Immortalized Rat Embryonic Fibroblasts (18IM)
| CAT.NO | UNIT | PRICE |
|---|---|---|
| T0354 | 1x106 cells / 1.0 ml | $0.00 |
| Description | Immortalized Rat Embryonic Fibroblasts (18IM) are derived from rat embryonic fibroblasts isolated from 13 to 14 day old fetuses which were transfected with a plasmid containing S18-2. Transfected cells were selected for with 0.5 mg/mL G418. 18IM cells display embryonic stem cell-like properties after immortalization and subsequent loss of mesodermal markers thereafter. Loss of contact inhibition was also observed and cells were also capable of showing anchorage-independent growth. Immunostaining showed the presence of stem cell markers, such as SSEA-1, and the loss of mesodermal markers, such as vimentin and smooth muscle actin. RT-PCR also confirmed the loss of mesodermal markers, FUT4 and vimentin, and an up-regulation of stem cell markers, Sox2 and Oct3/4. This cell line may be useful as a model for the role of S18-2 in other cell types and further studies in the binding partners of S18-2 in differentiated tissues. |
|---|---|
| SKU | T0354 |
| Species | Rat (R. norvegicus) |
| Tissue/Organ/Organ System | Reproductive |
| Cell Morphology | Polygonal |
| Applications | For Research Use Only |
| Seeding Density | 20,000 – 100,000 cells/cm2; Split Ratio: 1:2 or 1:3 |
| Population Doubling Time | 46-56 hours |
| Immortalization Method | Transfection of plasmid encoded with GFP-fused S18–2 (GFP-S18–2) carrying a neomycin resistance gene |
| Growth Properties | Adherent |
| Expression Profile | SSEA-1, Sox2 and Oct3/4 |
| Propagation Requirements | Use of PriCoatTMT25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. Grow cells inECM-coated culture vessels unless otherwise specified in the Propagation Requirements below. The base medium for this cell line is Prigrow V medium available at abm (TM015). To make the completed growth medium, add the following components to the base medium: fetal bovine serum (TM999)* to final concentration of 5%, 0.5mg/ml Geneticin (Antibiotic G418) (G271) and Penicillin/Streptomycin Solution (G255) to a final concentration of 1%. Carbon dioxide (CO2): 5%, Temperature: 37.0 |
| Freeze-Thaw Recovery | 1. Pre-warm complete media in a 37°C waterbath. 2. Remove the cryopreserved vial from the liquid nitrogen storage tank. 3. Thaw the cells quickly by placing the lower half of the vial into the 37°C water bath and remove after 60 seconds. There should still be a few ice crystals left after thawing. It is important not to over-thaw the cryovials as the presence of DMSO is toxic to the cells. 4. Re-suspend the cells in the vial and transfer the cell suspension into a 15mL sterile conical tube containing 5mL complete media. 5. Centrifuge cells at 200x g for 3 minutes to pellet. 6. Aspirate out the media, leaving cell pellet undisturbed. 7. Re-suspend pellet in fresh culture medium and plate in new culture vessel. 8. Incubate cultures at 37°C, 5% CO2. Note: Where applicable, we recommend selection drug addition after cells recovered from thawing. Directly adding selection drug while thawing could lead to stressed cells and lower viability. |
| Subculture Protocol | 1. Remove and discard culture medium. 2. Add 2.0mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed. Recommended step: Cells are difficult to detach may be placed at 37°C for 2 minutes after adding Trypsin-EDTA to facilitate dispersal. 3. Centrifuge cells at 1500rpm for 3 minutes to pellet. 4. Aspirate out trypsin, leaving pellet undisturbed. 5. Resuspend pellet in fresh culture medium and plate in new culture vessel. 6. Incubate cultures at 37°C. |
| Preservation Protocol | 1. Freeze Medium: Complete growth medium with 20% FBS and 10% DMSO. 2. Storage Temperature: Liquid nitrogen vapour phase. |
| Unit quantity | 1x106 cells / 1.0 ml |
| QC | 1) Western blot analysis for presence of S18-2 in cells; 2) Immunostaining for expression of markers ; 3) Reverse Transcription PCR for determination of presence of markers |
| Disclaimer |
1. For for-profit organizations and corporations, please contact [email protected] for pricing of this item.
2. Sale of this item is subjected to the completion of a Material Transfer Agreement (MTA) by the purchasing individual/institution for each order. If you have any questions regarding this, please contact us at [email protected].
3. All test parameters provided in the CoA are conducted using abm's standardized culture system and procedures. The stated values may vary under the end-user's culture conditions. Please verify that the product is suitable for your studies by referencing published papers or ordering RNA (0.5 μg, Cat.# C207, $450.00) or cell lysate (100 μg, Cat.# C206, $600.00) to perform preliminary experiments, or alternatively use our Gene Expression Assay Service (Cat# C138). All sales are final.
4. We recommend live cell shipments for ease of cell transfer and this option can be requested at the time of ordering. Please note that the end-user will need to evaluate the feasibility of live cell shipment by taking into account the final destination's temperature variation and its geographical location. In addition, we thoroughly test our cell lines for freeze-thaw recovery. If frozen cells were received and not recovered in your lab under the exact, specified conditions (using recommended culture vessel, media, additional supplements, and atmospheric conditions), a live cell replacement is possible at a cost (plus shipping).
