MDCK-SIAT1 Cells
General information
Description | The MDCK-SIAT1 cell line is a modified version of the Madin-Darby Canine Kidney (MDCK) cells, engineered to express higher levels of human 2,6-sialyltransferase (SIAT1). This enzyme is responsible for the addition of sialic acid in an alpha-2,6 linkage to galactose on glycoproteins and glycolipids. The modification was performed to increase the expression of alpha-2,6-linked sialic acids, which are the primary receptors for human influenza viruses. This enhancement is critical as it makes the MDCK-SIAT1 cells more similar to the human airway epithelium, which naturally has a high concentration of these receptors. As a result, these cells offer a more physiologically relevant model for studying human influenza viruses and their interactions with potential antiviral compounds. One of the significant applications of MDCK-SIAT1 cells is in the assessment of influenza virus sensitivity to neuraminidase inhibitors (NAIs), such as oseltamivir. Due to the increased presence of alpha-2,6-linked sialic acids, the MDCK-SIAT1 cells demonstrate improved sensitivity to NAIs compared to unmodified MDCK cells. This makes them an excellent tool for detecting resistance to these inhibitors, especially in low-passage-number clinical isolates of human influenza viruses. The MDCK-SIAT1 cell line allows for more accurate in vitro studies of drug efficacy and viral receptor interactions, providing valuable insights into the development of antiviral therapies and resistance mechanisms. |
---|---|
Organism | Canine |
Tissue | Kidney |
Characteristics
Age | Adult |
---|---|
Gender | Female |
Morphology | Epithelial |
Growth properties | Adherent |
Identifiers / Biosafety / Citation
Citation | MDCK-SIAT1 (Cytion catalog number 602281) |
---|
Expression / Mutation
Protein expression | Transfected with ST6 beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1, SIAT1) |
---|
Handling
Culture Medium | DMEM, w: 4.5 g/L Glucose, w: 4 mM L-Glutamine, w: 1.5 g/L NaHCO3, w: 1.0 mM Sodium pyruvate (Cytion article number 820300a) |
---|---|
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) |
Handling of cryopreserved cultures |
|
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. |
---|
Benötigte Produkte
What sets DMEM apart from other media is its unique composition. It contains an impressive fourfold increase in amino acid and vitamin concentration compared to the original Eagle's Minimal Essential Medium. Initially developed with low glucose (1 g/L) and sodium pyruvate, DMEM is frequently employed with higher glucose levels, either with or without sodium pyruvate. Notably, DMEM does not contain proteins, lipids, or growth factors, necessitating supplementation. To achieve optimal growth, a common approach is to supplement DMEM with 10% Fetal Bovine Serum (FBS). Additionally, DMEM employs a sodium bicarbonate buffer system (3.7 g/L), requiring a 5-10% CO2 environment to maintain a physiological pH.
Dulbecco's Modified Eagle Medium (DMEM) is highly regarded among defined media for cell and tissue culture, catering to the growth needs of various adherent cell phenotypes. It surpasses the original Eagle's Medium, developed in the 1950s for cultivating chicken cells, through the enhanced supplementary formulation known as Dulbecco's modification. This modification significantly elevates the content of select amino acids and vitamins up to fourfold compared to the original medium.
In the field of cell culture, DMEM plays a vital role as a growth medium for different cell types, including primary cells, stem cells, and transformed cells. Researchers also employ the modified version of DMEM for a wide array of research applications, such as drug discovery, tissue engineering, and the study of cell signaling pathways.
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 anhydrous
200,00
Iron (III) nitrate x 9H2O
0,10
Magnesium sulfate anhydrous
97,66
Potassium chloride
400,00
Sodium chloride
6.400,00
Sodium dihydrogen phosphateanhydrous
108,69
Other Components
D(+)-Glucose anhydrous
4.500,00
Sodium pyruvate
110,00
Phenol red
15,00
NaHCO3
1.500,00
Amino acids
L-Arginine x HCl
84,00
L-Cystine x 2HCl
62,58
L-Glutamine
584,00
Glycine
30,00
L-Histidine x HCl x H2O
42,00
L-Isoleucine
104,80
L-Leucine
104,80
L-Lysine x HCl
146,20
L-Methionine
30,00
L-Phenylalanine
66,00
L-Serine
42,00
L-Threonine
95,20
L-Tryptophan
16,00
L-Tyrosine x Na
103,79
L-Valine
93,60
Vitamins
D-Calcium pantothenate
4,00
Choline chloride
4,00
Folic acid
4,00
myo-Inositol
7,00
Nicotinamide
4,00
Pyridoxine x HCl
4,00
Riboflavin
0,40
Thiamine x HCl
4,00
- 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.