Immortalized Drosophila Embryonic Cells (R3)

Cat. No.
T0702
Unit
1x10⁶ cells / 1.0 ml
Price
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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.

Organism Insect (Insecta)
Tissue Embryo
Donor History Fruit Fly (D. melanogaster)
Growth Properties Adherent, spindle-shaped
Cell Type Immortalized Cells
Unit 1x10⁶ cells / 1.0 ml
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 Use of PriCoat™ T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. Schneider's Insect Medium + 10% heat-inactivated FBS + 1% Penicillin/Streptomycin Solution (G255), 25.0°C Thawing and Reviving Frozen Drosophila Cell Lines 1. Sterilize the hood by wiping the working surface with 70% ethanol. Dispense 5 mL of the appropriate medium (Table 1) into a 25 cm2 T-flask (T-25). 2. Remove the cryovial/ampule from liquid N2 or dry ice. Wipe the cryovial with 70% ethanol, carefully loosen and unseal the ampule. 3. Using a Pasteur pipette, withdraw 1 mL of room temperature (RT) media from the T-25 flask. Slowly add the media into the cryovial and gently mix to thaw the frozen cells, ensuring that the cell suspension does not overflow. 4. Transfer the entire volume of the thawed cell suspension from the ampule into the T-25 flask. Repeat to ensure the cell suspension has been completely transferred. 5. Place the flask in a 25°C incubator (no CO2), allowing the cells to settle and adhere for at least 2 h. Examine the cells under a microscope to ensure that most cells have settled on the growing surface. Gently remove old media and replace with 5 mL of fresh media. Return the flask into the incubator. 6. On the following day, gently remove the old media and replace with 5 mL of fresh media. Return the culture to the incubator. Some cell lines may take considerable time to begin growing. If the cells are not yet ready to be transferred within a week, withdraw half the medium and replace it with fresh medium. Repeat this step as necessary. WARNING: Freshly thawed cells are often accompanied by a substantial amount of debris resulting from cells damaged during storage and/or transit. The debris includes mitochondria, which look a lot like bacteria. Do NOT simply declare that the culture is contaminated and throw it out. Instead, watch the culture for a few days; if there actually is bacterial contamination, it will increase dramatically overnight. If the little black specks that you see are cellular debris, they will increase little if at all, and may even disappear over the course of a few days. If there is debris, but there are also healthy cells, the healthy cells should grow and take over the culture. Subculturing Adherent Cells 1. Transfer all the medium from the plate to a new sterile flask. Save the medium. 2. Rinse cells by slowly adding 1 mL of 0.5% trypsin-EDTA to the plate. Swirl gently to ensure the trypsin solution covers the entire growth surface. Discard the trypsin solution. 3. Gently add 1 mL of 0.5% trypsin-EDTA to the plate. Incubate the plate at 25°C between 5−10 min while monitoring for visible signs of the cell layer detaching and sliding off the growing surface. 4. Add 9 mL of the saved medium to the plate to stop trypsin activity. Mix the cell suspension to dissociate cell clumps. 5. Transfer the entire cell suspension into a 15 mL or 50 mL conical tube. Collect the cells by centrifugation at 1,000 x g for 5 min and discard the supernatant. 6. Resuspend the cell pellet in fresh media and transfer into a new flask at the desired dilution. 7. Place the flask back in a 25°C incubator.
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. Thaw cells quickly in a 37°C water bath while agitating gently (maximum 2 minutes). The vial cap should be kept above the water level to minimize the risk of contamination.

2. Decontaminate the vial by spraying and wiping the exterior of the vial with 70% ethanol. From this point onwards, all operations should be strictly carried out inside a biological safety cabinet using aseptic conditions.

3. Transfer the cell suspension into a 15ml sterile conical tube containing 5ml of pre-warmed, complete growth media. Centrifuge cells at 125xg for 5-7 minutes.

4. Aspirate the supernatant without disturbing the cell pellet. Re-suspend the cell pellet in the recommended pre-warmed, complete growth media and dispense into a T25 culture flask.

5. Incubate the cells at the recommended conditions.

Subculture Protocol

Volumes given below are for a T75 flask; proportionally increase or decrease the volume as required per culture vessel size. Subculture cells once the culture vessel is 80% confluent.

1. Aspirate the culture media, and add 2-3ml of pre-warmed 0.25% Trypsin-EDTA to the culture vessel.

2. Observe the cells under a microscope to confirm detachment (typically within 2-10 minutes). Cells that are difficult to detach can be put in 37°C, for several minutes to facilitate detachment.

3. Neutralize Trypsin-EDTA by adding an equal volume of the complete growth media into the culture vessel.

4. Transfer the culture suspension into a sterile centrifuge tube, and centrifuge at 125xg for 5 minutes. The actual centrifuge duration and speed may vary depending on the cell type.

5. Aspirate the supernatant, and re-suspend the pellet with pre-warmed fresh complete growth media. Add appropriate aliquots of the cell suspension to new culture vessels, as desired.

6. Incubate the cells at the recommended conditions.

Cryopreservation Cryopreservation Medium (TM024), or complete growth media with 10% DMSO.
Population Doubling Time (h) 24 - 48
Immortalization Method Serial passaging of primary cultures from Act5C > UAS-Rasᵛ¹² embryos
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.
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. All test parameters provided in the CoA are conducted using abm's standardized culture system and 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.

2. We recommend live cell shipments for ease of cell transfer and this option can be requested at the time of order placement. 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.

3. 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 or any other non-RUO application(s).

4. abm makes no warranties or representations as to the accuracy of the information on this site. Citations from literature are provided for informational purposes only. abm does not warrant that such information has been shown to be accurate.

5. 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
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
Print & Download Datasheet
  • 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

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