Isolation and FACS Analysis on Mononuclear Cells from CNS Tissue

Materials and Reagents

1. Mice (adult >6 weeks, any strain, e.g. C57BL/6 used for MOG-induced EAE)
   
2. Sodium pentobarbital (euthatal) (Merial)
   
3. Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: D8537 )
   
4. 10x PBS (Sigma-Aldrich, catalog number: D1408 )
   
5. RPMI solution(Sigma-Aldrich, catalog number: R0883 )
   
6. Penicillin-streptomycin (Sigma-Aldrich, catalog number: P4333 )
   
7. L-glutamine (Sigma-Aldrich, catalog number: G7513 )
   
8. FBS (Sigma-Aldrich, catalog number: F9665 )
   
9. Hank’s balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: H9394 ) supplemented with 3% FBS
   
10. Collagenase D (Roche, catalog number: 11088858001 )
    
11. DNase I (Sigma-Aldrich, catalog number: D4263 )
    
12. Percoll Plus (Sigma-Aldrich, catalog number: 17-5445-01 )
    
13. Cell permeabilisation kit (contains IntraStain Reagent A and B) (Dako, Denmark, catalog number: K2311 )
    
14. Phorbol myristate acetate (PMA) (Sigma-Aldrich, catalog number: P1585 )
    
15. Ionomycin (Sigma-Aldrich, catalog number: I0634 )
    
16. Brefeldin A (BFA) (Sigma-Aldrich, catalog number: B7651 )
    
17. LIVE/DEAD^® Fixable Aqua Dead Cell Stain kit (Life Technologies, catalog number: L34957 )
    
18. CD16/CD32 FcγRIII (BD Biosciences, catalog number: 553141 )
    
19. FACS antibodies (as appropriate)
    
20. Propidium iodide (PI) (Sigma-Aldrich, catalog number: P4864 )
    
21. Complete RPMI solution (see Recipes)
    
22. FACS buffer (see Recipes)
    
23. Stock isotonic percoll (SIP) (see Recipes).

Equipment

1. 70 μm nylon mesh filter (Corning, catalog number: 352350 )
   
2. Shaker capable of 200 rpm at 37 °C
   
3. Tissue culture facilities including class II laminar flow hood
   
4. Bench-top centrifuge, preferably refrigerated (any model)
   
5. Flow cytometer

Software

1. Summit software (Dako)
   
2. FlowJo software (Tree Star)

Procedure

1. Anaesthetise mice with sodium pentobarbital (40 μl i.p. ) and perfuse intracardially with sterile ice-cold PBS (20 ml). This is achieved by slowly and steadily injecting the ice-cold PBS into the left ventricle of the heart using a 20 ml syringe.
   
2. Dissect out brains by cutting away the skull with a sharp scissors, gently removing the brain with a forceps and place in 1 ml complete RPMI solution (or HBSS supplemented with 3% FBS can be used).
   
3. Prepare a single cell suspension by forcing the tissue through a sterile 70 μm nylon mesh filter using the plunger of a 5 ml syringe, wash with complete RPMI solution and centrifuge at 280 x g for 5 min.
   
4. Remove the supernatant and the remaining pellet and resuspend in complete RPMI (2 ml) containing collagenase D (1 mg/ml) and DNAse I (10 μg/ml), and incubate for 1 h at 37 °C with agitation.
   
5. Wash cells with complete RPMI and centrifuge at 280 x g for 5 min. Discard supernatants and resuspend cells in 40% Percoll [5 ml; 40% stock isotonic percoll (SIP) in PBS; 1.052 g/ml].
   
6. Carefully layer cell suspension on top of 70% Percoll (5 ml; 70% SIP in PBS; 1.088 g/ml).
   
7. Centrifuged at 600 x g for 20 min with the brake of the centrifuge switched off.
   
8. Remove mononuclear cells from the 1.088:1.052 g/ml interface by aspiration with sterile plastic pipette, wash twice in complete RPMI and count.
   
9. Samples are centrifuged at 280 x g for 5 min and cells are incubated in sterile FACS tubes at 37 °C in complete RPMI in the presence of PMA (10 ng/ml), ionomycin (1 μg/ml) and BFA (5 μg/ml) for 5 h.
   
10. After 5 h wash the cells by centrifuging the cells at 280 x g for 5 min, ready for intracellular staining using a cell permeabilisation kit.
    
11. Resuspend in 50 μl PBS with 1:1,000 LIVE/DEAD^® Fixable Aqua Dead Cell Stain kit for 20 min at 4 °C.
    
12. Wash cells in FACS buffer and resuspend in 50 μl FACS buffer containing CD16/CD32 FcγRIII (1: 100) and incubate at 4 °C for 10 min to block low-affinity IgG receptors.
    
13. Incubate cells in 50 μl/sample FACS buffer containing the appropriate FACS antibodies for 15 min at 4 °C.
    
14. Fix cells using IntraStain Reagent A (50 μl/sample) for 15 min at RT, wash twice with FACS buffer and centrifuge at 280 x g for 5 min.
    
15. Permeabilise cells with IntraStain Reagent B (50 μl/sample) including intracellular antibodies for 15 min at room temperature in the dark.
    
16. Wash cells twice in FACS buffer and centrifuge at 280 x g for 5 min.
    
17. Mononuclear cells which were surface stained only are blocked for 10 min, incubated with the appropriate antibodies for 15 min at 4 °C as described above, washed twice in FACS buffer and centrifuged at 280 x g for 5 min. PI can be used as a live/dead stain (with surface staining only) by adding 1:100 immediately before reading the samples on a flow cytometer.
    
18. Perform flow cytometric analysis on a flow cytometer and acquire data using Summit software. Analyse results using FlowJo software. Representative data can be viewed in References 1 and 2 below.

Recipes

1. Complete RPMI solution
   RPMI solution supplemented with 1% penicillin-streptomycin, 1% L-glutamine and 10% FBS
   Note: HBSS supplemented with 3% FBS can be used as a substitute for complete RPMI solution.
   
2. FACS buffer
   PBS supplemented with 2% FBS
   
3. Stock isotonic Percoll (SIP)
   9 parts Percoll plus (from the bottle), 1 part 10x PBS

Acknowledgments

This work was funded by a PI grant to Kingston Mills from Science Foundation Ireland.

References

1. Browne, T. C., McQuillan, K., McManus, R. M., O'Reilly, J. A., Mills, K. H. and Lynch, M. A. (2013). IFN-gamma production by amyloid beta-specific Th1 cells promotes microglial activation and increases plaque burden in a mouse model of Alzheimer's disease. J Immunol 190(5): 2241-2251.
   
2. Sutton, C. E., Lalor, S. J., Sweeney, C. M., Brereton, C. F., Lavelle, E. C. and Mills, K. H. (2009). Interleukin-1 and IL-23 induce innate IL-17 production from gammadelta T cells, amplifying Th17 responses and autoimmunity. Immunity 31(2): 331-341.