Single-cell dissociation of Drosophila melanogaster pupal tarsi

Collect white prepupae. Individuals should meet the P1 aging criteria laid out by Bainbridge and Bownes (1981): the pupae should be white or cream coloured, have stopped moving completely, and display everted anterior spiracles.

Identify individuals of the correct sex. Place white prepupae in a glass well with water under a dissection microscope. At this developmental stage the male gonads are visible on either side of the posterior ~1/3rd of the pupae.

Fold a kimwipe in half and then in half again a further two times. Place inside a Petri dish and wet the kimwipe with 500µL of water. Then transfer the pupae from the glass well to the kimwipe using a paintbrush. Cover the Petri dish and move to an incubator set to 25°C.

At n - 1 hours, remove the Petri dish from the incubator. Using a wet paintbrush, gently remove the pupae from the by now dry kimwipe. Wetting the paintbrush helps to loosen the pupae from the wipe. Transfer the pupae to a dry kimwipe and gently pat them down to further dry them. Then transfer dorsal side up to a piece of sticky tape (sticky side up) that is secured to a flat surface ( e.g. a piece of plastic or microscope slide).

Using forceps, gently remove the pupal casing. Begin by removing the operculum and then cut laterally down the side of the case, working from anterior to posterior and gently peeling away the cut casing to expose the pupa. Then transfer the de-cased pupa to a kimwipe soaked through with water in a Petri dish.

Add 100µL of Dulbecco's PBS (DPBS) to a glass well on ice under a dissection scope.

Use forceps to transfer several pupae, starting with the first that were de-cased, to the same tape-covered surface used for the de-casing. Place pupae ventral side up ( i.e. legs facing up).

For each pupa, begin by pinching the base of the abdomen to release fluid. This prevents the release of large amounts of fluid while pinching the leg, which interferes with the dissection. Gently press on the head to push a small amount of fluid out.

To dissect forelegs, pull the second and third legs away from the body such that they point ~90° away from the body. This exposes the foreleg tarsus. The tibia/tarsus joint should be visible approximately in line with or just below the base of where the leg connects to the thorax (directly below the mouthparts).

Using forceps, pinch just proximally to the joint and pull away from the body of the pupa. The tarsus should come away.

Place the tarsus down on the tape and cover with 10µL of DPBS.

Using a Micro Knife, slice at approximately the midsection of the tarsal segment adjacent to the segments that are being targeted i.e., if targeting the first tarsal segment slice at the midsection of the second tarsal segment. A slice can be made on both the proximal and distal side of the targeted region to isolate a subset of segments. After making the slice(s), use forceps to gently ease the targeted region out from the pupal cuticle.

Equipment

Value	Label
Micro Knives - Plastic Handle	NAME
Ultra-thin dissecting blade	TYPE
Fine Science Tools	BRAND
10318-14	SKU

Using a freshly BSA-coated 10ul tip, pipette the first tarsal segment and transfer to the well of DPBS on ice.

Repeat until the well contains 65-70 dissected tarsal segments. Two trained personnel working simultaneously can generally complete this within 1h 30m 0s

Using a 10ul tip, remove all of the DPBS from the well. Avoid removing dissected tissue.

Add 100µL of dissociation buffer, which consists of 10x TrypLE with a final concentration of 2mg/mL collagenase.

Note

The buffer can be premade and stored in individual aliquots at -20°C.

Cover the well with Nunc Aluminium Seal Tape and submerge inside of a bead bath at 37°C for 0h 35m 0s.

Equipment

Value	Label
Lab Armor Beads	NAME
Non-uniform metal beads	TYPE
Gibco	BRAND
A1254301	SKU

Equipment

Value	Label
Auminum Seal	NAME
Auminum Seal	TYPE
Nunc	BRAND
232698	SKU

Remove from the bead bath and then remove ~85µL of dissociation buffer using a 10ul tip.

Add 50µL of room temperature DPBS to the well.

Pipette the solution up and down 20x using a widebore, BSA-coated, lowbind tip. Aim for ~1.5 pipettes up and down per second, moving the tip around in the well to ensure an even dissociation throughout the suspension. Avoid introducing bubbles into the suspension. Use a 200ul pipette set to 40ul.

Equipment

Value	Label
ART 200ul Wide Bore Filtered Pipette Tips	NAME
Pipette tips	TYPE
ART	BRAND
2069G	SKU

Pipette the solution up and down a further 20x using a BSA-coated, flame-rounded 200ul tip. Again, make sure to move the tip around in the well.

Slowly pipette the solution up and down a further three times using the same flame-rounded tip before taking up 40µL and transferring to a 2mL low-bind Eppendorf tube on ice.

Add a further 20µL of DPBS to the well, pipette up and down three times using the flame-rounded tip, and add 25µL to the same Eppendorf on ice.

Preview 10µL of suspensions using a hemocytometer or automated cell counter. Concentrations and singlet rates should be checked against the requirements of the single-cell technology being used.