ECV-304 Cells
General information
Description | The ECV-304 cell line is a spontaneously transformed cell line derived from human umbilical vein endothelial cells (HUVEC). Originally characterized and utilized as a model of endothelial cell biology, further genomic analysis revealed that ECV-304 cells were contaminated and are genetically identical to the T24 bladder carcinoma cell line. This revelation has significant implications for the interpretation of research data, particularly studies conducted under the assumption that ECV-304 was a true endothelial cell model. Despite its endothelial mischaracterization, ECV-304 has been widely used in studies related to tumorigenesis, cytotoxicity, and drug screening, primarily due to its robust growth characteristics and the ease with which it can be cultured. The cells exhibit epithelial morphology and possess the ability to grow in a monolayer, making them a suitable in vitro model for studying various aspects of cancer biology, including cell proliferation, migration, and the cellular response to therapeutic agents. However, caution must be exercised in the interpretation of past studies where ECV-304 was used as a model of endothelial cells. Given the genetic identification with the T24 cell line, researchers should consider ECV-304 as a bladder carcinoma model rather than an endothelial cell line. This understanding is crucial for the design and interpretation of experiments aimed at investigating cellular behaviors that are relevant to cancer research rather than endothelial cell function. |
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Organism | Human |
Tissue | Bladder |
Disease | Carcinoma |
Synonyms | ECV 304, ECV304, ECV, E304, T24(ECV304) |
Characteristics
Age | 82 years |
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Gender | Female |
Morphology | Epithelial-like |
Growth properties | Adherent |
Identifiers / Biosafety / Citation
Citation | ECV-304 (Cytion catalog number 300452) |
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Biosafety level | 1 |
Expression / Mutation
Handling
Culture Medium | Medium 199, w: 2.7 mM stable Glutamine, w: 2.2 g/L NaHCO3, w: EBSS (Cytion article number 820101a) |
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Medium supplements | Supplement the medium with 10% FBS |
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) or CM-ACF (Cytion catalog number 806100) |
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,x
CSF1PO: 12
D13S317: 12
D16S539: 9
D5S818: 10
D7S820: 10,11
TH01: 6
TPOX: 8,11
vWA: 17
D3S1358: 16
D21S11: 29
D18S51: 16,18
D8S1179: 14
FGA: 17,22
D2S1338: 20,23
D12S391: 18
D19S433: 13,14
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Required products
Medium 199 offers a range of applications in the field. It can effectively maintain the cumulus-oocyte complex (COC) and support the in vitro maturation of oocytes. Additionally, it is employed in the rinsing of aspiration lines during ovum collection from German Holstein cows. Moreover, Medium 199 serves as an excellent medium for the culture of cardiac endothelial cells derived from rats. These applications demonstrate the versatility and adaptability of Medium 199 to various experimental needs.
History
The development of Medium 199 in the 1950s marked a significant advancement in tissue culture media. Prior to its introduction, many culture media relied on animal-derived products and tissue extracts. However, Morgan and colleagues revolutionized the field by formulating a completely defined nutritional source for cell cultures. Through their experiments involving different combinations of vitamins, amino acids, and other factors, they discovered the exceptional growth-promoting properties of Medium 199.
Quality control
pH = 7.2 +/
- 0.02 at 20-25°C.
Each lot has been tested for sterility and absence of mycoplasma and bacteria.
Maintenance
Keep refrigerated at +2°C to +8°C in the dark. Freezing and warming up to +37° C minimize the quality of the product.
Do not heat the medium to more than 37° C or use uncontrollable sources of heat (e.g., microwave appliances).
If only a part of the medium is to be used, remove this amount from the bottle and warm it up at room temperature.
Shelf life for any medium except for the basic medium is 8 weeks from the date of manufacture.
Composition
Components
mg/L
Inorganic Salts
Calcium chloride x 2H2O
264,92
Iron (III) nitrate x 9H2O
0,72
Magnesium sulfate
97,67
Potassium chloride
400,00
Sodium acetat x 3H2O
82,95
Sodium chloride
6,800.00
Sodium dihydrogen phosphate x H2O
140,00
Other Components
Adenine sulfate
10,00
AMP
0,20
ATP
1,00
Cholesterol
0,20
2‘-Deoxyribose
0,50
D(+)-Glucose anhydrous
1,000.00
Glutathione (red.)
