TITLE: SOP for Immune cells isolation mouse adult brain with CD45 beads
AUTHORS: Malu G Tansey

[DESCRIPTION]
SOP for Immune cells isolation mouse adult brain with CD45 beads

[STEPS]
SECTION: A. Brain dissociation
7. Remove mice brains and put them in pre-prepared 15mL falcon tubes filled with 3mL cold D-PBS.
SECTION: A. Brain dissociation
8. Add 1900 ul of buffer Z in each C-tube.
SECTION: A. Brain dissociation
9. Cut brain in ~8 equal pieces and add them in C-tubes (do not cut too much or viability will be affected).
SECTION: A. Brain dissociation
10. Add 50 ul of enzyme P in each C-tube.
SECTION: A. Brain dissociation
11. Add 30 ul of enzyme mix 2 in each C-tube.
SECTION: A. Brain dissociation
12. Close lids tightly until lid clicks into place
SECTION: A. Brain dissociation
13. Place C-tubes in GMHO machine-make sure all the brain pieces are at the bottom of the tubes; otherwise, they will not be properly dissociated.
SECTION: A. Brain dissociation
14. Start program: 37_ABDK_01 for 1 brain >100 mg; 37_ABDK_02 for smaller pieces 20-100 mg 

Tip: if any piece is stuck in the upper part, stop the program, mix brain pieces and start the program again.
SECTION: A. Brain dissociation
15. Centrifuge briefly or mix up and down manually to ensure all the sample is at the bottom of the tube.
SECTION: A. Brain dissociation
16. Prepare 50 ml tube with 70 um filters. Tip: add 2ml of PBS to the filter before.
SECTION: A. Brain dissociation
17. Add the cell suspension into the C-tube.
SECTION: A. Brain dissociation
18. Add 10 ml of cold D-PBS to the C-tubes, close the C-tubes, shake gently, and apply the cell suspension into a 50 ml falcon with a 70 um filter.
19. Add an extra 10 ml of D-PBS in the C-tube to make sure everything is taken and transfer again to the 50 ml falcon tube with the 70um filter. Optional: Use an extra 10 ml of D-PBS to dissociate small pieces that remain in the filter as much as possible.
20. Centrifuge 300xg for 10 min at 4°C.  B. Debris removal
SECTION: B. Debris removal
21. Remove supernatant, resuspend the cell pellet with 3100 ul of D-PBS and add 900 ul of debris removal solution.
SECTION: B. Debris removal
22. Transfer cell solution to 15 ml tubes. If there are a lot of samples to one at a time. Avoid having the samples with the debris removal solution sitting on ice for long periods of time.
SECTION: B. Debris removal
23. Overlay with 4 ml of D-PBS (debris removal solution is critical to have the overlay). The overlay volumes vary when using 1 or 2 brains:
 

