Immortalized Drosophila Embryonic Cells (R3)
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Sale of this item is subjected to the completion of a Material Transfer Agreement (MTA) by the purchasing institution.
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| Cat. No. | T0702 |
| Name | Immortalized Drosophila Embryonic Cells (R3) |
| Description |
The immortalized Drosophila Embryonic Cells (R3) was established by serial passaging of primary cultures from Act5C > UAS-RasV12 embryos. The RasV12 immortalized cell lines have significantly up-regulated cell-cycle and cell-division gene expression including gene targets of the E2 promoter binding factor/retinoblastoma protein (E2F/RB) pathway. The cell lines are in a proliferative undifferentiated progenitor-like state associated with increasing levels of Polycomb Group (PcG) transcripts during the immortalization process. Related to adult muscle precursors (AMPs), a stem cell-like population contributing to adult muscles and sharing properties with vertebrate satellite cells, the immortalized R3 cell line retained the capacity for myogenic differentiation when treated with the steroid hormone ecdysone. The cell line is recommended as a tool for Drosophila embryogenesis and muscle cell differentiation and proliferation. Express upregulated Cyc A, stg, dap, cdc2c, tum, sti, pav, stg, dup, dap, Orc2, and Mcm2. Note: This cell line is sensitive to cryopreservation; if recovery exceeds seven days, perform a 50% medium change weekly until the culture reaches sufficient confluence for subculture. |
| Organism | Insect (Insecta) |
| Tissue | Embryo |
| Donor History | Fruit Fly (D. melanogaster) |
| Growth Properties | Adherent, spindle-shaped |
| Storage Condition | Vapor phase of liquid nitrogen, or below -130°C. |
| Shipping Conditions | Ship with dry ice. |
| Product Format | Frozen |
| Intended Use | This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. |
| BioSafety | II |
| Certificate of Analysis | For batch-specific test results, refer to the applicable certificate of analysis that can be found at www.abmgood.com. |
| Growth Conditions |
For optimal cell culture, we recommend using PriCoat™ T25 Flasks (G299) or coating your preferred vessels with Applied Cell Extracellular Matrix (G422). Schneider's Insect Medium + 10% heat-inactivated FBS + 1% Penicillin/Streptomycin Solution (G255), 25.0°C, no CO₂ required. |
| Unpacking and Storage Instructions |
1. Visually examine the packaging containers for signs of leakage or breakage. 2. Immediately transfer frozen cells from dry ice packaging to a temperature below -130°C, preferably in liquid nitrogen vapor phase storage, until ready for use. To ensure the highest level of viability, thaw the vial and initiate culture as soon as possible upon receipt. If continued storage is desired, the vial should only be stored below -130°C or in liquid nitrogen vapor phase. Do not store at -70°C, as it will result in loss of viability. |
| Thawing Protocol |
1. Decontaminate the vial by spraying and wiping the exterior of the vial with 70% ethanol. 2. Transfer 1 ml of room-temperature medium into the cryovial to gently thaw cells. 3. Move the full cell suspension into a T-25 flask, rinsing to ensure complete transfer. 4. Incubate at 25°C (no CO₂) for at least 2 hours to allow attachment, then replace with 5 ml fresh medium and return to incubator. 5. Next day, replace medium with fresh 5 ml. If growth is slow, partially refresh medium as needed (replace half every few days). Note: Freshly thawed cultures may contain debris that can resemble contamination. Monitor for rapid overnight increase (likely contamination) versus stable or declining debris (normal recovery). Healthy cells should gradually overtake debris if viable. |
| Subculture Protocol |
1. Transfer culture medium from the plate to a sterile flask and retain it. 2. Rinse cells with 1 ml of 0.25% trypsin-EDTA, swirl gently, and discard. 3. Add 1 ml fresh trypsin-EDTA and incubate at 25°C for 5-10 min, monitoring detachment. 4. Neutralize trypsin with 9 ml of saved medium and gently dissociate clumps. 5. Transfer to a conical tube and centrifuge at 1,000 × g for 5 min; discard supernatant. 6. Resuspend pellet in fresh medium and seed into a new flask at the desired dilution. 7. Return flask to a 25°C incubator. |
| Cryopreservation |
We recommend using serum-free CryoGuard™ Freezing Media (TM078) or, if serum is preferred, Cryopreservation Medium (TM024). |
| Population Doubling Time (h) | 24 - 48 |
| Immortalization Method |
Serial passaging of primary cultures from Act5C > UAS-Rasᵛ¹² embryos |
| Warranty | 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”. |
| Disclaimer |
1. 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 licensing@abmgood.com. 2. 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. 3. 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). 4. 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. 5. 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. 6. 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." |
| Depositor | Harvard University |
| QC |
(1) Inducing muscle cell differentiation by treating cells with ecdysone and perform immunofluorescence staining and qPCR to assess the upregulated expression of mhc and Tropomyosin (Tm). |
| Application | Research Use Only. |
| Material Citation | If use of this material results in a scientific publication, please cite the material in the following manner: Applied Biological Materials Inc, Cat. No. T0702 |
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How should I handle live cells once I receive them? |
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Please refer to our Cell Handling and Thawing Guidelines for detailed instructions on receiving, thawing, and culturing live cells: |
| 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? | |
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We do not perform extensive downstream characterization, or gene expression profiling of our cell lines. However, you can check the ‘References’ tab to see if a publication is available, or you can contact us to see if RNA or cell lysates are available for your cell line of interest. Please directly contact us for further information at quotes@abmgood.com.
