NRK-Pom121-EGFP3 Cells
General information
Description | The NRK-Pom121-EGFP3 cell line is derived from normal rat kidney (NRK) cells and is genetically engineered to express the Pom121-EGFP3 fusion protein. Pom121 is a transmembrane nucleoporin that is an integral component of the nuclear pore complex (NPC), playing a crucial role in nuclear envelope assembly and NPC function. The inclusion of the enhanced green fluorescent protein (EGFP3) tag facilitates the visualization and study of Pom121 dynamics, localization, and interactions within live cells through fluorescence microscopy. This makes the NRK-Pom121-EGFP3 cell line a valuable tool for investigating nuclear transport mechanisms and NPC architecture. NRK cells, the parental line of NRK-Pom121-EGFP3, are commonly used in various research applications due to their stable growth characteristics and epithelial morphology. The modification to express Pom121-EGFP3 provides researchers with a robust model to examine the molecular mechanisms underlying nucleocytoplasmic transport, the structural organization of the NPC, and its regulation during cell division and differentiation. Additionally, this cell line can be used to study the effects of various genetic and pharmacological perturbations on NPC function, offering insights into diseases associated with nuclear transport defects, such as cancer and neurodegenerative disorders. Overall, the NRK-Pom121-EGFP3 cell line represents a sophisticated tool in cell biology and molecular research, providing high-resolution insights into the dynamic processes governing nucleocytoplasmic interactions. Its ability to allow real-time observation of NPC components in a live cellular context makes it invaluable for advancing our understanding of cellular transport mechanisms and their implications in health and disease. |
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Organism | Rat |
Tissue | Kidney |
Synonyms | NRK Pom121-EGFP3, NRK Pom121-3EGFP, NRK-Pom121-3EGFP |
Characteristics
Morphology | Fibroblast-like cells with fusiform shape |
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Growth properties | Monolayer, adherent |
Identifiers / Biosafety / Citation
Citation | NRK-Pom121-EGFP3 (Cytion catalog number 500669) |
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Biosafety level | 1 |
Depositor | Dr. J. Ellenberg, EMBL Heidelberg |
Expression / Mutation
Receptors expressed | Epidermal growth factor (EGF), multiplication stimulating activity (MSA) |
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Protein expression | Pom121-EGFP3: Location/Gene: 1..589 / Pcmv, 653..4250 / Pom121, 4251..4287 / null, 4318..6546 / 3EGFP, 7780..8574 / KanR/NeoR |
Products | Epidermal growth factor (EGF), multiplication stimulating activity (MSA), POM121, Transmembrane, Nucleoporin, CMV Promotor, Neomycin, Phosphotransferase |
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) |
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Medium supplements | Supplement the medium with 10% FBS, 0.5 mg/mL G418 |
Passaging solution | Accutase |
Subculturing | Discard the old medium and wash the cells with PBS. Add a freshly prepared 0.025% trypsin/0.02% EDTA solution heated to 37 degrees Celsius and wait until the cells detach, which usually takes about 5 minutes. Neutralize the trypsin by adding fresh medium, then transfer the cell mixture to a tube and centrifuge. After centrifugation, remove the supernatant, resuspend the cell pellet in fresh culture medium, and transfer the suspension to new flasks. Incorporate G418 into the culture medium to achieve a final concentration of 0.5 mg/ml |
Split ratio | A ratio of 1:3 to 1:4 is recommended |
Seeding density | 2 to 4 x 10^4 cells/cm^2 |
Fluid renewal | 2 to 3 times per week |
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 |
Rat_D1Wox31: 96,1
Rat_D2Wox37: 156
Rat_D19Wox11: 220
Rat_D10Wox8: 266,27
Rat_D4Wox7: 153,157
Rat_D2Wox27: 211
Rat_D5Rat33: 116,138
Rat_D10Wox11: 156
Rat_D1Wox23: 210,214
Rat_D12Wox1: 402,406
Rat_D6Wox2: 104,124
Rat_D8Wox7: 185
Rat_D6Cebr1: 221,233
SRY: x,Y
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Required products
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
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