Isolation of Microparticles Derived from Apoptotic T Lymphocytes In Vitro

Abstract

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

Protocol

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.
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Materials

List of materials used in this article

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