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

Prepare PMA+Ionomycin 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.

Store stimuli-loaded plates in 37°C incubator while thaw cells in next step.

A	B	C
anti-CD40
Antibody	Clone/vendor/catalog
anti-CD40 1.5 ug/mL per 10million cells	RF8B2/BD/740266
HR5
Total Volume

Obtain indicated number of vial(s) of PBMCs.

For each donor, prepare sterile 50ml 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.

Add CD40 antibody solution to all wells already containing stimulus.

Keep plate in incubator at 37°C while thawing/preparing cells in the following steps.

Obtain indicated number of vial(s) of PBMCs.

For each donor, prepare sterile 50ml 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 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 20-24 HR, add 50µL Intracellular Transport Blocking solution to each well and incubate for an additional 4h 0m 0s at 37°C/5% CO₂.

A	B	C	D
Intracellular Transport Blocking Solution
Reagent	Vendor/catalog	Minimum (μl)
Golgi Plug	BD/#555029	4
Golgi Stop	BD/#554724	4
HR5		992
Total Volume		1000

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 LIVE/DEAD and FC block mix. Prepare as follows:

A	B	C
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

Incubate at 4°C for 0h 30m 0s, protected from light. Wrap plate in aluminum foil and place in fridge.

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

Resuspend cells in 100µL of surface antibody mix and incubate at 4°C for 0h 30m 0s, protected from light.

A	B	C	D	E
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

After incubation, add 100µL FACS buffer and spin plate at 2000rpm,4°C.

Wash plate.

Wash plate using 200µL FACs buffer at 2000rpm,4°C. (1/2)

Wash plate using 200µL FACs buffer at 2000rpm,4°C. (2/2)

Add 200µL 4% PFA to each well, pipette to mix, cover and incubate at 4°C for 0h 10m 0s.

After incubation spin plate at 2000rpm.

Wash 1X with 200µL PBS at 2000rpm. Meanwhile, prepare saponin buffer as follows:

A	B	C	D	E	F	G
	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	1 mL	100μL of 1% azide	8.9 mL	10 mL	100 μL+ 900 μL SB

Wash 1X with 200µL of saponin buffer at 2000rpm. Meanwhile, prepare blocking buffer.

Add 50µL blocking buffer to each well and incubate protected from light at 4Room temperature for 0h 5m 0s.

Add 50µL of prepared intracellular stain to each well. Incubate protected from light at 4Room temperature for 0h 30m 0s.

A	B	C	D	E
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

Wash 1X with 100µL saponin buffer at 2000rpm.

Wash 1X with 200µL PBS at 2000rpm.

To store plate 0h 30m 0s, add 200µL FACS buffer. Wrap in foil and store at 4°C until analysis.