HaCaT-ras II-4 cells are a remarkable and extensively studied cellular model in biological science. These cells are derived from spontaneously immortalized human skin keratinocytes, known as HaCaT cells, which were modified through transfection with the c-Ha-ras (EJ) oncogene. The selection of these cells was based on their resistance to G418, a selective antibiotic, as described in the comprehensive study conducted by Boukamp et al. in 1990.One notable characteristic of HaCaT-ras II-4 cells is their tumorigenic nature. When these clonal cells are injected into Balb/c-nu/nu mice, they exhibit a fascinating behaviour by forming highly differentiated and locally invasive squamous cell carcinomas. This unique property allows researchers to explore tumour development and progression mechanisms within a controlled experimental environment.HaCaT-ras II-4 cells are predominantly derived from the Caucasian population, ensuring relevance to a specific ethnic group in scientific investigations. Their origin and characteristics make them an invaluable resource for researchers interested in studying various skin biology and differentiation aspects.These cells possess a partially to fully differentiated phenotype under typical culture conditions. This phenotype is attributed to the abundant presence of calcium in both traditional media and fetal bovine serum, which provides an ideal environment for the cells to exhibit characteristics resembling those of mature skin cells. This feature allows researchers to investigate the intricate processes involved in skin development, wound healing, and epidermal differentiation.With their tumorigenic nature and the ability to replicate skin biology in vitro, HaCaT-ras II-4 cells offer a unique opportunity to explore the molecular pathways associated with skin cancer and other skin-related disorders. By utilizing this exceptional cellular model, researchers can gain deeper insights into the underlying mechanisms of tumorigenesis, invasive potential, and therapeutic interventions.HaCaT-ras II-4 cells are a vital tool for biological science research, specifically in skin biology and differentiation studies. Their origin from spontaneously immortalized human skin keratinocytes, modification with the c-Ha-ras (EJ) oncogene, and subsequent tumorigenic behaviour in mice make them invaluable for investigating skin-related diseases and therapeutic approaches. By harnessing the unique characteristics of HaCaT-ras II-4 cells, researchers can unlock a deeper understanding of skin biology and contribute to advancing medical knowledge and treatment options for various skin disorders.
Synonyms
HaCaT-ras clone II-4, HaCaT II-4, II-4
Gender
Male
Age
62 years
Ethnicity
Caucasian
Growth Properties
Adherent
Tissue
Skin
Tissue Specificity
Skin
Biosafety level
1
Citation
HaCaT-ras II-4 (CLS catalog number 300495)
Depositor
DKFZ, Heidelberg
Notes
This cell line is licensed to CLS by a university or institute. The sale of this item requires the conclusion of a Material Transfer Agreement (MTA). Please get in touch with us for further information.
Karyotype
Aneuploid (hypotetraploid)
Protein Expression
P53 (+), CEA (+),
Tumorigenic
Formation of highly differentiated, locally invasive squamous cell carcinoma in Balb/c-nu/nu mice.
Culture Medium
DMEM
Medium Supplements
10% FBS, w: 4.5 g/L Glucose, w: 4 mM L-Glutamine, w: 1.5 g/L NaHCO3, w: 1.0 mM Sodium pyruvate
Passaging Solution
The 1:1 mixture of EDTA (stock. 0.05%) and trypsin (stock: 0.1%) must be prepared each time ahead of detaching the cells using PBS without Ca2+ and Mg2+ to provide a physiologic osmolarity. Ready-to-use mixtures of trypsin/EDTA are not recommended, as this may result in cell clumps. As an alternative, TrypLETM Express (Life Technologies) instead of trypsin/EDTA can be used. The protocol of the manufacturer should be followed.
Freeze Medium
CM-1 (CLS catalog number 800100) or CM-ACF (CLS catalog number 806100)
Handling of Cryopreserved Cultures
The cells come deep-frozen shipped on dry ice. Please make sure that the vial is still frozen. If immediate culturing is not intended, the cryovial must be stored below -150 degree Celsius after arrival. If immediate culturing is intended, please follow the below instructions: Quickly thaw by rapid agitation in a 37 degree Celsius water bath within 40-60 seconds. The water bath should have clean water containing an antimicrobial agent. As soon as the sample has thawed, remove the cryovial from the water bath. A small ice clump should still remain and the vial should still be cold. From now on, all operations should be carried out under aseptic conditions. Transfer the cryovial to a sterile flow cabinet and wipe with 70% alcohol. Carefully open the vial and transfer the cell suspension into a 15 ml centrifuge tube containing 8 ml of culture medium (room temperature). Resuspend the cells carefully. Centrifuge at 300 x g for 3 min and discard the supernatant. The centrifugation step may be omitted, but in this case the remains of the freeze medium have to be removed 24 hours later. Resuspend the cells carefully in 10 ml fresh cell culture medium and transfer them into two T25 cell culture flasks. All further steps are described in the subculture section.
