the original tumor. The cells demonstrate a limited ability to undergo further mesenchymal differentiation, such as forming muscle-like cells under specific conditions. Proteomic analyses of Wilms8 have revealed the activation of multiple receptor tyrosine kinases (RTKs), including PDGFRβ and AXL, which are involved in key processes such as cell survival, migration, and proliferation. The activation of downstream signaling pathways, particularly the MAPK and PI3K/AKT pathways, further contributes to the aggressive characteristics of Wilms8 cells. Overall, the Wilms8 cell line serves as an essential tool for investigating the molecular basis of Wilms tumor driven by WT1 loss and aberrant Wnt signaling. Its genetic and phenotypic features make it a robust platform for studying the interaction between these critical pathways and for identifying potential therapeutic targets in Wilms tumors with a stromal component. |
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Organism | Human |
Tissue | Kidney |
Disease | Wilms tumor |
Applications | In vitro cell culture model. Biochemical studies |
Characteristics
Age | 8 months |
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Gender | Male |
Ethnicity | Caucasian |
Morphology | Spindle-shaped |
Cell type | Wilms cells |
Growth properties | Adherent |
Identifiers / Biosafety / Citation
Citation | Wilms8 (Cytion catalog number 300416) |
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Biosafety level | 1 |
Depositor | B. Royer-Pokora |
Expression / Mutation
Mutational profile | WT1 mutation status: homozygous c.1168C>T, p.390x, LOH: , CTNNB1 mutation status: heterozygous TCT>GCT, p.S45A |
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Handling
Culture Medium | MSCGM kit (from Lonza) |
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Passaging solution | Accutase |
Subculturing | Remove the old medium from the adherent cells and wash them with PBS that lacks calcium and magnesium. For T25 flasks, use 3-5 ml of PBS, and for T75 flasks, use 5-10 ml. Then, cover the cells completely with Accutase, using 1-2 ml for T25 flasks and 2.5 ml for T75 flasks. Let the cells incubate at room temperature for 8-10 minutes to detach them. After incubation, gently mix the cells with 10 ml of medium to resuspend them, then centrifuge at 300xg for 3 minutes. Discard the supernatant, resuspend the cells in fresh medium, and transfer them into new flasks that already contain fresh medium. |
Freeze medium | CM-1 (Cytion catalog number 800100) |
Handling of cryopreserved cultures |
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Quality control / Genetic profile / HLA
Sterility | Mycoplasma contamination is excluded using both PCR-based assays and luminescence-based mycoplasma detection methods. To ensure there is no bacterial, fungal, or yeast contamination, cell cultures are subjected to daily visual inspections. |
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STR profile |
Amelogenin: x,y
CSF1PO: 11,12
D13S317: 8,9
D16S539: 13,13
D5S818: 12,13
D7S820: 8,1
TH01: 8,8
TPOX: 8,9
vWA: 18,18
D3S1358: 16,18
D21S11: 29,33.2
D18S51: 12,12
Penta E: 12,17
Penta D: 10,12
D8S1179: 8,13
FGA: 20,21
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HLA alleles |
A*: 02:01:01, 03:01:01
B*: 15:01:01, 37:01:01
C*: 04:01:01, 06:02:01
DRB1*: 08:01:01G, 11:01:01
DQA1*: 04:01:01, 05:05:01
DQB1*: 03:01:01, 04:02:01
DPB1*: 03:01:01, 06:01:01
E: 01:03:02
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Required products
- A Gentle Alternative to Trypsin
Accutase is a cell detachment solution that is revolutionizing the cell culture industry. It is a mix of proteolytic and collagenolytic enzymes that mimics the action of trypsin and collagenase. Unlike trypsin, Accutase does not contain any mammalian or bacterial components and is much gentler on cells, making it an ideal solution for the routine detachment of cells from standard tissue culture plasticware and adhesion coated plasticware. In this blog post, we will explore the benefits and uses of Accutase and how it is changing the game in cell culture.
Advantages of Accutase
Accutase has several advantages over traditional trypsin solutions. Firstly, it can be used whenever gentle and efficient detachment of any adherent cell line is needed, making it a direct replacement for trypsin. Secondly, Accutase works extremely well on embryonic and neuronal stem cells, and it has been shown to maintain the viability of these cells after passaging. Thirdly, Accutase preserves most epitopes for subsequent flow cytometry analysis, making it ideal for cell surface marker analysis.
Additionally, Accutase does not need to be neutralized when passaging adherent cells. The addition of more media after the cells are split dilutes Accutase so it is no longer able to detach cells. This eliminates the need for an inactivation step and saves time for cell culture technicians. Finally, Accutase does not need to be aliquoted, and a bottle is stable in the refrigerator for 2 months.
Applications of Accutase
Accutase is a direct replacement for trypsin solution and can be used for the passaging of cell lines. Additionally, Accutase performs well when detaching cells for the analysis of many cell surface markers using flow cytometry and for cell sorting. Other downstream applications of Accutase treatment include analysis of cell surface markers, virus growth assay, cell proliferation, tumor cell migration assays, routine cell passage, production scale-up (bioreactor), and flow cytometry.
Composition of Accutase
Accutase contains no mammalian or bacterial components and is a natural enzyme mixture with proteolytic and collagenolytic enzyme activity. It is formulated at a much lower concentration than trypsin and collagenase, making it less toxic and gentler, but just as effective.
Efficiency of Accutase
Accutase has been shown to be efficient in detaching primary and stem cells and maintaining high cell viability compared to animal origin enzymes such as trypsin. 100% of cells are recovered after 10 minutes, and there is no harm in leaving cells in Accutase for up to 45 minutes, thanks to autodigestion of Accutase.
In summary
In conclusion, Accutase is a powerful solution that is changing the game in cell culture. With its gentle nature, efficiency, and versatility, Accutase is the ideal alternative to trypsin. If you are looking for a reliable and efficient solution for cell detachment, Accutase is the solution for you.
Phosphate-buffered saline (PBS) is a versatile buffer solution used in many biological and chemical applications, as well as tissue processing. Our PBS solution is formulated with high-quality ingredients to ensure a constant pH during experiments. The osmolarity and ion concentrations of our PBS solution are matched to those of the human body, making it isotonic and non-toxic to most cells.
Composition of our PBS Solution
Our PBS solution is a pH-adjusted blend of ultrapure-grade phosphate buffers and saline solutions. At a 1X working concentration, it contains 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4. We have chosen this composition based on CSHL protocols and Molecular cloning by Sambrook, which are well-established standards in the research community.
Applications of our PBS Solution
Our PBS solution is ideal for a wide range of applications in biological research. Its isotonic and non-toxic properties make it perfect for substance dilution and cell container rinsing. Our PBS solution with EDTA can also be used to disengage attached and clumped cells. However, it is important to note that divalent metals such as zinc cannot be added to PBS as this may result in precipitation. In such cases, Good's buffers are recommended. Moreover, our PBS solution has been shown to be an acceptable alternative to viral transport medium for the transport and storage of RNA viruses, such as SARS-CoV-2.
Storage of our PBS Solution
Our PBS solution can be stored at room temperature, making it easy to use and access.
To sum up
In summary, our PBS solution is an essential component in many biological and chemical experiments. Its isotonic and non-toxic properties make it suitable for numerous applications, from cell culture to viral transport medium. By choosing our high-quality PBS solution, researchers can optimize their experiments and ensure accurate and reliable results.
Composition
Components
mg/L
Inorganic Salts
Potassium chloride
200,00
Potassium dihydrogen phosphate
200,00
Sodium chloride
8,000.00
di-Sodium hydrogen phosphate anhydrous
1,150.00