TITLE: Immunophenotyping for NHPs, containment protocol
AUTHORS: Jonathan  Audet, Courtney  Meilleur

[DESCRIPTION]
This is a protocol used to perform immunophenotyping of 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 AGM only). It allows the characterization of T cells (CD69/CD25 for activation; CCR7/CD45RA for memory phenotype; α/β  vs γ/δ ), B cells, Monocytes, Neutrophils, Basophils, Eosinophils, an potentially NK cells (CD56 does not work for AGM).

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 African green monkey whole blood and BAL fluid. CD56 does not stain AGM NK cells. All antibodies should cross-react with cynomolgus and rhesus macaques 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
1. 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
4. Put 5 µl of TruStain FcX in FACS tubes (1 tube per sample).  

 

SECTION: Surface Staining
5. Add 100 µl of whole blood (EDTA blood) or BAL cells to the correct tube. Mix.100 µL 

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

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

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

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


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

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

SECTION: RBC Lysis
14. Decant supernatant.
SECTION: RBC Lysis
15. Add 2 ml of PBS. Vortex.2 mL 
  

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: Sample Inactivation
18. Resuspend in Cytofix/Cytoperm.   

SECTION: Sample Inactivation
18.1. 100 µl per 5 x 105 cells.
SECTION: Sample Inactivation
18.2. Use at least 400 µl for easy decanting.
SECTION: Sample Inactivation
19. Incubate at least 30 min at RT in the dark.30 min Room temperature 

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

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

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

 

SECTION: Sample Inactivation
25. 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: Final Wash &amp; Run
26. Give tubes a quick spin in a tabletop centrifuge.
SECTION: Final Wash &amp; Run
27. Ensure all tubes have at least a few hundred microliters of Cytofix/Cytoperm.
SECTION: Final Wash &amp; Run
28. Transfer the samples into 1 ml of PBS in FACS tubes.  

SECTION: Final Wash &amp; Run
29. Spin at 500 x g for 8-10 min.500 x g, 8 min, 20 °C 

SECTION: Final Wash &amp; Run
30. Decant supernatant.
SECTION: Final Wash &amp; Run
31. Resuspend in 200 µl of PBS.  

SECTION: Final Wash &amp; Run
32. Run on FACSymphony A5.
SECTION: Preparations
2. 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: Preparations
3. Prepare a 1:100 dilution of viability dye combining 495 µL  and  5 µL   
For whole blood, use the Ghost Dye undiluted.
SECTION: Surface Staining
7. Add 5 µl of the diluted   
If staining whole blood: Add 5 µl of undiluted Ghost Dye.
SECTION: Surface Staining
8. Incubate 20 min at RT in the dark.20 Room temperature 
20 min 
SECTION: Sample Inactivation
24. Transfer tubes from containment space to CL2 space according to the facility's approved protocols/SOPs.
SECTION: Final Wash &amp; Run
33. Gating strategy for whole blood: