Flow cytometry dissociation protocol for Individual Drosophila brains

Preparing individual adult Drosophila brains for Flow Cytometric

analysis of fluorescent protein expression, cellular ploidy and cell death

L. Buttitta, Olga Grushko and Shyama Nandakumar

Established 2013, Modified Aug., 2015

Prepare staining solution. You will need 500uL per sample

Staining solution (1mL):

900uL 10X Trypsin (aliquoted stored in -20oC to minimize freeze/thaw cycles)

100uL 10X PBS pH 7.2

2uL DyeCycle Violet (note this is 2X the concentration we use for other samples)

2.25 uL of 10mg/mL Propidium Iodide staining solution. (This is an exclusion assay, PI+ cells are dead/dying)

Dissect adult brain in 1X PBS. Remove as much trachea and adipose tissue as possible. Dissections should be performed within 20 minutes to limit cell death.

Using forceps, transfer the dissected brain to an eppendorf cap containing 100uL of the staining solution. Incubate for 20 min.

Using a plastic P200 tip, pressed against the plastic cap, triturate to dissociate the brain. 

Transfer the 100uL from the cap with cells to the remaining 400uL of staining solution in the eppendorf tube. This makes a 500uL total

Incubate in the dark, (no mixing/no vortexing) for 45min – 1 hour. (Note: less than 45 min will lead to DNA understaining and smearing artifacts in FACS, over 1 hour leads to overdigestion and excessive cell death.)

 Prior to running, add 500uL of 1X PBS, 0.1% BSA to the sample. This stops the trypsinization and dilutes the sample. 

Do not pipet to mix (cells stick to the plastic pipet at this point). The addition of the PBS will mix the sample and dilute it to a concentration appropriate to maximize individual nuclei passing the laser.  Run the sample set to take 800uL on high sensitivity at flow rate of 100 within 10 min. of adding the PBS/BSA solution. 

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Trypsin used is from SIGMA ALDRICH  catalogue # T4174

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