In our hands, probably no more than 10% of the GFP positives cells can be collected as single cells. A lot of GFP positive cells are lost during the single cell suspension prep and FACS sorting, if you are going to do bulk experiments, you will get more cells from the sorting.

Fluorescent cell sorting from Adult Drosophila Brains

Rosbash lab at Brandeis University

1. Prepare Following regents

SM-active Bis-Tris Media
Dissecting Saline (10X stock, dilute to 1X)
Papain (L-cysteine activated papain from Worthington, 50 U/ml, 50 μl aliquots in -80°C). Reactivate aliquot at 37 °C for 10 min (optional), then place at room temperature until needed.
a. Dissolve papain to 50 U/ml in dissecting saline (2 ml dissecting saline per vial of Worthington papain)
b. Incubate at 37 °C for 10 min to activate
c. Make 50 μl aliquots and store at -80 °C
Neural Inhibitors (aliquots in -80 °C)
a. DNQX: Sigma#D0540-25 mg, 100 mM in DMSO, 5000X stock
i. AMPA and kainate receptor antagonist
ii. Dissolve one 25 mg vial in 1 ml DMSO
iii. Aliquot, keep in -80 °C
b. TTX: Sigma #T8024-1mg, 0.1mM in H2O, 1000X stock
i. Voltage-gated sodium channel blocker
ii. Dissolve one 1mg vial in 30 ml H2O
iii. Aliquot, keep in -80 °C
c. APV (AP5): Sigma #A8064-5mg (or Tocris #0106-10mg), 25 mM in H2O, 500X
i. NMDA receptor antagonist
ii. Dissolve one 5 mg vial in 1 ml H2O
iii. Aliquot, keep in -80 °C

2. Gather Following materials

Flame-rounded pipette tips (large, medium, small openings)

3. Prepare single cell suspension

1) Add neural inhibitors to dissecting saline and SM-media. Keep on ice.
For every 10 ml of SM-media or 10 ml dissecting saline add:

DNQX (100 mM): 2 μl
TTX (0.1 mM): 10 μl
APV (25 mM): 20 μl

2) Use a syringe and filter to sterilize media.

3) Put 300 μl of media + inhibitors in 1.5 ml Eppendorf tube. Keep on ice

4)Get flies and place on ice to put sleep.

5) Dissect brains in dissecting saline (with inhibitors) and use forceps to place in Eppendorf of media on ice.

6) After dissection, spin brains at room temperature for 2 min at 1000 rpm.

7) Remove supernatant.

8) Wash brains with 300 μl cold dissecting saline. Spin again as before, remove supernatant.

9) Add papain, ~ 2 μl/brain. Leave at RT for 20-30 min, mix occasionally by pipetting with 20 ul tip (don’t suck up brains)

10) Add 400 μl cold SM-media to stop papain digestion.

11) Spin at RT for 2 min at 1000 rpm.

12) Remove supernatant (leave 100-150 behind, do not disturb cell/brain material).

13) Add cold SM-media up to a total volume of 350 μl (This is what we use for 50 brains, if there are fewer brains, could probably use less media)

14) Triturate brains with flame-rounded tips (use P1000 set at 1ml, suck up liquid/brains until almost no liquid remains, try not to create bubbles).

a. 30 times up and down with large pre-wetted tip. Place brains on ice for 2 min before moving to medium tip.

b. 15-20 times with medium tip. Place on ice for 2 min every 10X.

c. ~ 10 times with small tip. Place on ice for 2 min every ~3X.
Note: The trituration is not exact, and will be different depending on the number of brains, the small tip should be used until the liquid easily goes through with nothing getting stuck. It is possible to over triturate, which will cause your cells to die.

15）Filter the cell suspension through 5 ml round-bottom tube with cell-strainer cap.

