Isolation of Microparticles Derived from Apoptotic T Lymphocytes In Vitro

Cellular microparticles are submicron-sized membrane-bound vesicles that enclose biomolecules released from the plasma membrane of eukaryotic cells undergoing apoptosis.

To generate T lymphocyte-derived microparticles, LMPs in vitro, take a culture flask containing T lymphocyte culture. Add actinomycin-D, an antibiotic. Incubate for an appropriate duration.

Actinomycin-D enters the lymphocytes and translocates to the nucleus. It intercalates between specific DNA base pairs, inhibiting transcription. This leads to cell cycle arrest and activates the apoptotic pathway.

At the onset of apoptosis, the activated caspase-3 cleaves and activates rho-associated kinase, ROCK1. ROCK1, in turn, phosphorylates its downstream target proteins, causing actomyosin contraction, leading to membrane blebbing and the subsequent release of LMPs containing the cytosolic and nuclear components into the medium.

The cells eventually undergo apoptosis.

Post-incubation, transfer the culture medium to a fresh tube. Centrifuge to pellet the T lymphocytes. Transfer the supernatant to a fresh tube.

Centrifuge to remove large cell debris. Transfer the supernatant containing LMPs to a bottle. Perform high-speed ultracentrifugation to pellet the LMPs. Resuspend the LMPs in a buffer. Store at low temperatures until further analysis.

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.