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Overview

This is a protocol designed for T-regulatory assay for Mouse allergen (Mus m1) study.

Materials

• List reagents, supplies and equipment necessary to perform the protocol here. For those materials which have their own OWW pages, link to that page. Alternatively, links to the suppliers' page on that material are also appropriate.

Isolation of Mononuclear Cells	PBMC Antigen Stimulation Assay	Collection of Supernatant	T-Regulatory Cells Surface Staining
On average, 18mL of whole heparinized blood/patient will be given.	8 x 10^6 (8 million) cells for 7 day culture (for measurement of frequency of PBMC-derived, Mus M1-specific CD4+ CD25+ FoxP3+ T cells).	Sterile 5mL polystyrene round-bottom tubes.	Phosphate buffered saline (PBS)
Ficoll Paque Plus [endotoxin tested], room temperature.	CFSE (5μM aliquots).	Staining buffer (PBS + 5g BSA/L + 2mM EDTA)	Staining buffer (PBS + 5g BSA/L + 2mM EDTA)

Isolation of Mononuclear Cells

• This is a sterile procedure and all steps should be performed in a hood.

1. Turn on the hood. Bring Ficoll and PBS to room temperature in the hood.

1. Obtain whole blood specimens collected in sodium heparin (green top) collection tubes and record subject information, i.e. ID #, date collected, date received.

1. If performing Basophil Activation assay on this sample, set aside 3mL of whole blood.

1. Dilute remaining blood at 1:1 with PBS in 50 mL conical tubes, adding PBS first followed by blood.

1. Place 15 mL of Ficoll in a 50 mL conical tube. Overlay with up to 30mL of diluted blood, adding it very slowly to make sure that the blood doesn’t mix with the Ficoll layer.

1. Centrifuge at 500 g for 30 minutes at room temperature (slow acceleration, deceleration off to ensure no disruption of the density gradient).

1. Using a sterile transfer pipette, aspirate the buffy coat (peripheral blood mononuclear cells [PBMCs]) into a new 50 mL conical tube. (Avoid aspirating the Ficoll.) Add PBS to bring to a minimum of 2x the volume, inverting up and down to mix.

1. Centrifuge at 500 g for 20 minutes at room temperature (maximum acceleration and deceleration).

1. Aspirate and discard the supernatant. Resuspend the cell pellet first by tapping the tube until no clumps are visible, then adding 1 mL of PBS. Set aside a 10 µL aliquot of cells for counting as follows: place the 10 µL of cells into 90 µL of PBS in a small sterile eppendorf tube, and add 100 µL of Trypan Blue (0.2%) into the eppendorf tube. Mix well, and let it sit for 1 minute before counting.

1. Add 19 mL of PBS to the cells in 990 µL to make a total volume of 19.99 mL and centrifuge at 300 g for 15 minutes at room temperature (maximum acceleration and deceleration).

1. In order to determine the volume to use for resuspending the PBMCs after the wash, the total number of cells in the sample must be determined.

1. Carefully introduce 10 µL of the stained cells into the notch of a hemocytometer and record cell counts using a hand-held counter. Calculate the number of cells, taking into account the dilution factors and sample volume used. Or place 20 µL of the stained cells onto a disposable slide and count using the automated cell counter.

1. After centrifugation is completed, aspirate and discard the supernatant. Resuspend the cell pellet by tapping the tube until no clumps are visible. Suspend PBMCs with PBS at 10 x 10^6/ml in 15 ml PP conical tube.

*This is a sterile procedure and all steps should be performed in a hood.

1. Remove stimulants from the freezer and thaw.
2. Label 24 well plate with specimen ID and date (this is for the 7 days cell culture). Label each well with the appropriate stimulant condition, ordered by priority (for cases where there are insufficient cells to test all stimulants).
   1. Musm1 (allergen): @ 200μg/mL purified Musm1 protein in Aim-V.
   2. AIM-V + IL-2 (negative control): AIM-V + IL-2 medium alone.
   3. Beads (positive control): 1μg/mL anti-Cd3/Cd28 beads.
   4. Tetanus: 200μg/mL tetanus in AIM-V.
3. Add an equal volume (1:1 dilution) of freshly prepared 10μM CFSE (in PBS) to the tube of cells. To make 1.5mL of PBS + CFSE (2x solutin): add 3μL of stock CFSE (5mM) into 1.5mL of PBS.
4. Incubate in 37deg;C water bath for 10 minutes.
5. After incubation, wash the CFSE stained cells in 10mL of AIM-V @ 300g for 10 minutes. Aspirate supernatant after centrifugation.
6. Resuspend CFSE stained cells in AIM-V medium @ 4 million cells/mL. For plating, each well should contain 2 milliion cells/mL.

• Begin the stimulation process by preparing AIM-V medium + a 2X solution of IL-2 by adding 2μL of IL-2 per mL of medium in a 15mL conical tube. For 5 stimulant conditions, you will need atleast 2.5mL of AIM-V + IL-2. Vortex gently.