4. Cell sorting by BD melody FACS machine.

a. Before turning anything on, empty the waste tank and fill the sheath tank with fresh NERL saline up to the fill line. After filling the sheath tank, ensure the top is securely tightened before moving on to the next step.

b. Turn on all 4 machines in the FACS workstation (from compressor to the computer).
To turn the cooling system on, press the power button and then the arrow button located on the front of the machine. Next, turn on the FACS machine by pressing the power button located directly in the front of the machine. Finally, turn on the computer connected to the FACS.

c. After all 4 components have been turned on, open the “FACSMelody” application located on the right of the desktop. Login to sorting account.

d. Once logged in, click “Run Daily Fluidics Startup.” If FACS machine has not been run in the past 24 hours, click “Run Extended Fluidics Startup” instead. The machine prompts you with the recommended startup by highlighting Daily or Extended in orange.

e. After fluidics startup is complete, click Continue to see the cleaning options.

f. Click “Flow Cell Clean” and follow instructions on screen.

g. Remove the closed-loop nozzle (the one with the wires attached to it) from the bottom of the flow cell cuvette and place in the top hole immediately to the right of the flow cell.

h. Insert the sort nozzle (individual nozzle) straight into the bottom of the flow cell cuvette.

i. Insert the sort nozzle into the bottom of the flow cell cuvette (where closed-loop nozzle used to be) with “TOP” facing up. Ensure the red nozzle O-ring is enters first into the cuvette. Turn the nozzle-locking lever clockwise to the 12:00 position and click Continue.

j. Prior to conducting Cytometer Setup, ensure the lot number for your BD CS&T RUO beads matches that of the cytometer. If the two lot numbers differ, change the lot number on the cytometer such that it matches that of the BD CS&T RUO beads.

k. Prepare a tube of BD CS&T RUO beads by adding one drop of beads to 500 μl of NERL in a 5 mL Falcon tube. Shake or vortex beads briefly before use.

l. Load the CS&T bead solution into the FACS machine and follow the prompts on the screen.

m. Prepare a tube of BD FACSTM Accudrop beads by adding one drop of beads to 500 μl of NERL in a 5 mL Falcon tube. Shake or vortex beads briefly before use. Bead mixture should appear light blue after vortexing.

n. Load the Accudrop bead solution into the FACS machine and follow the prompts on the screen.

o. After passing drop delay, you are ready to setup the experiment and sort. Select the appropriate experimental template or design one to fit your particular needs.

p. There are three sorting modes on BD melody, yield, purity and single cell mode. Purity mode is recommended for bulk experiments, single cell mode is used for single cells experiments.
Note: For single cell sorting, it is very important to align the plate stage every time before the sorting.

q. Once the sorting is done, spin the tube or plate in 4°C and keep it in -80 °C.

Recipes:

1. Dissecting saline
1X working solution:

Reagents	Final concentration
HEPES-KOH, pH7.4	9.9mM
NaCl	137mM
KCl	5.4mM
NaH₂PO₄	0.17 mM
KH₂PO₄	0.22 mM
glucose	3.3mM
sucrose	43.8mM

2. SM-active + BisTris

1) Mix following:

Regents	Final concentration
KH₂PO₄	4.18 mM
CaCl₂	1.05 mM
MgSO₄.7H₂O	0.7 mM
NaCl	116mM
NaHCO₃	8 mM
glucose	200 mg/100 ml
trehalose	200 mg/100 ml
a-ketoglutaric acid (Sigma,K1128-5G)	35 mg/100 ml
Fumaric acid (Sigma, F8509-100G)	6 mg/100 ml
Malic acid (Sigma, M0750-5G)	60 mg/100 ml
Succinic acid (Sigma, S9512-100G)	6 mg/100 ml
Yeast extract (BD, BBL yeastextract)	200 mg/100 ml
H₂O	to 318 ml

2) Add FCS
Add Non heat-inactivated FBS 80 ml.
3）Filter sterilize.
4）Keep it in dark for 3 days at 26 °C.

5）Add insulin, Bis-tris

Regents	Final concentration
insulin	2 ug/ml
Bis-Tris pH6.8 (Sterilized)	5 mM

Reference:

Abruzzi, Katharine, Xiao Chen, Emi Nagoshi, Abby Zadina, and Michael Rosbash. 2015. “RNA- Seq Profiling of Small Numbers of Drosophila Neurons.” Methods in Enzymology 551: 369–86.