Name: isolation of microparticles derived from apoptotic t lymphocytes in vitro
Uploaded: 2023-08-31T09:44:28.000+00:00
Duration: 1 min 53 s
Description: Source: Yang, C. et al., Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells. J. Vis. Exp. ...

isolation of microparticles derived from apoptotic t lymphocytes in vitro

Isolation of Microparticles Derived from Apoptotic T Lymphocytes In Vitro

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.

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1.&#160;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.
<ol>
	<li>Stimulation and Collection of MPs
	<ol>
		<li>Thaw an aliquot of 10 million CEM T cells in a 37 &#176;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.</li>
		<li>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 &#176;C with 5% CO2.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Add actinomycin D (dissolved in DMSO at 2 mg/ml) to the medium at a final concentration of 0.5 &#181;g/ml and incubate for 24 hr.</li>
		<li>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.</li>
		<li>Transfer the supernatant into a 250 ml bottle and ultracentrifuge at 12,000 x g for 50 min. Discard the supernatant and collect pellets.</li>
		<li>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.</li>
		<li>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 &#176;C (to avoid multiple free-thaw cycles).</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>CEM T cells</td><td>ATCC</td><td>CCL-119</td><td></td></tr><tr><td>X-VIVO 15 medium</td><td>Cambrex, Walkersville</td><td>04-744Q</td><td></td></tr><tr><td>Flask T75</td><td>Sarstedt</td><td>83.1813.502</td><td></td></tr><tr><td>Flask T175</td><td>Sarstedt</td><td>83.1812.502</td><td></td></tr><tr><td>Actinomycin D</td><td>Sigma Chemical Co.</td><td>A9415-2mg</td><td></td></tr><tr><td>PBS</td><td>Lifetechnologies</td><td>14190-144</td><td></td></tr><tr><td>0.22&amp;micro;m filter</td><td>Sarstedt</td><td>83.1826.001</td><td></td></tr><tr><td>Cell incubator</td><td>Mandel</td><td>Heracell 150</td><td></td></tr><tr><td>Low speed centrifuge</td><td>IEC</td><td>Centra8R</td><td></td></tr><tr><td>High speed centrifuge</td><td>Beckman</td><td>Avanti J8</td><td></td></tr><tr><td>High speed rotor for 250ml bottle</td><td>Beckman</td><td>JLA16.250</td><td></td></tr><tr><td>High speed rotor for 50ml tube</td><td>Beckman</td><td>JA30.50</td><td></td></tr></tbody></table>

Source: Yang, C. et al., <a href="https://app.jove.com/v/52651">Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells.</a> J. Vis. Exp. (2015)
This video demonstrates the generation and isolation of T lymphocyte-derived microparticles, LMPs, from T lymphocytes following treatment with actinomycin-D.

Source: Yang, C. et al., Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells. J. Vis. Exp. ...

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.

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Isolation of Microparticles Derived from Apoptotic T Lymphocytes In Vitro

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Isolation of Microparticles Derived from Apoptotic T Lymphocytes In Vitro