Immortalized Drosophila Embryonic Cells (R3)

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.