Purification of cells by FACS

UTRICLE DISSOCIATION/FACS ISOLATION AND RNA-SEQUENCING FOR ADULT UTRICLE HAIR CELLS

Utricle dissociation

1. Before starting the experiment, pre-warm the DNase-containing Papain (To make this solution, please follow the instruction for the Papain Dissociation system from Worthington Biochemical Corporation (Catalog Number LK003150) in 37-degree incubation chamber.  Prepare CMF-PBS (calcium and magnesium-free PBS) with 2% FBS. 
2. If using cultured utricles, remove the utricle-containing culture membrane to a slide. Use a transfer pipette with PBS to apply water pressure and then detach the utricle from the culture membrane. Then, use the transfer pipette to transfer the utricle to 1.5 mL Eppendorf tube. Note, pre-coat the transfer pipette with FBS or any type of serum to prevent utricle attach to the pipette during transfer. 
3. After collecting all the utricles, wash them with CMF-PBS twice. Then, wash the utricles with 100 µl of papain. Depending on the quantity of the utricle, add 200-800 µl of papain (For example, add 500 µl to approximately 20 utricle). Rotate the Eppendorf tube for 10 times then, incubate the tube at 37°C with 200-500 rpm shaking for 50 minutes. 
4. Put the reagents and the tubes on ice. Remove the papain solution and rinse the utricle with ice-cold CMF-PBS with 2% FBS twice. A brief centrifuge can help settle down the utricles. 
5. Add 400 µl of the ice-cold CMF-PBS with 2% FBS into the Eppendorf tube. Place the Eppendorf on ice. Turn the 1000 µl pipette to 300 µl, gently triturate the utricle for as many times as needed, typically around 100-200 times. For the first couple of times, make sure to take a couple of microliters and check under the microscope for complete dissociation. 

FACS isolation of specific cell group

1. Filter the cells through a 5 mL Polystyrene Round-Bottom Tube with Cell-strainer Cap (75 mm style) on ice
2. Add DAPI (with a final concentration of 0.1 µg/mL) to the cell-containing-tube to sort out dying cells
3. Place the cell containing tube into the BD FACS Aria cell sorting flow cytometer with a 100-µm nozzle. Remember to always bring a negative control (dissociated utricle without fluorescence). Try to sort the cells within one hour of dissociation. 

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