Activation Induced Marker (AIM) Staining Protocol

Label U-bottom plate with donor, stimulation solution, name and date.

Prepare PHA and DMSO mix separately

Prepare and arrange the remaining stimulation solution. Mix thoroughly by pipetting up and down before adding to the experimental plate.

Add appropriate stimulus solution to each well in 96-well U-bottom plates.

After adding stimulation solutions, prepare an additional solution of anti-CXCR5 antibody as described in below table.

A	B	C	D
Antibody	Fluorochrome	Clone/vendor/catalog	Amount per well(50ul) (uL)
CXCR5	BUV395	RF8B2/BD/740266	1
HR5			49

Add anti-CXCR5 antibody solution to all wells already containing stimulus.

Keep plate in incubator at 37°C until cells are ready to be added.

Obtain indicated number of vial(s) of PBMCs.

For each donor, prepare sterile 50 ml tubes with 10mL HR5 and 20µL Benzonase per vial to be thawed.

Thaw PBMC vials.

Centrifuge @ 1200rpm.

Resuspend cells in HR5 and determine cell number.

Centrifuge @ 1200rpm.

While sample(s) spinning, prepare the CD40 antibody solution the stock for all donors.

Resuspend each donor at 1.5 per 10 million cells per ml in prepared 1.5 CD40 antibody solution.

Incubate the tube for 0h 15m 0s at 37°C/5% CO₂.

Add 100µL of CD40 antibody-treated PMBCs to each well already containing stimulus.

Incubate plate for a total of 20-24 hours at 37°C/5% CO₂.

After incubation, spin plate at 1400rpm,4°C.

Wash plate by adding 200µL PBS and spinning at 1400rpm,4°C.

Resuspend cells in 100µL of antibody mix and incubate at 4°C for 0h 30m 0s, protected from light. Wrap plate in aluminum foil and place in fridge.

After incubation, add 100µL MACS buffer and spin plate at 1400rpm,4°C.

Wash 1X plate using 200µL MACs buffer at 1400rpm,4°C..

Wash 1X plate using PBS μL MACs buffer at 1400rpm,4°C.

Resuspend in 120µL PBS.

Wrap in foil and store at 4°C until analysis.