In vitro efferocytosis

1. Plate macrophages (MΦs) onto 24-well tissue culture plates in 500 μL of media overnight.
   1. 150K MΦs per well
2. Spin down Jurkat cells at 600g for 5 min.
3. Resuspend pellet in 1X PBS and spin down Jurkat cells again at 600g for 5 min.
4. Carry out PKH67 staining on Jurkat cells (catalog numbers MINI67, MIDI67, or PKH67GL from Sigma-Aldrich) as follows.
   1. Prepare a 2x Cell Suspension by adding 1 mL of Diluent C to the cell pellet and resuspend by gentle pipetting
      1. Do not vortex or let cells remain in Diluent C for extended periods of time
   2. Prepare a 2x Dye Solution in Diluent C by adding 4 μL of the PKH67 dye to 1 mL of Diluent C in a polypropylene centrifuge tube and mix well.
   3. Add the 1x cell suspension to the 1 mL of 2x dye solution and immediately mix by continuous pipetting for 2-3 minutes.
      1. Final cell concentration should be NO MORE than 1 x 107 cells/mL
   4. Terminate the staining by adding 2 mL of 1% BSA or FBS and incubate for 1 min to allow binding of excess dye.
   5. Centrifuge Jurkat cells at 600g for 5 min and resuspend the cell pellet in complete media.
   6. Spin cells once more and resuspend in 1X PBS.
5. Transfer stained cells onto petri dish.
6. Remove the dish lid and place dish 4-5 inches from the UV source and expose to UV light for 15 min in a tissue culture hood with hood UV light on.
   1. UV 254 nM irradiation: EL Series Ultraviolet Hand Lamp attached to a lamp stand from UVP-Model UVS-18 assembly 8W bulb (95-0200-01) 115V/60Hz/0.16Amp.
7. Add dish lid back to plate and place the dish containing fluorescently-labeled Jurkat cells in a cell culture incubator for at least 2 hr.
   1. Check for morphological features of apoptosis (blebbing) every 30 min.
8. Once apoptotic, spin cells and resuspend to desired concentration.
9. Add apoptotic cells to MΦs to reach at 3:1 apoptotic cell: MΦ ratio for 45 min.
   1. This is 450K apoptotic cells to a well of 150K macrophages from Step 1.
10. Remove media and rinse twice with 1X PBS then fix with 4% formaldehyde for 20 min.
11. Rinse twice with 1X PBS and capture images on an epifluorescence microscope.
12. Determining the percent of efferocytosis is calculated as the number of PKH67+ MΦs divided by the number of total MΦs.

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