Isolation of Microparticles Derived from Apoptotic T Lymphocytes In Vitro

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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).

자료

Name	Company	Catalog Number	Comments
CEM T cells	ATCC	CCL-119
X-VIVO 15 medium	Cambrex, Walkersville	04-744Q
Flask T75	Sarstedt	83.1813.502
Flask T175	Sarstedt	83.1812.502
Actinomycin D	Sigma Chemical Co.	A9415-2mg
PBS	Lifetechnologies	14190-144
0.22µm filter	Sarstedt	83.1826.001
Cell incubator	Mandel	Heracell 150
Low speed centrifuge	IEC	Centra8R
High speed centrifuge	Beckman	Avanti J8
High speed rotor for 250ml bottle	Beckman	JLA16.250
High speed rotor for 50ml tube	Beckman	JA30.50