Handling of Proliferating Cultures
One or two cell culture flasks come filled with cell culture medium. Collect the entire medium in 1 or 2 x 50 ml centrifuge tubes, respectively. Carefully add 5 ml of cell culture medium to each T25 cell culture flask. Control the cell morphology and confluency under the microscope. Incubate at 37 degree Celsius for a minimum of 24 hours. Spin down the collected medium at 300 x g for 3 minutes to collect the cells which may have detached during transit. If a cell pellet is visible, resuspend the cells in 5 ml of cell culture medium and transfer to a T25 cell culture flask. Incubate at 37 degree Celsius for a minimum of 24 hours.
Subculturing
Remove the culture medium and rinse the adherent cells using PBS without calcium and magnesium (3-5 ml PBS for T25, 5-10ml for T75 cell culture flasks). Add freshly prepared 0.05% EDTA solution, 1-2ml per T25, 2.5ml per T75 cell culture flask, the cell sheet must be covered completely, and incubate at 37 degree Celsius for 10 min. Add freshly prepared trypsin/EDTA, 0.05%/0.025% solution *2 (1ml per T25, 2.5ml per T75 cell culture flask), the cell sheet must be covered completely. The cells must detach within 1-2 minutes. Stop the trypsin activity using FBS-containing cell culture medium. Dispense into new flasks which contain fresh cell culture medium.
Seeding Density
1 x 10^4 cells/cm^2
Fluid Renewal
2 times per week
Split Ratio
A ratio of 1:5 to 1:10 is recommended
Sterility
Mycoplasma contamination was excluded through PCR-based and luminescence-based mycoplasma assays. Bacterial or fungal contaminations are detected through daily visual cell monitoring.
This cell line is available in the U.S. For non-profit users, please sign and return the Supply Agreement to [email protected] when placing an order. For commercial users, please complete the CLEAR Form as additional usage fee may apply based on the given intended usage. For further details, please contact us at [email protected].
Material Safety Data Sheets (MSDS)
The MSDS are available in the download section of the product pages. Please, navigate to the product section and the product of interest to obtain the MSDS. If you cannot find the MSDS in the download section, please get in contact with us to request the respective document. Product sheets
The product sheets are available in the download section of the product pages. Please, navigate to the product section and to the product of interest to obtain a product sheet. If you cannot find the product sheet in the download section, please get in contact with us to request the document. Licensed items
If a cell line is licensed to CLS by a partnering university or research institute our customers cannot purchase the cell line through the online shop. The cell line may be subject to individual terms and licensing fees as required by the licensor. Please contact us for further information. Transfer of ownership, limitation of rights of use
1. Orders for Cells and other materials from CLS (“CLS Material”) are regularly provided under a Supply Agreement or Material Transfer Agreement which for the Customer constitutes the right to use the CLS Material as specified therein. By delivering the CLS Material to the Customer, the Customer is therefore only permitted to use the CLS Material in accordance with the regulations in the respective supply contract and under no circumstances does CLS transfer ownership of the CLS Material to the Customer. Only if expressly agreed, orders delivered by CLS constitute purchase contracts. In the case of such purchase contracts, CLS retains title to the Deliveries until full payment by the Customer (retention of title). The use of the products is limited to the contractually agreed purposes. The scope of the permitted use shall be determined by the contract concluded with the respective order. If the products are provided to the customer for use only for a limited period of time, transfer of ownership shall not take place. Commercial transfer to third parties is not permitted unless CLS and - unless CLS is the unrestricted owner of all rights to the Products - other holders of rights to the Products have given their express prior written consent thereto.
2. The retention of title shall apply until full payment of all claims of CLS arising from the business relationship with the Customer.
3. The type and scope of permissible use shall follow from the respective Material Transfer Agreement to be concluded with the Client. The Customer is obligated to return or destroy the product after expiry of the period of use. The details follow from the respective Material Transfer Agreement. If third parties are entitled to rights to delivered products, the customer must in such cases fulfill the conditions communicated to him upon conclusion of the agreement in order to obtain their consent to use.
4. CLS has the right to be identified and have its products mentioned in any Publication, in the research of which such products were used, and the Customer shall expressly name CLS and its respective products in any such Publication.
4.1 For this purpose, Publication shall mean any publications, in whatever form, in particular, but not limited to all academic or scientific papers, patent applications or other research and experimental descriptions.
4.2 Material provided by CLS shall be deemed used in research, if the Publication is based on any research or experiments in which products supplied by CLS were used, regardless of whether and to what extent the material was relevant to the outcome of such research or experiment.
4.3 The right to be identified and have material mentioned in any Publication further applies to any supplier which provides materials to CLS, if such material is used in the underlying research. In case a Publication refers to such Material, CLS is entitled to inform the provider of the Publication and to pass on the Customer's data to the provider for this purpose.
