Isolation of Microparticles Derived from Apoptotic T Lymphocytes In Vitro
Transcrição
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.