SECTION: B. Debris removal
24. Centrifuge 3000xg for 10 min at 4°C with full acceleration and brake of 7. Tip1: use the centrifuge on the right, close to the external entrance of the lab. Place the tubes on the inside of the centrifuge, the positions close to the rotor. Tip2: if after the centrifugation the debris layer is in the bottom of the tube with the cells, remove the upper layers, add again 900 ul of debris removal solution, overlay with respective D-PBS, and centrifuge again. Optional: If centrifuges give suboptimal centrifugation, the acceleration and brake can be reduced.
SECTION: B. Debris removal
25. Three phases should be formed; aspirate the two top phases and discard them.
SECTION: B. Debris removal
26. Add D-PBS up to 15 ml and gently invert the tube three times- do not vortex!
SECTION: B. Debris removal
27. Centrifuge 1000xg for 10 min at 4°C with full acceleration and full brake.  C. Red blood cell removal
SECTION: C. Red blood cell removal
28. Prepare red blood cell removal (RBCR; prepare fresh every time). RBCR: 1:10 RBCR solution (10x) with ddH2O (i.e., 1 ml of RBCR with 9ml of cold ddH2O).
SECTION: C. Red blood cell removal
29. Resuspend the cell pellet with the appropriate RBCR volume:
SECTION: C. Red blood cell removal
30. ml for 100-1000 mg (one mouse brain).
SECTION: C. Red blood cell removal
31. 5 ml for 20-100 mg.
SECTION: C. Red blood cell removal
32. Incubate the cells for 10 min at 2°C -8°C (fridge at 4°C).
SECTION: C. Red blood cell removal
33. Add PB buffer:
SECTION: C. Red blood cell removal
34. ml for 100-1000 mg (one mouse brain)
SECTION: C. Red blood cell removal
35. ml for 20-100 mg
SECTION: C. Red blood cell removal
36. Centrifuge 300xg for 10 min at 4°C. If need to count cells: resuspend in 10 ml of Beads Buffer take an aliquote to count cells centrifuge 300g 10 min at 4°C.  D. CD45 beads isolation
SECTION: D. CD45 beads isolation
37. Prepare Eppendorf tubes to incubate cells with CD45 microbeads.
SECTION: D. CD45 beads isolation
38. Resuspend the cell pellet in 90 ul of Beads Buffer and 10 ul of CD45 microbeads for 107 cells Tip: cells should be counted to resuspend the cell pellet with appropriate vol of beads buffer and beads; however, we never expect more than 107 total cells per brain, thus directly resuspend the pellet with min vol to speed up the protocol.
SECTION: D. CD45 beads isolation
39. Incubate the cells in the fridge (4°C) for 15 min. Tip: mix up and down after 7 min.
SECTION: D. CD45 beads isolation
40. Add 2 ml of Beads Buffer (for 107 total cells) to each sample and centrifuge 300xg for 10 min. Tip: no temperature recommended, but I would use at 4°C.
SECTION: D. CD45 beads isolation
41. Prepare the MACS MultiStand and magnetic field under the hood.
SECTION: D. CD45 beads isolation
42. Place the columns (make sure they are correctly placed) and 15 ml tubes for trash.
SECTION: D. CD45 beads isolation
43. Pre-wash columns with 500 ul Beads Buffer (MS columns).
SECTION: D. CD45 beads isolation
44. Resuspend cell pellet with 500 ul of Beads Buffer (up to 108 cells). Tip: make sure the columns don’t dry from now on.
SECTION: D. CD45 beads isolation
45. Transfer the cell suspension to the columns (CD45+ cells are attached in the magnetic part of the column).
SECTION: D. CD45 beads isolation
46. Wash the columns x3 times with 500 ul of Beads Buffer, add buffer only when column is empty each time.
SECTION: D. CD45 beads isolation
47. Place columns in new 15 ml tubes.
SECTION: D. CD45 beads isolation
48. Add 1 ml (MS) or 5 ml (LS) of Beads Buffer to each column and flush out the columns by pushing the plunger into the column.
SECTION: D. CD45 beads isolation
49. Take an aliquot to count cells.
SECTION: D. CD45 beads isolation
50. Centrifuge 300xg for 10 min at 4°C.
SECTION: D. CD45 beads isolation
51. Resuspend according to the follow-up experiment.
SECTION: Before:
1. EVERYTHING DONE ON ICE!!!
SECTION: Before:
2. Label all the tubes and make sure all the material is available.
SECTION: Before:
5. Prepare the hood and check if there is enough material in the tissue culture room.
SECTION: Before:
4. Set up centrifuge at 4°C (use centrifuge on the right; close to the external entrance of the lab).
SECTION: Before:
3. Make sure all the buffers are prepared and cold!! Can be done the day before, or day of.
SECTION: Before:
6. Prepare enzyme mixes and store in the fridge:
*Tip: buffer Z can be added first in C-tubes-then start cutting brains-add them in C-tube-add enzyme P.
Enzyme mix 1*: 50 ul of enzyme P and 1900 ul of buffer Z per sample/C tube.
Enzyme mix 2: 10 ul of enzyme A and 20 ul of buffer Y per sample/C tube.