1. LMPs Production and Characterization

NOTE: To prevent contamination, ensure that all materials used in this experiment are sterile or autoclaved. Perform all steps at RT in a biological safety cabinet under sterile conditions, unless otherwise indicated.

1. Stimulation and Collection of MPs
   1. Thaw an aliquot of 10 million CEM T cells in a 37 °C water bath. Dilute in 10 ml pre-warmed serum-free hematopoietic medium such as X-VIVO, in a 15 ml sterile tube and centrifuge at 200 g x 5 min. Aspirate the supernatant and resuspend cells in a 5 ml pre-warmed medium.
   2. Transfer cells into a T75 tissue culture flask (for suspension cells) with 15 ml pre-warmed hematopoietic medium such as X-VIVO and incubate for 4 days in a humidified incubator at 37 °C with 5% CO₂.
   3. After 4 days, transfer all the culture medium and cells into a T175 tissue culture flask containing 100 ml fresh medium. Continue incubating the cells for about 72 hr under the same conditions until they have grown to a density of 2 million cells/ml.
   4. Evenly split cells between four T175 flasks each containing 150 ml fresh medium and continue cell culture until cells have grown (approximately 48 hr incubation) to a density of 2 million/ml.
   5. Collect cells from each flask by centrifugation at 200 x g for 5 min and resuspend 300 x 10⁶ cells into a new T175 flask containing 150 ml fresh medium, to maintain the 2 million/ml cell density.
   6. Add actinomycin D (dissolved in DMSO at 2 mg/ml) to the medium at a final concentration of 0.5 µg/ml and incubate for 24 hr.
   7. Transfer all the culture medium into 50 ml conical tubes and spin down the cells at 750 x g for 5 min. Transfer the supernatant into 50 ml conical tubes and centrifuge at 1,500 x g for 15 min to remove large cell fragments.
   8. Transfer the supernatant into a 250 ml bottle and ultracentrifuge at 12,000 x g for 50 min. Discard the supernatant and collect pellets.
   9. Wash LMPs-enriched pellets with 40 ml sterile PBS in a 50 ml tube by centrifugation at 12,000 x g for 50 min. Repeat this step twice.
   10. Collect the last wash supernatant; it will be used as vehicle control. Suspend the LMP pellets in 1 ml of PBS and transfer them into a 1.5 ml sterile microtube. Aliquot and store isolated LMPs at -80 °C (to avoid multiple free-thaw cycles).