TITLE: Brain Single Cell Isolation for Flow Cytometry
AUTHORS: Jhodi Webster, Ashley Harms, Vickie Yang, Aidan Miller

[DESCRIPTION]
This protocol is used to isolate single cells from mouse brains and process these samples for flow cytometry analysis. The protocol can be optimized/altered to account for staining of intracellular cellular markers.

[STEPS]
SECTION: SETUP
1. Make reagents needed:
Flow media: RPMI 1640 + 10%FBS + 1% Penicillin/Streptomycin + 1% L-Glutamine
10X Stock Enzyme: 1g Collagenase IV in 100mL serum-free media (RPMI 1640). Aliquot into 5mL and store at -20 °C until use. To use (working concentration) thaw on ice, QS to 38mL using serum-free media. Final concentration with 2mL sample is ~1mg/mL Collagenase IV.
90% Percoll: 18mL percoll + 2mL 10X PBS
70% Percoll: 7mL 90% + 3mL 1X PBS
Staining buffer: 2% FBS in 1X DPBS without calcium and magnesium 
SECTION: SETUP
2. Pre-heat water bath to 37C and pre-cool centrifuge to 4C. Label and set-up 6-wel plates, 50mL and 15mL conical tubes. 
SECTION: PROCEDURE
3. Isolate brain tissue using standard perfusion protocols and dissection techniques based on desired brain regions.
SECTION: PROCEDURE
4. Place tissue in flat-bottom-6-well cell culture plate with 2mL flow media until tissue collection is complete.
SECTION: PROCEDURE
5. Prepare enzyme and add 6mL working concentration to labeled 15mL conical tubes.
SECTION: PROCEDURE
6. Mince isolated tissue with small scissors and transfer using pre-cut 1000μL pipette tips to conical tubes with enzyme.
SECTION: PROCEDURE
7. Shake in water bath at 37C for 20 minutes: at 10 minutes, vortex and pipette up and down with Pasteur pipette to break up tissue then continue timer.
SECTION: PROCEDURE
8. Transfer to 70μm cell strainers placed in 50mL tubes and smash tissue using the plunger of a 5mL syringe, rinsing with DPBS or flow media.
SECTION: PROCEDURE
9. Spin at 1800RPM for 8min at 4C (brake on high)
SECTION: PROCEDURE
10. Decant supernatant and re-suspend pellet in 7mL of flow media in 15mL conical tube.
SECTION: PROCEDURE
11. Vortex cell suspension with 3mL 90% Percoll.
SECTION: PROCEDURE
12. Inject 1.5mL of 70% Percoll under the 90% mixture SLOWLY. It is recommended to take-up 2mL of Percoll to inject, in order to prevent bubbles or disturbing the layer.
SECTION: PROCEDURE
13. Spin at 1500RPM for 30min at 4C (no brake). DO NOT DISTURB GRADIENT.
SECTION: PROCEDURE
14. Check for cells - should be suspended between pink and clear layer. Vacuum supernatant and fat to about 7mL line.
SECTION: PROCEDURE
15. Transfer cells to a clean 15mL conical tube and fill rest of the way with PBS. DO NOT TAKE PELLET!
SECTION: PROCEDURE
16. Vortex and spin at 1800RPM for 8min at 4C (brake on high).
SECTION: PROCEDURE
17. Aspirate and leave 200uL liquid with pellet. Pipette up and down to break up pellet and transfer to round-bottom 96-well plate for staining. Label plate appropriately.
Splenocytes: wells for single color control and unstained control (and optional: well for mastermind + splenocytes) - see steps below for splenocyte isolation
Brain samples: mastermix only

SECTION: PROCEDURE
18. Spin plate at 1500RPM for 5 min. Decant supernatant.
SECTION: PROCEDURE
19. Re-suspend cells (only brain samples) in 100μL Fc Block (1:100 in staining buffer (FBS/PBS)) OR 100uL of staining buffer to splenocytes. Incubate on ice for 15 minutes, then add 100uL staining buffer on top for volume.
SECTION: PROCEDURE
20. Spin plate at 1500RPM for 5min. Decant supernatant.
SECTION: PROCEDURE
21. Make up antibody cocktail (mastermix) and single colour controls:

To make mastermix: add anitbodies at their specific concentrations to at least 500uL FBS/PBS (or based on how many samples you have)

To make single colour controls: use volume of 2% FBS/PBS that will allow for 1μL of each antibody. (i.e if CD45 is 1:200, use 200μL of FBS/PBS)
SECTION: PROCEDURE
22. Re-suspend cells in 50μL antibody cocktail in staining buffer. Incubate 20-30 minutes on ice or at 4C.
SECTION: PROCEDURE
23. Add 150uL staining buffer on top for 200μL total volume.
SECTION: PROCEDURE
24. Spin 1500RPM for 5min. Decant supernatant.
SECTION: PROCEDURE
25. Re-suspend cells in 200μL 2%PFA. Incubate on ice or at 4C.
SECTION: PROCEDURE
26. Spin 1500RPM for 5min. Decant supernatant. Re-suspend in 200μL FBS/PBS.
SECTION: PROCEDURE
27. For storage: foil and parafilm and place plate in 4C. Before running samples on flow cytometer, filter samples with a strainer cap into 5mL FACS tubes. 
SECTION: SPLENOCYTE ISOLATION
28. Isolate spleen from perfused mouse and place in 70μm strainer in 50mL conical tube. 
SECTION: SPLENOCYTE ISOLATION
29. Smash spleen with media and spin down at 1500RPM for 5min.
SECTION: SPLENOCYTE ISOLATION
30. Decant media and add 5mL ACK lysis buffer into tube for 3-5 minutes. Neutralize with 10mL 1X DPBS.
SECTION: SPLENOCYTE ISOLATION
31. Spon 1500RPM for 5min. Decant and resuspend in 10mL DPBS.
SECTION: SPLENOCYTE ISOLATION
32. Spin 1500RPM for 5min. Aspirate and resuspend in 10mL flow media.