TITLE: Intracellular Cytokine (ICS) Staining Protocol
AUTHORS: Gregory P. Williams, cecilia

[DESCRIPTION]
This protocol details about intracellular cytokine (ICS) staining.

[STEPS]
SECTION: Stimulus Solution
1. Label U-bottom plate with donor, stimulation solution, name and date.
SECTION: Stimulus Solution
2. Prepare PMA+Ionomycin and DMSO mix separately.
SECTION: Stimulus Solution
3. Prepare and arrange the remaining stimulation solution. Mix thoroughly by pipetting up and down before adding to the experimental plate.
SECTION: Stimulus Solution
4. Add appropriate stimulus solution to each well in 96-well U-bottom plates.
SECTION: Stimulus Solution
5. Store stimuli-loaded plates in 37 °C incubator while thaw cells in next step.
 
      anti-CD40      Antibody  Clone/vendor/catalog    anti-CD40 1.5ug/mL per 10million cells  RF8B2/BD/740266    HR5        Total Volume    
 
SECTION: PBMC Counting and Stimulus Preparation
6. Obtain indicated number of vial(s) of PBMCs.
SECTION: PBMC Counting and Stimulus Preparation
7. For each donor, prepare sterile 50ml tubes with 10 mL HR5 and 20 µL Benzonase per vial to be thawed.

SECTION: PBMC Counting and Stimulus Preparation
8. Thaw PBMC vials.
SECTION: PBMC Counting and Stimulus Preparation
9. Centrifuge @ 1200 rpm, 7 min.

SECTION: PBMC Counting and Stimulus Preparation
10. Resuspend cells in HR5 and determine cell number.
SECTION: PBMC Counting and Stimulus Preparation
11. Centrifuge @  1200 rpm, 7 min.
SECTION: PBMC Counting and Stimulus Preparation
12. While sample(s) spinning, prepare the CD40 antibody solution the stock for all donors.
SECTION: PBMC Counting and Stimulus Preparation
13. Resuspend each donor at 1.5 1809 per 10 million cells per ml in prepared  1.5 1809 CD40 antibody solution.

SECTION: PBMC Counting and Stimulus Preparation
14. Add CD40 antibody solution to all wells already containing stimulus.
SECTION: PBMC Counting and Stimulus Preparation
15. Keep plate in incubator at 37 °C while thawing/preparing cells in the following steps.

SECTION: PBMC Counting and Stimulus Preparation
16. Obtain indicated number of vial(s) of PBMCs.
SECTION: PBMC Counting and Stimulus Preparation
17. For each donor, prepare sterile 50ml tubes with 10 mL HR5 and 20 µL Benzonase per vial to be thawed.

SECTION: PBMC Counting and Stimulus Preparation
18. Thaw PBMC vials.
SECTION: PBMC Counting and Stimulus Preparation
19. Centrifuge @  1200 rpm, 7 min.
SECTION: PBMC Counting and Stimulus Preparation
20. Resuspend cells in HR5 and determine cell number.
SECTION: PBMC Counting and Stimulus Preparation
21. Centrifuge @  1200 rpm, 7 min.
SECTION: PBMC Counting and Stimulus Preparation
22. While sample(s) spinning, prepare the CD40 antibody solution the stock for all donors.
SECTION: PBMC Counting and Stimulus Preparation
23. Resuspend each donor at 10 million cells per ml in prepared 1.5 1809 CD40 antibody solution.

SECTION: PBMC Counting and Stimulus Preparation
24. Incubate the tube for 15 min at 37 °C/5% CO2.

SECTION: PBMC Counting and Stimulus Preparation
25. Add 100 µL of CD40 antibody-treated PMBCs to each well already containing stimulus.

SECTION: PBMC Counting and Stimulus Preparation
26. Incubate plate for a total of 20-24 hours at 37 °C/5% CO2.

SECTION: PBMC Counting and Stimulus Preparation
27. After 20-24 HR, add 50 µL Intracellular Transport Blocking solution to each well and incubate for an additional 240 min at 37 °C/5% CO2.
 
      Intracellular Transport Blocking Solution    Reagent   Vendor/catalog   Minimum (μl)    Golgi Plug  BD/#555029  4      Golgi Stop  BD/#554724  4      HR5    992      Total Volume  1000    
 
SECTION: PBMC Counting and Stimulus Preparation
28. After incubation, spin plate at 1400 rpm, 2 min, 4 °C.

SECTION: PBMC Counting and Stimulus Preparation
29. Wash plate by adding 200 µL PBS and spinning at  1400 rpm, 2 min, 4 °C.