4.4 The Customer will subject any third-party which is designated to conduct research with materials provided by CLS to an obligation to the same extent as under this clause
4.5. If applicable, the Customer will obtain the consent of any such Transferee for his data to be transferred to a Provider. For the avoidance of doubt, this does not grant the Customer any right to transfer the material to third parties; this is only permitted if and insofar as it has been expressly authorized by CLS under the Material Transfer Agreement. Transfer of risk, obligation to inspect and give notice of defects
1. The risk shall pass to the Customer depending on the agreed terms of delivery. In the event of delivery by handing over the goods to a carrier, the risk shall pass to the Customer as soon as CLS hands over the goods to the carrier.
2. The Customer shall immediately inspect the goods for defects upon delivery in the ordinary course of business. The Customer must give notice of any defects discovered by him without undue delay. The notice of defects must be precise and must be in writing.
What products or services do you offer?
We offer high-quality cell lines including human cells, animal cell lines, fluorescence-tagged cell lines, iPSC lines, as well as different human primary cells (human mesenchymal stem cells, fibroblasts, follicular dendritic cells, HUVEC, oral cells).
In addition, you can acquire more than 30 different serum-containing media and 10 serum-free media for the cultivation and maintenance of various cell types.
Our service includes viral testing, mycoplasma detection service, cell line authentication, cell banking and species identification. Moreover, we offer bulk and custom manufacturing.
Where can I find references from other researchers that have used your products?
If available, you will find the references with the respective product.
How to determine the concentration of cells in a suspension?
Multiply the average cell count from each of the hemocytometer's 16 corner squares by 10,000. Final viability counts per milliliter of sample are obtained by multiplying the initial count by 5, to account for the 1:5 dilution caused by the addition of trypan blue. Example:
(50 + 40 + 45 +52) ÷ 4 = 46.75
75 x 10,000 (10^4) = 467,500
467,500 x 5 = 2,337,500 live cells/mL in original cell suspension
Alternatively use an automated cell counting device such as the CellDrop from DeNovix or the NucleoCounter NC-200 from Chemometec.
How to store the purchased cells?
We strongly recommend to store the cells after receipt on dry ice for a very short time (1 day at the most) at -80°C and start culturing as soon as possible. If immediate culturing is not possible, the cryovial(s) must be stored at temperatures below -150°C. The quality of the permanent cell lines should be controlled within 60 days after receipt. Approach:
Thaw the cells
Determine cell viability
Culture the cells
Make up your own master stock.
How to store the purchased media and supplements?
All media available in our shop must be stored at 4 °C in the refrigerator. The supplements available have different storage conditions. Please refer to the information on the data sheets supplied.
How do I establish a culture from cryopreserved cells?
Bring water to a temperature of 37 °C in a beaker.
Remove a vial of cells from liquid nitrogen storage with care, making sure to shield your hands and eyes.
Loosen the vial's cap for 10 seconds to allow any liquid nitrogen to escape the threads, and then re-tighten the top.
To thaw, just submerge the vial's lower half in the 37 °C water for four minutes.
Wipe the outside of the vial with disinfectant solution and place it in a laminar flow culture hood of Class II, type A, once the contents have thawed until just a little piece of ice remains.
To disperse the cells, open the vial and pipette the suspension up and down with a 1 mL pipette.
Take out 20 µl, then add 20 µl of trypan blue solution to the cell suspension (e.g., Cat. No. 15250-061).
Use a hemocytometer or an automatized cell count system to calculate the number of viable cells per milliter.
Dilute the vial's contents (1 mL) to the concentration suggested in the product instructions.
After inoculation, agitate the medium in the flasks to disperse the cells. Numerous cell types adhere rapidly to culture surfaces; If the medium is not evenly distributed immediately after inoculation, the cells may grow in an uneven pattern.
Maintain the cultures in a cell culture incubator at 37 degrees Celsius with 5% carbon dioxide and 95% air humidity. Ideally, the culture should be left alone for at least 24 hours after it has been started.
How do I know what kind of cell culture medium i need to use for my specific cell line?
The information on the cell culture medium we do recommend for a specific cell line is given on the product page for the cell line as well as in the product information. The most common cell culture media are available as basic as well as ready-to-use (containing e.g., FBS and other supplements) formulations.
Some cell lines such as NFS-60 or TF-1 need special ingredients in their culture medium. Our Conditioned media are a good supply of these factors.
In order to cryopreserve the cells in liquid nitrogen, we recommend to use either CM-1, which contains serum, or the serum free freeze medium CM-ACF.
What are living or growing cultures?
Our product range also includes living cell cultures. Depending on the cell type, these are adherent or suspended cells.
How often should I change the media?
