Isolation of Microparticles Derived from Apoptotic T Lymphocytes In Vitro
Transcription
To begin, culture CEM T cells in four T175 flasks, each containing 150 milliliters of fresh medium, and incubate at 37 degrees Celsius to a density of 2 million cells per milliliter.
Collect the cells from each flask by centrifugation at 200 times g for 5 minutes and resuspend 300 times 10 to the 6 cells into a new T175 flask containing 150 milliliters of fresh medium, to maintain the 2 million cells per milliliter density. Add 37.5 microliters of actinomycin D to the medium at a final concentration of 0.5 micrograms per milliliter, and incubate at 37 degrees Celsius for 24 hours.
Transfer all the culture medium into 50-milliliter conical tubes, and spin down the cells at 750 times g for 5 minutes. Then, transfer the supernatant into 50-milliliter conical tubes and centrifuge at 1,500 times g for 15 minutes to remove large cell fragments.
Next, transfer the supernatant into a 250-milliliter bottle, and ultracentrifuge at 12,000 times g for 50 minutes. Then, discard the supernatant and collect the pellets. Use 40 milliliters of sterile PBS to wash the LMP-enriched pellets in a 50-milliliter tube and centrifuge at 12,000 times g for 50 minutes. Repeat this step twice.
Collect the supernatant from the last wash to be used as a vehicle control, with 1 milliliter of PBS. Suspend the pellets and transfer into a 1.5-milliliter sterile microtube. Aliquot and store isolated LMPs at negative 80 degrees Celsius.
