1. Pick up 4 blood tubes from ACRC processing lab (M1334). Keep tubes at RT. Drop off 1 tube of Buffer 1.
2. Dilute blood 1:1 with room temperature PBS.
3. Carefully layer 2 volumes of diluted blood on top of 1 volume of lymphoprep in 50ml falcon tubes (ex: 25ml blood on top of 12.5ml lymphoprep).
4. Place tubes in aerosol containment buckets, and spin at 800xg (~1900rpm) for 20 minutes at room temp. Turn the brake OFF.
5. Collect the PBMC layer at the interface between plasma and lymphoprep by slowly pipetting up in swirling motion with a glass Pasteur pipette on a pipet-aid.
6. Collect PBMCs in a 50ml falcon tube. Fill with PBS to wash. Spin at 300xg for 10 minutes, room temp.
7. Aspirate the supernatant and resuspend the cells in 10mls Buffer 1.
8. Count cells with 1:10 dilution in trypan blue.
   1. PBMC count: ______________
9. Spin cells again to wash. Aspirate buffer and resuspend at 100 million cells/ml in Buffer 1 for staining.
10. Pick up BAL cells when ready from the Woodruff lab. Record cell count and measure volume using a pipette.
    1. BAL cell count: _______________
11. Take 2ul of PBMCs for each compensation control in V-bottom 96-well plate.
12. Make 2x antibody mix to stain both PBMC and BAL equal to total volume of PBMC + BAL:

1. Stain compensation controls in 50ul each in V-bottom plate using the following antibodies:

1. Stain for 30 minutes on ice in the dark.
2. Wash cells twice with buffer 1- 100ul washes for comp controls and 2-5ml washes for blood and BAL.
3. Transfer comp controls to small FACS tubes in 150ul buffer 1.
4. Strain blood and BAL cells through blue-capped mesh FACS tubes. Spin BAL cells to get them through the mesh. Resuspend by light vortexing after the spin.
5. Keep cells on ice until sorting.
6. Make 8 tubes to sort into with 300ul media + 50% FCS. Keep on ice for sorting.

Sorting:

1. Bring:
   1. PBMCs and BAL cells
   2. PI for live/dead staining
   3. 8 tubes to sort into
   4. Notes with antibodies
   5. Flash drive to transfer data
   6. 4 extra FACS tubes
2. Run/save compensation controls and calculate compensation.
3. Switch to Global Sheet 1.
4. Run/save 10,000 events from PBMCs before adding PI. Adjust gates for sorting.
5. Add PI 1:20 to PBMCs. Vortex lightly to mix, or pipette up and down.
6. Check that the test streams are going into the center of the eppendorf tubes.
7. Vortex and label tubes with media to coat the sides of the tube. Open and place in tube holder for sorting in order.
8. Open Sort Layout 1 (under Global Sheet 1). Sort naïve T cells, Th1, Th2, and Th17. Click “Sort” and “OK” to move the waste drawer and turn on voltage plates.
9. Run/save 1,000,000 events from PBMCs while sorting. Sort 250,000 cells into each. When you reach 250k in one of the tubes, pause the sort, turn that sorting stream to 0, then resume the sort.
10.  When you have 250k cells for each tube, stop the sort and click ‘yes’ to save sort report. Record the cell counts for each cell type.
11. Take the tubes out of the tube holder. Lightly vortex to get droplets off the sides of the tube. Keep on ice.
12. Transfer 50ul from each of the sorted tubes to each of the 4 empty FACS tubes. Run/save 5,000 events from each for purity.
13. Switch to Global Sheet 2.
14. Run/save 10,000 events from BAL before adding PI. Adjust gates for sorting.
15. Add PI 1:20 to BAL. Vortex lightly to mix, or pipette up and down.
16. Return the sorting streams to their original values. Check that the test streams are going into the center of the eppendorf tubes.
17. Vortex and label tubes with media to coat the sides of the tube. Open and place in tube holder for sorting in order.
18. Open Sort Layout 2 (under Global Sheet 2). Sort naïve T cells, Th1, Th2, and Th17. Click “Sort” and “OK” to move the waste drawer and turn on voltage plates.
19. Run/save 1,000,000 events from BAL while sorting. Sort entire volume of BAL. Stop sort and click ‘yes’ to save sort report.
20. Record the cell counts for each cell type.
21. Do not need to run cells for purity from BAL.
22. Proceed with machine washing/shutdown. Check the calendar- if someone is after you do “In between users” protocol. If not, do “Fluidics shutdown” if it’s a Friday, and “Easy shutdown” otherwise