An In Vitro Technique for UV Irradiation-Mediated Generation of Apoptotic T Cells

1. Preparation of Jurkat T cell line (Day 2)

NOTE: All procedures should be conducted in a class II biological safety cabinet.

1. Culture Jurkat T cells in 24 mL of basal cell culture medium + 10% FBS + 5% penicillin/streptomycin at 37 °C + 5% CO₂ (Table of Material). Jurkat T cells are a suspension cell line that can be maintained through passaging 1:6-1:8 into pre-warmed culturing media every 3 days. Do not shake.
2. To prepare apoptotic cells, grow them to 90% confluency in each flask (which takes 3-4 days to achieve after passaging). For this study, use cells from five T75 flasks to obtain a sufficient number of cells used in this protocol.
   NOTE: A confluent flask contains about 20-24 million cells.
3. Pipette up cells (which is the entire flask) from each flask (approximately 24 mL) and transfer cells to a sterile 50 mL conical tube using a serological pipette. Use multiple conical tubes for multiple flasks.
4. Count cells by removing an 11 µL aliquot of cells from the 50 mL conical tube and mix with 11 µL of trypan blue stain and pipette 11 µL onto hemocytometer slides.
5. Insert the slide into an automated cell counter and record the number of live cells to calculate the total cell count in each flask by multiplying the number of live cells by 24, as each flask contains 24 mL of media.
6. Centrifuge the cell suspension at 271 x g for 5 min at room temperature (RT) to pellet cells.
7. Discard the supernatant by aspiration and resuspend the cell pellet in media to obtain 3.0 x 10⁶ cells per mL.
8. Aliquot 5 mL of cells in 100 mm x 20 mm tissue culture dishes (approximately nine dishes will be used; the total amount of cells in each dish should be ~15 x 10⁶).
9. Use one dish for control/unexposed, and the remaining dishes will be exposed to UV.
10. Set the UV crosslinker to the correct energy level, press the energy button, and enter "600" using the number pad, which the machine will read as 600 µJ/cm² x 100.
    NOTE: UV crosslinker energy units are in µJ/cm² x 100; therefore, to achieve 60 millijoules/cm², convert units to match the UV crosslinker.
11. Irradiate all dishes with cells, not including the control, at 60 millijoules (mJ)/cm² using the UV crosslinker. Remove the top cover of the tissue culture dishes during UV exposure, as UV light will not penetrate the plastic cover.
12. Incubate all the dishes in a cell culture incubator, including unexposed control, at 37 °C at 5% CO₂ for 4 h.
13. Confirm apoptosis by flow cytometry using an apoptosis assay detection kit containing annexin V and propidium iodide (PI) (markers for apoptosis and necrosis, respectively) after 4 h of incubation, per the manufacturer's instructions.
    NOTE: Irradiating Jurkat T cells in the UV crosslinker at an energy level of 600 µJ/cm², following a 4 h incubation will lead to ≥75% of apoptotic (both early and late) cells having more early apoptotic phenotype than the late apoptotic phenotype. This makes it easier for alveolar macrophages to recognize them and engulf them as their membranes are uncompromised, unlike late apoptotic cells, leading to a higher efferocytic index and more accurate imaging of alveolar macrophage efferocytosis in this study.
    1. Pool 333 µL (1 x 10⁶ cells) of Jurkat T cells from several dishes (both "no UV" and "UV-exposed") together to use for compensation analysis tubes.
    2. Aliquot 333 µL of Jurkat T cells in an unstained, annexin V single stain, PI single stain, no UV control, and 600 µJ/cm² UV-exposed labeled flow cytometry tubes.
    3. Centrifuge tubes at 188 x g for 5 min at RT and decant the supernatant.
    4. Wash cells by resuspending in 500 µL of cold, 1x phosphate-buffered saline (PBS).
    5. Centrifuge and pellet cells at 188 x g for 5 min at RT. Discard the supernatant after centrifugation.
    6. Prepare 400 µL of 1x binding buffer per flow tube by diluting 10x binding buffer with distilled water while cells are centrifuging.
    7. Prepare annexin V and PI incubation reagent (100 µL per sample/tube) per the manufacturer's instructions.
    8. Decant the supernatant after centrifugation and gently resuspend all tubes in 400 µL of 1x binding buffer, then add 100 µL of annexin V incubation reagent to each sample tube. Lastly, add 100 µL of annexin V single stain and PI single stain to their respective tubes, but do not add anything beyond the 1x binding buffer to the unstained tube.
    9. Incubate tubes in the dark for 15 min at RT.
    10. Centrifuge all cells at 188 x g for 5 min at RT and decant supernatant.
    11. Resuspend cells in 400 µL of 1x binding buffer, then analyze samples for apoptosis by flow cytometry. Collect at least 10,000 events per tube to allow accurate representation of staining.
14. Combine all the irradiated cells from dishes into a 50 mL conical tube and pellet cells by centrifugation at 271 x g for 5 min at RT.
15. Discard the supernatant from the tube by aspiration and resuspend cells in 24 mL of sterile phosphate-buffered saline (PBS) and pellet cells by centrifugation at 271 x g for 5 min at room temperature.
16. Discard the supernatant from the tube by aspiration and resuspend cells in the amount of PBS used for dosing mice approved by IACUC. The dose used is between 5-10 x 10⁶ cells/50 µL per mouse; therefore, for 10 mice, resuspend in 500 µL (the number of cells in each dose varies depending on how many cells are cultured for irradiation).
    NOTE: Make at least two additional doses to account for any liquid that may stick to the sides of the pipette tip resulting in the loss of cells.