Analyzing Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants

Abstract

Source: Naomi Keliinui, C., et al. Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants. J. Vis. Exp. (2022).

This video demonstrates a protocol to identify exogenous factors that initiate the reactivation of Drosophila brain neural stem cells from their quiescent state. Freshly hatched larvae are dissected to isolate their brains, which are then cultured in media with or without a test hormone to evaluate the hormone's potential to induce neural stem cell reactivation and proliferation.

Protocol

1. Culture media and tool preparation

1. Spray the bench and work area with 70% ethanol and let dry.
2. Spray the dissection tools, forceps, and two glass watch dishes, with 70% ethanol and let them dry on the bench.
3. Make the supplemented Schneider's media (SSM, Table 1) and place it on ice.
4. Pipette 1 mL of SSM into each of the glass watch dishes.
5. Using a micropipette with a sterile tip, transfer the freshly hatched larvae from the plate of PBS to the SSM in the first glass watch dish. Using a micropipette with a sterile tip, transfer the freshly hatched larvae to the SSM in the second glass watch dish.

2. Dissections and brain cultures

1. Once the larvae are in the second glass watch dish with SSM, dissect the brains out of the larvae using forceps and a dissecting microscope. Adjust the magnification as needed.
2. Use one forceps to grab the mouth hooks and with the other, gently grab the body halfway down and pull in the opposite direction (Figure 1) to split the larva into two pieces.
   NOTE: The brain will be located right behind the mouth hooks. Note that there may be other tissues surrounding the brain. Be very careful when removing these tissues as it can result in damaging the brain.
3. Once 15-20 brains have been dissected, add 1 mL of SSM into one well of a sterile 12-well culture tray. Transfer the freshly dissected brains into the SSM using a micropipette and a sterile tip (Figure 2A).
4. Place the brains in the SSM media in the 12-well culture tray into an incubator at 25 °C for 24 h (Figure 2A).

Table 1: Recipe for making supplemented Schneider's media (SSM). The table lists the volume of all ingredients required for preparing 5 mL of SSM.

Ingredient (starting concentration)	To make 5 mL	Final concentration
Schneider's Drosophila media	3.89 mL
Fetal bovine Serum (100%)	500 µL<	10%
Glutamine (200 mM)	500 µL	20 mM
Penicillin (5000 units/mL), Streptomycin (50,000 µg/mL)	100 µL	1000 U/mL Pen, 1 mg/mL Strep
Insulin (10 mg/mL)	10 µL	0.02 mg/mL
glutathione (50 mg/mL)	5 µL	0.05 mg/mL
In a sterile hood, using sterile technique, add all ingredients together in a 14 mL conical tube. Place it on ice until use.

Results

Figure 1: Drosophila larvae in a glass watch dish with SSM. Forceps are properly positioned for dissection. The location of the larval brain (dark grey) is posterior to the mouth hooks (black), and both are shown inside the larva.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 µL Pipette tips	Denville Sci	P2102
1000 µL Pipette tips	Denville Sci	P2103-N
1000 µL Pipettor	Gilson	P1000
24-well multiwell culture plates	Fisher Scientific	50-197-4477
35 mm Petri dishes	Fisher Scientific	08-757-100A	Grape Plate Ingredients
Dissecting microscope	Zeiss	Stemi 2000
Ethanol 200 proof (100%), Decon Labs, 1 gallon bottle	Fisher Scientific	2701	Used to wash off the larvae before the 24 hr hold in culture medium
Fetal Bovine Serum (10%)	Sigma	F4135-100ML	Supplement for cell culture media.
Fine forceps for dissection	Fine Science Tools	11295-20	Forcepts used in disections. They work best when sharpened.
Glass Dissection Dish (3 well)	These are no longer available
Glutathione	Sigma	G6013	Provides oxidative protection during cell culture.
Incubator	Thermo Fisher Scientific		Ensures that the temperature, humidity, and light exposure is exactly the same throughout experiment.
Insulin	Sigma	I0516	Independant variable of the experiment
Laminar flow hood			For aliquoting culture media
L-Glutamine	Sigma	G7513	Provides support during cell culture
Pen-Strep	Sigma	P4458-100ml	Antibiodics used to prevent bacterial contamination of cells during culture.
Phosphate Buffer, pH7.4	Made in house	Made in house	Solvent used to wash the brains after fixing and staining steps
Pick	Fine Science Tools	10140-01	Used to pick larvae off of the grape plate
Schneiders Culture Medium	Life Tech	21720024	Contains nutrients that help the cells grow and proliferate
Sterile Water	Autoclave Milli-Q water made in house		Needed for Solutions