SECTION: PBMC Counting and Stimulus Preparation
30. Resuspend cells in 100 µL of LIVE/DEAD and FC block mix. Prepare as follows: 
      Reagent  Clone/Vendor/Catalog/Peak  Amount per well (uL)    Fixable Live/Dead Blue  Thermo/L23105/UV6  0.2    Human FC Block  BD/564220  5    PBS    94.8    *Total   Volume  100  
 
SECTION: PBMC Counting and Stimulus Preparation
31. Incubate at 4 °C for 30 min, protected from light. Wrap plate in aluminum foil and place in fridge.

SECTION: PBMC Counting and Stimulus Preparation
32. After incubation, add 100 µL PBS buffer and spin plate at 1400 rpm, 2 min, 4 °C. Decant.
SECTION: PBMC Counting and Stimulus Preparation
33. Resuspend cells in 100 µL of surface antibody mix and incubate at 4 °C for 30 min, protected from light.
 
      Surface Stain (100μl per well)        Membrane Antibody  Fluorochrome  Clone/vendor/catalog  Amount per well (uL)    CD3  BUV395  UCHT1/BD/563546  1      CD8  BUV661  RPA-T8/BD/750699  0.5      CD16  BV510  3G8/Biolegend/302048  0.5      CD14  BV510  63D3/Biolegend/367124  0.5      CD20  BV510  2H7/Biolegend/302340  0.5      CD45RA  BV570  HI100/Biolegend/304132  2      CD4  BV711  RPA-T4/BD/740769  1      CCR7  PE-Cy7  G043H7/Biolegend/353226  1      FACS Buffer  83        BSB plus    BD Horizon/566385  10      *Total   Volume    100    
 
SECTION: PBMC Counting and Stimulus Preparation
34. After incubation, add 100 µL FACS buffer and spin plate at 2000 rpm, 1 min, 4 °C.

SECTION: PBMC Counting and Stimulus Preparation
35. Wash plate. 

SECTION: PBMC Counting and Stimulus Preparation
36. Add 200 µL 4% PFA to each well, pipette to mix, cover and incubate at 4 °C for 10 min.
SECTION: PBMC Counting and Stimulus Preparation
37. After incubation spin plate at 2000 rpm, 5 min.
SECTION: PBMC Counting and Stimulus Preparation
38. Wash 1X with 200 µL PBS at  2000 rpm, 5 min. Meanwhile, prepare saponin buffer as follows: 
        Saponin Buffer  Blocking Buffer            Saponin powder  10% BSA  0.01% azide  Vol. PBS  Total Vol.  Blocking buffer (10% Human serum in SB)    Example  0.05 g  1mL  100μL of 1% azide  8.9mL  10mL  100 μL+ 900 μL SB  
 
SECTION: PBMC Counting and Stimulus Preparation
39. Wash 1X with 200 µL of saponin buffer at  2000 rpm, 5 min. Meanwhile, prepare blocking buffer. 
 


SECTION: PBMC Counting and Stimulus Preparation
40. Add 50 µL blocking buffer to each well and incubate protected from light at 4 Room temperature for 5 min.

SECTION: PBMC Counting and Stimulus Preparation
41. Add 50 µL of prepared intracellular stain to each well. Incubate protected from light at 4 Room temperature for 30 min. 
 
      Intracellular Stain (50μl per well)      IC Antibody  Fluorochrome  Clone/vendor/catalog  Amount per Well (μL)    IL-4  BUV737  MP4-25D2/BD/612835  0.5      IL-17  BV785  BL168/Biolegend/512338  1      TNFa  eFluor450  Mab11/eBioscience/48-7349-42  0.2      IFNg  FITC  4S.B3/eBioscience/11-7319-82  0.2      IL-2  BB700  MQ1-17H12 /BD/566405  0.5      IL-10  PE-Dazzle594  JES3-19F1/BioLegend/506812  1      CD40L  APC-ef780 Changed from percp-ef710  24-31/LifeTech/47-1548-42  2      PBS      34.6      BSB plus    BD Horizon/566385  10      *Total   Volume  50      
 
 
SECTION: PBMC Counting and Stimulus Preparation
42. Wash 1X with 100 µL saponin buffer at 2000 rpm, 5 min.
SECTION: PBMC Counting and Stimulus Preparation
43. Wash 1X with 200 µL PBS at  2000 rpm, 5 min.
SECTION: PBMC Counting and Stimulus Preparation
44. To store plate 30 min, add 200 µL FACS buffer. Wrap in foil and store at 4 °C until analysis.
SECTION: PBMC Counting and Stimulus Preparation
35.1. Wash plate using 200 µL FACs buffer at 2000 rpm, 1 min, 4 °C. (1/2)
SECTION: PBMC Counting and Stimulus Preparation
35.2. Wash plate using 200 µL FACs buffer at 2000 rpm, 1 min, 4 °C. (2/2)