[Historical Background] The first report of fixing and permeabilizing lymphocytes, then staining them with antibodies against IFN gamma, was made by Andersson et al. in 1989. In 1991, Sander et al. demonstrated improved methods, using paraformaldehyde to fix cells, saponin (an amphipathic glycoside) to permeabilize them, and fluorescently-labeled antibodies to stain intracellular cytokines for microscope examination. In 1993, Jung et al. extended this method for use with flow cytometry, included monensin (a polyether antibiotic ionophore which blocks intracellular protein transport) to inhibit secretion, so as to increase the intracellular signal of the cytokine molecules that would otherwise be released soon after synthesis. In 1995, Prussin and Metcalfe used directly-conjugated antibodies, and reported good results with 6 h incubations. Also in 1995, Picker et al. considerably enhanced the sensitivity and reproducibility of cytokine detection by using Brefeldin A (‘BfA’, a fungal lactone antibiotic) to block the cytokine-secretion apparatus, and by using a different permeant (Tween-20). This improved method was applied by Picker et al. in a 1997 report of the antigen-specific homeostatic mechanism in human HIV+ patients. In 2001, Schuerwegh et al. confirmed that BfA provides better cytokine signal in the assay than does monensin, though monensin is still used widely by others in this method.

Regarding non-human primate studies, two reports in 1989, one by Gardner and another by McClure, showed that Rhesus macaques were a useful model for studying HIV disease and AIDS. In 2002, Picker et al. reported the application of a specially-modified CFC assay to Rhesus macaques. In 2012, a consortium-appointed group aiming to establish standards for collaborating groups using CFC in Rhesus vaccine studies published their recommendations for a 96-well plate method with a 6 h total incubation (Donaldson et al., 2012; Foulds et al., 2012).

The general procedure reported here is that 2002 tube-format (see Note 1) method, now with a 9 h total incubation, and optimized especially for low-end sensitivity. The specific details here are the state of the art now practiced by the Picker Lab, at the Oregon Health and Science University, affiliated with the Oregon National Primate Research Center. These methods have been used in several of our recent publications (Hansen et al., 2013a; Hansen et al., 2013b, Fukazawa et al., 2012; Hansen et al., 2011; Hansen et al., 2009). It is important to note that in our hands, plate-format CFC is not as sensitive and reproducible for weak responses as is this tube-based method described here (unpublished observations). Until that difference is understood and solved, the tube-based method remains the most-sensitive format for CFC.

Materials and Reagents

1. Lymphocyte suspension from Rhesus macaques blood (Note 2) or bronchoaveolar lavage (BAL), or harvest from solid biopsy or necropsy tissue, cell density determined by a method accurate for the sample (Notes 3 and 4)
   1. Need ~1 x 10⁶ viable lymphocytes per test
      
   2. Freshly-obtained (Note 5)
      
   3. OR thawed cryopreserved sample (Note 6)
2. Antigen
   1. Negative control(s) (Note 9)
      
   2. Positive control:
      Superantigen Staphylococcus Enterotoxin B SEB (Note 10) (Toxin Technology, catalog number: BT202 ) (lyophilized powder, 100 μg; stock: 100 μg/ml in water; usage: 2 μl/test)
      
   3. Other positive control (experiment-specific)
      
   4. Peptide mixes (1-100 different peptides, at ≥ 2 μg/peptide/1 ml-test) 15 amino acid peptides (15 mers) overlapping by 11 amino acids 
      
3. Antibody
   1. Unconjugated antibody for costimulation during culture incubation (Note 11)
      Anti-CD28, pure unconjugated, clone CD29.2 (Note 12)
      Anti-CD49d, pure unconjugated, clone 9F10
      Stocks diluted to 0.5 mg/ml; use 1 μl per 1 x 10⁶ Ly
      
   2. Essential fluorophore-conjugated monoclonal antibodies
      The fluorophores you use are dependent upon the flow cytometer available to you. Many companies sell the appropriate fluorophore-conjugated antibodies, including BD, Beckman Coulter, Life Technologies, Invitrogen^TM, eBiosciences, and many others.
      Anti-CD3e, clones reactive with Rhesus (SP34-2, FN18)
      Anti-CD4 (L200, MT477))
      Anti-CD8a (SK1, RPA-T8)
      Anti-CD69 (FN50, CH/4, TP1.55.3) (Note 13)
      Anti-IFNg (B27)
      Anti-TNFa (MAB11)
      