0,05
Guanine x HCl
0,30
Hypoxanthine
0,30
Phenol red
10,00
D-Ribose
0,50
Thymine
0,30
Tween 80
4,90
Uracil
0,30
Xanthine
0,30
NaHCO3
2,200.00
Amino Acids
L-Alanine
25,00
L-Arginine x HCl
70,00
L-Aspartic acid
30,00
L-Cysteine x HCl x H2O
0,10
L-Cystine
20,00
L-Glutamine stable
149,00
L-Glutamic acid
67,00
Glycine
50,00
L-Histidine x HCl x H2O
21,88
L-Hydroxyproline
10,00
L-Isoleucine
20,00
L-Leucine
60,00
L-Lysine x HCl
70,00
L-Methionine
15,00
L-Phenylalanine
25,00
L-Proline
40,00
L-Serine
25,00
L-Threonine
30,00
L-Tryptophan
10,00
L-Tyrosine
40,00
L-Valine
25,00
Vitamins
4-Amino benzoic acid
0,05
Ascorbic acid
0,05
D(+)-Biotin
0,01
Calciferol
0,10
D-Calcium pantothenate
0,01
Choline chloride
0,50
Folic acid
0,01
myo-Inositol
0,05
Menadione
0,01
Nicotinic acid
0.025
Nicotinamide
0.025
Pyridoxal x HCl
0.025
Pyridoxol x HCl
0.025
Riboflavin
0,01
DL-α-Tocopherol phosphate disodium salt
0,01
Thiamine x HCl
0,01
Vitamine A acetate
0,14
Key features of Freeze Medium CM-1 include:
Broad Compatibility: Effective for a wide range of cell types, including primary cells, stem cells, and established cell lines.
High Viability: Optimized to maximize post-thaw cell recovery and viability, ensuring reliable experimental outcomes.
Ready-to-Use: Conveniently prepared and sterilized for immediate application, reducing preparation time and risk of contamination.
Enhanced Stability: Maintains consistent performance under standard cryopreservation conditions, ensuring reproducible results.
Long Shelf Life: CM-1 is a serum-containing, ready-to-use cryopreservation medium that can be stored in the refrigerator for up to one year.
Using CM-1 for Freezing Cells
To use CM-1 for freezing both adherent and suspension cells, follow these steps:
For adherent cells, wash and dissociate them from the culture substrate. For suspension cells, proceed directly to the next step.
Count the cells to ensure they are at the proper concentration.
Centrifuge the cells to pellet them, then resuspend in CM-1 freeze medium.
Transfer the resuspended cells into cryovials.
Use a slow-freezing method before transferring the cells to long-term storage.
🥶 Method
🔍 Description
💡 Steps
❄️
Manual Freezing
A step-by-step method involving gradual temperature reduction to ensure cell viability.
1️⃣ Place cells in freeze medium in a 4°C freezer for 40 minutes.
2️⃣ Transfer to a -80°C freezer for 24 hours.
3️⃣ Store cells in liquid nitrogen for long-term preservation.
🧊
Using Mr. Frosty
A convenient device that allows for controlled freezing rates without electrical power.
1️⃣ Prepare cells in cryovials with freeze medium.
2️⃣ Place cryovials in Mr. Frosty container.
3️⃣ Store at -80°C for 24 hours before transferring to liquid nitrogen.
🧬
Controlled-Rate Freezer
A high-precision freezer by Thermo Fisher or other manufacturers designed for controlled temperature reduction.
1️⃣ Program the device to gradually decrease the temperature.
2️⃣ Place prepared cells in the freezer.
3️⃣ After the freezing cycle, transfer cells to liquid nitrogen.
Store the cryovials at temperatures below -130°C or in liquid nitrogen for long-term preservation.
Ingredients
Contains FBS, DMSO, Glucose, Salts
Buffering capacity: pH = 7.2 to 7.6
Cytion’s Freeze Medium CM-1 offers a reliable solution for cryopreservation, ensuring high cell viability and functionality post-thaw for a wide range of research applications.
- 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