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| Does abm's PriGrow series medium contain antibiotics? | |
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No, the medium does not contain antibiotics and needs to be supplemented by the end-user as desired.
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| Do I need to use abm's media and Applied Cell Extracellular Matrix (G422) to culture my cells? | |
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We strongly encourage using the recommended media and culture conditions described in the "Growth Conditions" section as they have been optimized for cell growth. Other supplier's media and coating conditions have not been tested.
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| What is the difference between Applied Cell Extracellular Matrix (G422) and Collagen Coating? | |
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The main component of our Applied Cell Extracellular Matrix (G422) is type I collagen specifically. For more information on abm's Applied Cell Extracellular Matrix please visit: http://www.abmgood.com/Applied-Cell-Extracellular-Matrix-G422.html.
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| Do I have to use T25 ECM-coated flasks for growing the cells? | |
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We strongly recommend thawing cells in the T25 flasks specified on the cell-specific product page to ensure optimal post-thaw recovery of the frozen cells before testing other culture vessels.
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| If I want to plate these cells to multi-well plates (e.g. 96 well plates) or dishes, how should I prepare the plates? | |
| What percentage of Trypsin-EDTA should be used to subculture cells? Can I use Trypsin-EDTA containing phenol red? | |
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The standard concentration used for subculturing is 0.25% Trypsin-EDTA, available at abm (Cat. No. TM050). Yes, Trypsin-EDTA containing phenol red can be used as it does not interfere with trypsinization.
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| How often do I need to change the media? | |
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Media should be changed every 2-3 days or as specified within the recommended growth conditions.
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| Why are these cells classified as biosafety level II? | |
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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 desired.
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| How long can I store frozen vials? | |
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Cells that are properly frozen using an effective cryoprotective agent can be stored in liquid nitrogen vapor phase (or below -130°C) indefinitely without affecting viability.
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| What is the recommended storage temperature for cells? | |
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In general, if you received:
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| Can I substitute heat-inactivated FBS with FBS or vice versa? | |
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FBS and heat-inactivated FBS are different in their composition; they cannot be substituted for one another.
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| My cells are not detaching, what method do you recommend to trypsinize the cells? | |
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| How are live cells shipped? | |
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Live cells are shipped as a live culture in a T12.5 flask. Cells are seeded at an optimal confluency to avoid metabolic waste build up during transport. In addition, we thoroughly test our cell lines for functional freeze-thaw recovery. For more information on how to handle live cells, please view our detailed instructions here (https://www.abmgood.com/uploads/document/Cell_Handling_Instructions_Upon_Arrival_150623.pdf).
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| Why are cells not attaching and forming clumps after subculturing with trypsin? | |
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Cells form clumps and display attachment issues when the trypsinization agent is not effectively neutralized. To completely neutralize the trypsin agent, add an equal amount of complete growth media prior to centrifugation. Subtle changes in culture conditions, particularly in pH and the quality of serum used in the growth medium, may also affect the amount of clumping exhibited by the cells.
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| Why are my cells forming clumps after plating? | |
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It is important to ensure cell clumps are broken up through gentle re-suspension by pipetting up and down several times after pelleting cells by centrifugation. This will ensure a homogenous single cell suspension for even plating.
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| Do I need to add the selection drug to the complete growth medium? | |
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We recommend maintaining selection pressure using the drug concentration specified in the Growth Conditions section.
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| Where can I find the CoA for my product? | |
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Certificates of analysis can be found here (https://www.abmgood.com/CoA-Library.html).
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What is your warranty or return policy? |
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Our warranty and return policy is outlined in abm’s Terms and Conditions, including details on product quality, limitations, and claims. For additional questions, our Order team is happy to assist and can be reached at order@abmgood.com. |
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How many times can cells divide? |
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The number of times cells can divide depends on the cell type:
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What is the difference between spontaneous immortalization and tumor-derived cells? |
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Spontaneously immortalized cells arise when normal cells acquire the ability to proliferate indefinitely in culture without intentional genetic manipulation. These cells are not derived from tumors but may undergo genetic or phenotypic changes during immortalization.
Tumor-derived cells are established directly from cancerous tissue and typically retain characteristics associated with malignancy, such as altered growth control and genetic instability. |
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Do I need Applied Cell Extracellular Matrix (G422) if I am using PriCoat™ flasks? |
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Please refer to the Growth Conditions section of your specific product page. This section will indicate whether Applied Cell Extracellular Matrix (G422), PriCoat™ flasks, or both are recommended for optimal cell attachment and growth, as requirements may vary by cell type. |
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Dequéant, M. L., Fagegaltier, D., Hu, Y., Spirohn, K., Simcox, A., Hannon, G. J., & Perrimon, N. (2015). Discovery of progenitor cell signatures by time-series synexpression analysis during Drosophila embryonic cell immortalization. Proceedings of the National Academy of Sciences of the United States of America, 112(42), 12974–12979. https://doi.org/10.1073/pnas.1517729112