Immortalized Drosophila Embryonic Cells (R3)

Overview

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.

Key elements and design rationale

• Model identity: Immortalized Drosophila Embryonic Cells (R3) is supplied as an immortalized cell line derived from Insect embryo.
• Growth properties: Adherent, spindle-shaped
• 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.
• Product format: Frozen, BSL-2

This cell-based model is generally used in cell fate, differentiation, and expansion studies. Donor/background information is available for contextual interpretation.

Biological background

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. Donor/background information provided for this product: Fruit Fly (D. melanogaster).

Research relevance and current trends

• Cultured cell-line models remain central to in vitro studies of phenotype, signaling, and pathway regulation under controlled conditions.
• Researchers commonly compare morphology, growth rate, and marker expression across media formulations, treatments, or time courses.
• Interpretation is generally strengthened by using matched controls, consistent passage handling, and appropriate culture surfaces.

Common research applications

• Expansion and maintenance studies under defined growth conditions prior to downstream differentiation or phenotype analysis.
• Comparative experiments that examine changes in morphology, marker expression, or functional readouts over time.
• Assay development in culture systems where media composition and substrate selection influence interpretation.

Changes in morphology, growth rate, viability, or reporter signal are typically interpreted together with passage history, culture matrix, and the specified growth conditions for the model.

Notes for experimental interpretation

• Morphology, doubling behavior, and reporter or marker output can shift with passage number, substrate choice, and medium composition; these variables should be recorded alongside experimental readouts.
• Matched controls such as parental cells, untreated cultures, or parallel cultures maintained under identical conditions help distinguish background effects from biology of interest.

Culture and product details

• 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.
• Population Doubling Time (h): 24 - 48

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