5. All of abm's cell biology products are for research use ONLY and NOT for therapeutic/diagnostic applications. abm is not liable for any repercussions arising from the use of its cell biology product(s) in therapeutic/diagnostic application(s). Please contact a technical service representative for more information.
6. abm makes no warranties or representations as to the accuracy of the information on this site. Citations from literature and provided for informational purposes only. abm does not warrant that such information has been shown to be accurate. 7. abm warrants that cell lines shall be viable upon initiation of culture for a period of thirty (30) days after shipment and that they shall meet the specifications on the applicable abm Material Product Information sheet, certificate of analysis, and/or catalog description. Such thirty (30) day period is referred to herein as the "Warranty Period." |
Supporting Protocol
- Important Considerations for Immortalized Cells
- Immortalized Cell Handling Instructions Upon Arrival
- Subculturing Protocol
- Freezing Protocol
- Hepatocyte Instructions
- Thawing Protocol
MSDS
QC
Other
| I want to make sure these cells express my gene of interest before I decide to buy the cell line. Can you provide a sample so this can be tested? | |
We do not carry out downstream characterization or gene expression profiling of our cell lines. To facilitate your preliminary experiments we can provide an RNA extraction (0.5ug total RNA) or cell lysate (100ug/100ul provided in 62.5mM Tris‐HCl, 2% SDS, 10% Glycerol, 50mM DTT, 0.01% w/v Bromophenol Blue) for any of our immortalized cell lines for a small fee. Please inquire directly for more information. The lead time will be around 2 weeks from the time of placing an order (if the item is in stock). | |
| How often do I need to change the media? | |
The media should be changed every 2-3 days. | |
| Why do these cells need bio safety level II? | |
In order to be more cautious, we follow the CDC-NIH recommendations that all mammalian sourced products should be handled at the Biological Safety Level 2 to minimize exposure of potentially infectious products. This information can be found in 'Biosafety in Microbiological and Biomedical Laboratories' (1999). Your institution's Safety Officer or Technical Services will be able to make the call as to whether BioSafety Level I is possible with these cells at your site if required. | |
| Do you sell ECM coated T75 flasks? | |
Yes we can provide a coating service. Please inquire with [email protected] | |
| Is it necessary that we have to use the recommended T25 ECM-coated flasks for growing the cells? Can we use normal ECM coated 60mm/90mm petri plates for growing the cells? | |
We strongly recommend using G299 T25 flasks to ensure recovery of these cells before testing other plates. | |
| What can I coat a larger dish to subculture? | |
We also offer applied extracellular matrix (collagen type I) in liquid form, for the coating of larger flasks and other required plasticware: http://www.abmgood.com/Applied-Cell-Extracellular-Matrix-G422.html | |
| How long can I store frozen vials for? | |
Cells that are properly frozen using an effective cryoprotective agent can be stored in liquid nitrogen indefinitely without affecting their recovery. | |
| When are cells plated for live cell shipments? | |
1 day prior to shipping | |
| Should the cap of the flask be changed before starting the cell culturing step? | |
No, there is no need in sterile biosafety cabinets unless it has contacted any non-sterile condition (e.g. touching the contaminated tip, etc.). | |
| What is the recommended storage temperature? | |
In general, if you received:
Live cells: acclimatize for 3-4 hrs at at the recommended conditions stated for the cell line under the propagation section, and then change media afterwards.
Frozen cells: Immediately place cells in liquid nitrogen; -180C. | |
| How is cell density crucial for drug selection? | |
If antibiotic selection is applicable to the target cells, we suggest getting rid of all the background cells so that the cell density is kept lower (even 20-30%). However, once the clones are selected by clonal dilution, we don't need the drug to still be present. If needed, the cell density should be towards the higher end since cells are already selected. Any primary cells still present will be depleted as a result of senescence and the cell population that remains will be resistant to the specific antibiotic. | |
| My cells are not detaching, what method do you recommend to trypsinize the cells? | |
1. Incubate the coated plate containing trypsin solution at recommended temperature indicated in the propagation section for 3-5 min till the cells round up, monitoring from time to time under microscope.
2. Diluting G422 (1:1) with PBS and coating for lesser time. Sometimes the collagen content in G422 is higher and thus make stronger bonding with cells.
3. You can try reducing the incubation time as well for coating the plate to make a thinner layer.
| |
| Why is it important to determine the optimal seeding density? | |
The seeding density we recommend is for when cells are plated to a new vessel. The optimal seeding density should allow cells to attach to the surface and have room to proliferate.
If you seed too little, cells may not attach well to the surface (for adherent cells). Seeding density is important as many cells (adherent or suspension cells) need to be in close proximity for better growth. Cell-cell interactions allow cells to communicate with each other in response to changes in their microenvironment. This ability to send and receive signals is essential for the survival of the cell. In other cases, if the seeding density is too low, cells may attach but a retardation in cell growth is observed.
If you seed too high, the cells will attach but there is insufficient room for further proliferation and they will stop replicating. | |
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- Kashuba, E. et al. "MRPS18–2 protein immortalizes primary rat embryonic fibroblasts and endows them with stem cell-like properties" Proceedings of the National Academy of Sciences of the United States of America 106:19866-19871 (2009). DOI: 10.1073/pnas.0911545106.