TITLE: Whole blood T cell assay for NHPs, containment protocol
AUTHORS: Jonathan  Audet, Courtney Meilleur

[DESCRIPTION]
This is a protocol used to perform T cell assays using whole blood (collected in EDTA tubes) or cells from bronchoalveolar lavage (BAL). We have successfully used this protocol for rhesus macaques, cynomolgus macaques, and African green monkeys (although antibody mix provided was titrated on macaques). It allows the characterization of T cells into Th1 and Th2 (or Tc1 and Tc2) phenotypes. It technically offers 64 different activation profiles (4 cytokines + CD107a + CD69).

The nature and concentration of the stimulant is left unspecified as we have used different ones depending on reagent availability.

This protocol was designed to deal with samples coming from containment labs (> CL2) at our facility. If you are using this protocol at a different facility please ensure that proper testing and approvals are in place. The protocol can be used for experiments completed entirely in CL2, simply go from step 17 directly to 31.

[BEFORE_START]
The antibody mix described in the methods was tested on Cynomolgus macaque whole blood and BAL fluid. All antibodies should cross-react with rhesus macaques and African green monkeys but the panel might need to be re-titrated.

[GUIDELINES]
For BAL fluid, make sure to set FSC voltage much lower than for whole blood.

[STEPS]
SECTION: Preparations
6. Prepare the staining mix. (for 1 sample:)

 
              Supplier  Antibody  Clone  Channel  Volume per test    BD Biosciences  CD45  D058-1283  BUV395  1.25    BD Biosciences  CD3  SP34-2  BUV496  5    BD Biosciences  CD8  RPA-T8  BUV563  1.25    BD Biosciences  CD16  3G8  BUV737  5    BD Biosciences  CD45RA  5H9  BV421  0.625    BD Biosciences  CD49d  9F10  BV480  5    BD Biosciences  CD4  L200  BV605  0.625    BD Biosciences  CD14  M5E2  BV650  5    BD Biosciences  CD123  7G3  BV786  1.25    BD Biosciences  CD25  M-A251  BB515  5    Miltenyi Biotec  CD66abce  TET2  PerCP-Vio700  1    BD Biosciences  CD163  GHI/61  PE  20    BD Biosciences  CCR7  2-L1-A  PE-CF594  0.625    BD Biosciences  HLA-DR  L243  PE/Fire640  1.25    BD Biosciences  CD69  FN50  PE-Cy7  5    BD Biosciences  TCRgd  B1  APC  5      CD20  2H7  Alexa 700  0.625        Total      63.5        BD Brilliant Stain      36.5  
 
 

SECTION: Surface Staining
8. Put 5 µl of TruStain FcX in each tube/well.  



SECTION: Surface Staining
9. Incubate 10 min at Room Temperature (RT).10 min Room temperature 

SECTION: Surface Staining
12. Add stain mix. Mix.100 µL 

SECTION: Surface Staining
13. Incubate 20 min at RT in the dark.20 Room temperature 
20 min 

SECTION: RBC Lysis
14. Add 2 ml of 1X FACS Lysing solution. Vortex immediately, but gently.2 mL 


SECTION: RBC Lysis
15. Incubate no more than 12 min at RT in the dark.10 min 

SECTION: RBC Lysis
16. Spin at 300 x g for 5 min.300 x g, 5 min, 20 °C 

SECTION: RBC Lysis
17. Decant supernatant.
SECTION: RBC Lysis
18. Add 2 ml of PBS. Vortex.2 mL 
  

SECTION: RBC Lysis
19. Spin at 300 x g for 5 min.300 x g, 5 min, 20 °C 

SECTION: RBC Lysis
20. Decant supernatant.
SECTION: Sample Inactivation
21. Resuspend in Cytofix/Cytoperm.   

SECTION: Sample Inactivation
21.1. 100 µl per 5 x 105 cells.
SECTION: Sample Inactivation
21.2. Use at least 400 µl for easy decanting.
SECTION: Sample Inactivation
22. Incubate at least 30 min at RT in the dark.30 min Room temperature 

SECTION: Sample Inactivation
23. Spin at 500 x g for 8-10 min.500 x g, 8 min, 20 °C 

SECTION: Sample Inactivation
24. On a clean bench, decant supernatant.
SECTION: Sample Inactivation
25. Use same volume of Cytofix/Cytoperm as before to resuspend the cells.  