   3. Optional fluorophore-conjugated monoclonal antibodies
      Anti-CD45 (DO58-1283) (Note 14)
      Anti-IL2 (MQ1-17H12)
      Anti-MIP1b (D21-1351)
      Ant-CD107 (alpha: H4A3, beta: H4B4) (Note 15)
      Anti-CD95 (DX2)
      Anti- CD45RA (L48, 5H9, MEM-56, others) (Note 41)
      Anti-CCR7 (CD197) (Note 42)
      Anti-Ki67 (B56) (Note 38)
4. Brefeldin A (Sigma-Aldrich, catalog number: B-7651 )
   Vendors:  (Sigma-Aldrich, catalog number: B-7651; BioLegend, catalog number: 91850 )
   Working stock: 10 mg/ml, in DMSO (1.0 μl/test) (Note 16)
5. Benzonase (Merck KgaA, Novagen) (use at 50 U/ml)
   
6. 1x RPMI-1640 (w/o L-glutamine, 0.1 μm filtered) (e.g. HyCone, catalog number: SH30096.02 )
   
7. Fetal Bovine Serum (FBS, aka: 'FCS') (e.g. HyClone, catalog number: SH30070.03 ) (defined, heat-inactivated, 40 nm-filtered)
   
8. Penicillin+Streptomycin (P/S) Solution (e.g. Sigma-Aldrich, catalog number: P-0781 )
   
9. L-glutamine (200 mM) (e.g. Sigma-Aldrich, catalog number: G-7513 (100 ml)
   
10. Sodium pyruvate (SP) (e.g. Sigma-Aldrich, catalog number: S-8636 ) (100 ml)
    
11. Beta-Mercaptoethanol (bME) (e.g. Sigma-Aldrich, catalog number: M-7522 ) (100 ml) (Note 40)
    
12. Sterile-filtration apparatus (e.g. Corning, catalog number: 430769 ) (500 ml capacity 0.22 μm cellulose-acetate filter)
    
13. Dulbecco's Phosphate Buffered Saline (DPBS) (e.g. Thermo Fisher Scientific, Corning, catalog number: 55-031-PB )
    
14. Bovine serum albumin (BSA) (e.g. Thermo Fisher Scientific, catalog number: BP1605100 )
    
15. Sodium azide (preservative; NaN₃) (e.g. Thermo Fisher Scientific, catalog number: BP922-500 )
    
16. BD FACS Lysing Solution (10x concentrate) (BD, catalog number: 349202 )
    
17. Tween-20 (polyoxyethylenesorbitan monolaurate) (e.g. Sigma-Aldrich, catalog number: P-7949 )
    
18. Aqua LIVE/DEAD kit (Life Technologies, Invitrogen^TM, www.lifetechnologies.com, search 'LIVE/DEAD' for an evolving array of stains and kits) (Note 43)
    1. Concentrated dye stock (see Recipes)
       
    2. Staining Solution (made fresh) (see Recipes)
19. Tissue culture medium ('R10') (see Recipes)
    
20. 'PAB' wash buffer (see Recipes)
    
21. 'Lyse' fixation and RBC-lysing solution (see Recipes) (Note 17)
    
22. 'Perm' fixation and cell-permeabilizing solution (see Recipes) (Note 18)
    

Equipment

1. Tubes (Notes 1 and 7) polypropylene (PP) (round-bottom, 5 ml/12 x 75 mm, sterile) (e.g. Falcon^®, catalog number: 35-2054 )
   
2. Computer-generated printed labels for tubes (optional; must stick well to polypropylene)
   
3. Tube-holding racks (optional) (e.g. Thermo Fisher Scientific, No-Wire Grip Rack 10-13 MM 90 place) (Note 8)
   
4. Foam cosmetic wedges (or functional equivalent) (The ones we use are 2" long x ¾" high when lying on the long side.)
   
5. Laminar flow biosafety cabinet (for sterility, even if not working with pathogens)
   
6. Trapped vacuum aspirator
   
7. Appropriate fluid measuring dispensers, with appropriate disposables
   1. Electric pump pipettors for measurements between 1-50 ml
      
   2. Manual hand micropipettors for measurements between 0.5-1,000 μl
      
   3. Repeater-dispensers (e.g. from Eppendorf)
      
   4. Hand repeaters, for measurements between 0.5-2 ml
      
   5. Stationary pump dispensers, for measurements between 1-5 ml
8. Centrifuge with swing-buckets (capable of 800 x g) (e.g. Sorvall Legend T/RT)
   
9. Vortexer
   
10. Lab timer
    
11. Incubator for tissue culture (humidified, stable at 37 °C, 5% CO₂ atmosphere)
    Option: Standard water-jacketed T/C incubator (e.g. Thermo Fisher Scientific, Forma^TM, Series II, Model: 3110 )
    Option: UniBator (Tritech Research) DigiTherm CO₂ incubator with rapid cooling and bi-directional interface (Note 19)
    
12. Refrigerator at 4 °C
    
13. Flow cytometry analyser, 6-fluorescence detectors or more (e.g. BD, model: LSR-II )
    
14. A method of counting PBMC (e.g., Coulter counter, Guava, or hemocytometer)
    

Procedure