1. Prepare solutions for each stimulant condition in 5 mL PP tubes as follows:

1. 1. AIM-V+IL-2 medium alone: place 500μL of AIM-V+IL-2 in the appropriately labeled tube and add 500μL of cells in AIM-V+IL-2. Pipette up and down and plate.

1. 1. 1μg/mL anti-CD3, anti-CD28 beads in AIM-V+IL-2: place 500μL of AIM-V in the appropriately labeled tube and add 2.5μL of CD3, CD28 expander beads. Then add 500μL of cells in AIM-V+IL-2. Pipette up and down and plate.

1. 1. 200μg/mL MusM1 in AIM-V+IL-2: place 450μL of AIM-V+IL-2 in the appropriately labeled tube and add 50μL of allergen (MusM1). Then add 500μL of cells in AIM-V+IL-2 + allergen. Pipette up and down, and plate.

1. 1. 200μg/mL tetanus in AIM-V+IL-2: place 475μL of AIM-V+IL-2 in the appropriately labeled tube and add 25μL of tetanus. Then add 500μL of cells in AIM-V+IL-2. Pipette up and down and plate.

1. 1. Place the tissue culture plate in the 37°C CO2 incubator for 7 days.

COLLECTION OF SUPERNATANTS

1. Cell culture supernatants will be collected for cytokine measurement for 2 or 7 days cultures.

1. At day 7, collect cells carefully by pipetting up and down to resuspend cells in the well, and

transfer the total volume in each well into separate 5ml polystyrene tubes. Rinse each well

with 200μl of staining buffer and add to corresponding PS tubes. Tap gently to mix.

1. Centrifuge tubes at 300g for 5 minutes at 25°C temperature.

1. Obtain a cluster tube rack for the storage of supernatants.

1. Label cluster tubes as follows:

1. 1. Specimen ID

1. 1. Date

1. 1. Supernatants - 7 day

1. 1. Stimulant condition 1-4

1. 1. 1. Stimulant 1 = MusM1

1. 1. 1. Stimulant 2 = AIM-V

1. 1. 1. Stimulant 3 = Beads

1. 1. 1. Stimulant 4 = Tetanus Toxoid

1. 1. For each stimulant, using a 1000μL pipette tip, transfer 800μL of supernatant from the PS culture tube into each corresponding cluster tube in the cluster rack. Be careful not to disturb the cell pellets.

1. 1. Cap the cluster tubes and store in the –80oC freezer (by the freight elevators).

T REGULATORY CELL SURFACE STAINING

1. After the collection of supernatants according to the Collection of Supernatants Protocol. Resuspend cells in 80μL of cell separation buffer.

1. Add 1mL of staining buffer to each tube and vortex.

1. Wash cells at 300 g for 5 minutes at 4°C. Decant supernatant.

1. Add 1mL of staining buffer to each tube and vortex.

1. Wash cells at 300 g for 10 minutes at 4°C. Decant supernatant.

1. Prepare cocktail preparation according to the T REGULATORY CELL SURFACE STAINING; Materials. Add 50μL of cocktail to each tube.

1. Incubate @ 4°C for 20-30 minutes.

1. Add 3mL of staining buffer to each tube and vortex.

1. Wash at 300 g for 10 minutes at 4°C. Decant supernatant.

• At this point in the experiment, one can fix/freeze the cells or continue with Intracellular cytokine staining.

1. 1. To fix/freeze the cells, pipette 500μl of 1X Facs Lysis Buffer into each tube and incubate @ 25°C in the dark for 15 minutes. After the incubation, pipette 500μl of the 20% DMSO + PBS solution into each tube.

1. Transfer the 1ml of cells + 1X Facs Lysis Buffer + 20% DMSO/PBS solution into an appropriately labeled cluster tubes, and freeze at -80°C until when you are ready to conduct Intracellular cytokine staining..

1. To Intracellular Cytokine stain the cell with Fox P3, prepare a Fix/Perm working solution as follow: dilute the Fix/Perm Concentrate (1 part) into the Fix/Perm Diluent (3 parts) to the desired volume of working solution (1mL per tube).

1. Resuspend cell pellet with pulse vortex and add 1mL of Fix/Perm buffer to each sample tube.

Pulse vortex again.

1. Incubate at 4°C for 30-60 minutes in the dark (preferably for 60 minutes if possible).

Wash for 10 minutes at 800 g x 2 (4°C) with 2mL of 1X Perm Buffer (made from 10X solution using dH2O). Decant supernatant.

1. Add 20μL of Foxp3 antibody into 100μL of 1X Perm Buffer. Incubate for at least 30 minutes (45

minutes if possible) at 4°C.

1. Wash at 800 g for 10 minutes x 2 with 2mL 1X Perm Buffer. Decant supernatant. Vortex and acquire using the Flow machine at 17-40.

Notes

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References

Relevant papers and books

1. Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4. DOI:10.1073/pnas.78.11.6840 |
2. JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. DOI:10.1016/s0022-2836(61)80072-7 |
3. ISBN:0879697164

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