SECTION: Sample Inactivation
26. Transfer in a 2 ml screwcap tube. Shake the tube to cover all surfaces with Cytofix/Cytoperm.

 

SECTION: Sample Inactivation
28. Tubes can be opened in CL2 (in a BSC) no less than 30 min after the resuspension (step 17).
(Samples are generally processed the next day; keep at 4 C overnight, in the dark)
SECTION: Intracellular staining
29. Give tubes a quick spin in a tabletop centrifuge.
SECTION: Intracellular staining
30. Ensure all tubes have at least a few hundred microliters of Cytofix/Cytoperm.


SECTION: Intracellular staining
31. Transfer the samples into 1 ml of BD   in FACS tubes.

SECTION: Intracellular staining
32. Spin at 500 x g for 8-10 min.500 x g, 8 min, 20 °C 

SECTION: Intracellular staining
33. Decant supernatant.
SECTION: Intracellular staining
34. Repeat steps 31-33.
SECTION: Intracellular staining
36. Resuspend in 100 µl of intracellular staining mix.
SECTION: Preparations
7. Prepare the 1X FACS Lysing solution by diluting the 10X stock with Milli-Q water. You will need 2 ml of 1X solution for each sample.
  

SECTION: Surface Staining
10. Add 5 µl of the    
If staining BAL: Add 5 µl of 1:100 diluted Ghost Dye.
SECTION: Surface Staining
11. Incubate 20 min at RT in the dark.20 Room temperature 
20 min 
SECTION: Sample Inactivation
27. Transfer tubes from containment space to CL2 space according to the facility's approved protocols/SOPs.
SECTION: Intracellular staining
44. Gating strategy for whole blood:



SECTION: Stimulation
1. Aliquot 100 µL  into a tube or well.

SECTION: Stimulation
2. Add 200 µL containing 1.5 X the desired concentration of stimulant.
(Stimulant(s) and concentration depend on the experiment and can include anti-CD28 and anti-CD49d)

SECTION: Stimulation
3. Mix and incubate at 37 °C   .

SECTION: Stimulation
4. Add monensin and brefeldin A, assuming 1e6 cells.
Anti-CD107a BV421 can be added here as well (5 µL )

SECTION: Stimulation
5. Mix and incubate at 37 °C 360 min .
SECTION: Intracellular staining
35. Prepare the staining mix. (for 1 sample:)
(uses   )
 
              Supplier  Antibody  Clone  Channel  Volume per test    BD Biosciences  CD45  D058-1283  BUV395  1.25    BD Biosciences  CD3  SP34-2  BUV496  5    BD Biosciences  CD8  RPA-T8  BUV563  1.25    BD Biosciences  CD16  3G8  BUV737  5    BD Biosciences  CD45RA  5H9  BV421  0.625    BD Biosciences  CD49d  9F10  BV480  5    BD Biosciences  CD4  L200  BV605  0.625    BD Biosciences  CD14  M5E2  BV650  5    BD Biosciences  CD123  7G3  BV786  1.25    BD Biosciences  CD25  M-A251  BB515  5    Miltenyi Biotec  CD66abce  TET2  PerCP-Vio700  1    BD Biosciences  CD163  GHI/61  PE  20    BD Biosciences  CCR7  2-L1-A  PE-CF594  0.625    BD Biosciences  HLA-DR  L243  PE/Fire640  1.25    BD Biosciences  CD69  FN50  PE-Cy7  5    BD Biosciences  TCRgd  B1  APC  5      CD20  2H7  Alexa 700  0.625        Total      63.5        BD Brilliant Stain      36.5  
 
 

SECTION: Intracellular staining
37. Incubate 4 °C 30 min 

SECTION: Intracellular staining
38. Add 1 mL   .

SECTION: Intracellular staining
39. 500 x g, 8 min, 4 °C 

SECTION: Intracellular staining
40. Decant supernatant and blot tubes/plate.
SECTION: Intracellular staining
41. Repeat steps 38-40.
SECTION: Intracellular staining
42. Resuspend in PBS 1% formaldehyde.
SECTION: Intracellular staining
43. Run on flow cytometer.