After thawing and plating cryopreserved cells, the first medium change should be performed after 24 hours or overnight to remove any remaining DMSO and dead cells. The medium should then be replaced every 24-48 hours until the cells are ready to be passed. Protocols for each cell line contain more specific instructions.
Can I expand your cells and re-freeze them?
Our cryopreserved cell lines or growing cultures may be expanded and cryopreserved again. However, the process of cryopreservation may alter the growth performance of cells.
Do I need to spin the cells out of the cryopreservation medium to plate them?
We do not advise spinning cells out of cryopreservation medium before plating. When centrifugation is performed at too high speeds, it might cause damage to cells. In our cell culture labs, we have found that at low concentrations, DMSO is safe for the cells. We advise diluting the cells in culture medium until the final DMSO content is less than 0.4% (v/v).
Do I need a surface matrix to culture my cells in 2D?
For the most of our cells you do not need a special surface matrix for 2D culture. Only our induced pluripotent stem cells (hiPSCs) require a culture surface substrate that maintains pluripotency. Here we recommend to use iMatrix or Matrigel.
How can I dissociate my cells from the cell culture plates?
For the detachment of most of the cells provided by CLS (except for e.g., for some keratinocyte cells such as HaCaT), we recommend to use Accutase which gently detaches the cells from the bottom of the cell culture flask. Accutase works with most cell lines. In case of the need of alternative detachment solutions, these are indicated in the specific product information of the cell line.
Answers to frequently asked questions on cell lines, strains, and types
When cells are separated from a tissue to generate a primary culture, a confluent monolayer or dense cell suspension is formed due to the cells' ability to proliferate in vitro.
In accordance with the traditional concept, a cell line is created after the initial collection and subculturing of this population (Freshney, R.I. (1987). Methods used to grow new tissues from animal cells. (New York: Alan R. Liss, Inc.) A primer on the fundamentals. Over the course of a cell line's existence, those cells with the greatest growth potential will prevail, leading to some genetic and behavioral homogeneity.
With this terminology, a population of cells that has undergone a genetic alteration that allows for infinite growth is called a continuous cell line. Constant cell lines are usually aneuploid. Even while it is possible to culture continuous cell lines through a large number of subcultures, there is a risk that additional genotypic and, by extension, phenotypic changes will occur at these very high passage numbers. One can achieve immortality in a variety of ways, including naturally or with the help of viruses or chemicals. Understand that different research teams may use slightly different operational definitions of these concepts. Many researchers avoid calling a population a "cell line" until they can prove that it has undergone some sort of genetic mutation.
Positively selected from a culture, either by cloning or some other means, a small subset of cells is called a cell strain. Since its original parent lineage was established, most cell strains have experienced significant genetic modifications. Some subsets of cells may have become more tumorigenic than the original line, or the transfection process may have resulted in the creation of a new strain.
Cell types, which include keratinocytes and melanocytes, are collections of cells that have a common phenotype. That's why it's safe to say that every donor's keratinocytes are the same cell type.
What are stem cells?
Stem cells aid in the production of new cells in healthy tissues and may aid in the repair of injured or damaged tissues. They serve as the foundation for the distinct cell types that comprise each organ in the body. Stem cells are distinguished from other cells by their ability to self-renew, proliferate for an extended period of time, and, under specific conditions, differentiate into specialized cells with distinct functions (phenotypes), including but not limited to cardiac cells, liver cells, fat cells, bone cells, cartilage cells, nerve cells, and connective tissue cells. Multipotency is the capacity of cells to differentiate into a variety of other cell types. What scientists discover regarding the regulation of stem cell differentiation can serve as the foundation for the development of novel treatments for a variety of life-threatening diseases and injuries.
What are induced pluripotent stem cells?
Somatic (adult, non-germline) cells that have been reverted to an embryonic stem cell-like state give rise to induced pluripotent cells. As with embryonic stem cells, iPS cells are pluripotent because they can differentiate into any cell in the body. The process of creating these cells, commonly referred to as "reprogramming," involves the introduction of three to four genes for transcription factors into a somatic cell via retroviruses.
Recent techniques have replaced and reduced the number of genes required for transformation, utilized alternative gene delivery methods, or sought to replace genes with chemical factors. Patients with diseases such as ALS, Parkinson's, or cardiovascular disease can have their cells induced to form iPS cells. When differentiated into more specialized cell types, iPS cells have multiple applications, including the development of assays for studying disease processes, screening drug candidates for safety and efficacy, and use in regenerative medicine.
What is the shelf life of your culture media?
You can find the information about shelf life of our products in the material safety data sheets at the respective product as pdf-file under product information/downloads.
Is it possible to freeze and thaw your culture media and supplements?
It is not recommended to store our cell culture media products under 4 °C. Regarding our supplement products please use the individual safety data sheets to get information’s about storing.
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