TITLE: Activation Induced Marker (AIM) Staining Protocol
AUTHORS: Gregory P. Williams, cecilia

[DESCRIPTION]
This protocol details about activation induced marker staining.

[STEPS]
SECTION: Peptide Stimulation Solution
1. Label U-bottom plate with donor, stimulation solution, name and date.
SECTION: Peptide Stimulation Solution
2. Prepare PHA and DMSO mix separately
SECTION: Peptide Stimulation Solution
3. Prepare and arrange the remaining stimulation solution. Mix thoroughly by pipetting up and down before adding to the experimental plate.
SECTION: Peptide Stimulation Solution
4. Add appropriate stimulus solution to each well in 96-well U-bottom plates.
SECTION: Peptide Stimulation Solution
5. After adding stimulation solutions, prepare an additional solution of anti-CXCR5 antibody as described in below table.
 
      Antibody  Fluorochrome  Clone/vendor/catalog  Amount per well(50ul) (uL)    CXCR5  BUV395  RF8B2/BD/740266  1    HR5      49  
 
SECTION: Peptide Stimulation Solution
6. Add anti-CXCR5 antibody solution to all wells already containing stimulus.
SECTION: Peptide Stimulation Solution
7. Keep plate in incubator at 37 °C until cells are ready to be added. 

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

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

SECTION: PBMC Counting and Stimulus Preparation
12. Resuspend cells in HR5 and determine cell number.
SECTION: PBMC Counting and Stimulus Preparation
13. Centrifuge @  1200 rpm, 7 min.
SECTION: PBMC Counting and Stimulus Preparation
14. While sample(s) spinning, prepare the CD40 antibody solution the stock for all donors.
SECTION: PBMC Counting and Stimulus Preparation
15. 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
16. Incubate the tube for 15 min at 37 °C/5% CO2.

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

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

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

SECTION: PBMC Counting and Stimulus Preparation
21. Resuspend cells in 100 µL of antibody mix and 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
22. After incubation, add 100 µL MACS buffer and spin plate at  1400 rpm, 2 min, 4 °C.

SECTION: PBMC Counting and Stimulus Preparation
23. Wash 1X plate using 200 µL MACs buffer at  1400 rpm, 2 min, 4 °C..

SECTION: PBMC Counting and Stimulus Preparation
24. Wash 1X plate using PBS μL MACs buffer at  1400 rpm, 2 min, 4 °C.
SECTION: PBMC Counting and Stimulus Preparation
25. Resuspend in 120 µL PBS.

SECTION: PBMC Counting and Stimulus Preparation
26. Wrap in foil and store at 4 °C until analysis.