TITLE: Flow cytometry
AUTHORS: gurvir.virdi

[DESCRIPTION]
This protocol is for immunolabelling fixed midbrain dopaminergic neurons for flow cytometry analysis and downstream flow cytometry acquisition.

[STEPS]
SECTION: Sample preparation
1. Cells were washed once with PBS
SECTION: Sample preparation
2. The cells were incubated with Accutase (Gibco) to generate a single-cell suspension for 5 min at 37 °C 

SECTION: Sample preparation
3. A cell suspension of 500k/ml was prepared in media
SECTION: Sample preparation
4. Cells were then spun down at 200 x g, 5 min, and the supernatant was removed.

SECTION: Sample preparation
5. Cell pellet was resuspended gently in 4ml of 4% paraformaldehyde and briefly vortexed at a low speed before being spun on a rotation spinner for 10 min  at room temperature.

SECTION: Sample preparation
6. After fixation, samples were spun down and supernatant removed
SECTION: Sample preparation
7. Cells were resuspended in 2ml of 0.1% BSA (Sigma) in PBS.
SECTION: Sample preparation
8. After resuspension, cells were filtered through a 70μm strainer (Miltenyi Biotec) to filter out any cell clumps
SECTION: Sample preparation
9. Cells were then centrifuged 200 x g, 5 min , and the supernatant was removed. 

SECTION: Sample preparation
10. Cell pellets were then resuspended in 1ml of permeabilization/blocking buffer (0.1% Triton X-100, 1% BSA, 10% normal goat serum (Sigma) in PBS), and incubated on a rotation spinner for 30 min  at room temperature.

SECTION: Sample preparation
11. After permeabilization/blocking, cells were centrifuged 200 x g, 5 min  and the supernatant was removed. 

SECTION: Sample preparation
12. Cells were then resuspended in the primary antibodies (1:200) made up in 0.1% BSA in PBS, and incubated on the rotation spinner for 60 min  at room temperature.

SECTION: Sample preparation
13. After primary antibody incubation, cells were centrifuged 200 x g, 5 min , supernatant removed and washed once in 0.1% BSA in PBS.

SECTION: Sample preparation
14. Cells were then resuspended in the species-specific secondary antibodies (AlexaFluor 488, 647) at a dilution of 1:500 made up in 0.1% BSA in PBS and incubated in the dark on a rotation spinner for  30 min .

SECTION: Sample preparation
15. After incubation, cells were centrifuged 200 x g, 5 min , supernatant removed and washed once in PBS, followed by incubation with DAPI made up in PBS for 5 min .

SECTION: Sample preparation
16. The DAPI + PBS was then removed, followed by one wash in PBS, before being analysed on the flow cytometer.
SECTION: Cell sorting and analysis
17. The samples were run on the LSRii (BD) cell sorter. Scattering was initially used to discard debris as well as cell doublets and larger clumps.
SECTION: Cell sorting and analysis
18. The single-cell population was then gated to include DAPI positive only cells (negative control). 
SECTION: Cell sorting and analysis
19. The gating threshold for measured channels was determined using the control lacking the antibody of interest (Fluorescence minus one (FMO) control), for both channels being recorded. 
SECTION: Cell sorting and analysis
20. Once the parameters had been set, 10,000 cell events were recorded, and data were processed and analysed on the FlowJo software.