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Method Article
Cell membrane–shed microparticles (MPs) are active biological vesicles that can be isolated and their pathophysiological effects investigated in various models. Here we describe a method for generating MPs derived from T lymphocytes (LMPs) and for demonstrating their proapoptotic effect on airway epithelial cells.
Interest in the biological roles of cell membrane–derived vesicles in cell–cell communication has increased in recent years. Microparticles (MPs) are one such type of vesicles, ranging in diameter from 0.1 μm to 1 μm, and typically shed from the plasma membrane of eukaryotic cells undergoing activation or apoptosis. Here we describe the generation of T lymphocyte–derived microparticles (LMPs) from apoptotic CEM T cells stimulated with actinomycin D. LMPs are isolated through a multistep differential centrifugation process and characterized using flow cytometry. This protocol also presents an in situ cell death detection method for demonstrating the proapoptotic effect of LMPs on bronchial epithelial cells derived from mouse primary respiratory bronchial tissue explants. Methods described herein provide a reproducible procedure for isolating abundant quantities of LMPs from apoptotic lymphocytes in vitro. LMPs derived in this manner can be used to evaluate the characteristics of various disease models, and for pharmacology and toxicology testing. Given that the airway epithelium offers a protective physical and functional barrier between the external environment and underlying tissue, use of bronchial tissue explants rather than immortalized epithelial cell lines provides an effective model for investigations requiring airway tract tissue.
Microparticles (MPs) are biologically active submicron membrane vesicles released following cell activation or apoptosis. MPs are derived from both healthy and damaged cells and are implicated in many physiological and pathological processes.1 MPs have been detected not only in human plasma, but also in inflammatory and apoptotic tissue. The biological utility of cell membrane–derived MPs has been demonstrated in various settings, including cell signalling models and as pharmacological tools.2,3 We previously demonstrated that LMPs derived from T lymphocytes following actinomycin D stimulation (to induce apoptosis) suppress angiogenesis and inhibit endothelial cell survival and proliferation.4,5 The antiangiogenic effects of LMPs may vary significantly depending on the stimuli used to activate T lymphocytes in vitro.6
The airway epithelium functions as a protective physical and functional barrier. Increased numbers of T lymphocytes in the airway can contribute to cell damage and airway inflammation.7 We have shown that LMPs induce apoptosis of human bronchial epithelial cells,8 which indicated LMPs may change barrier function of bronchial epithelium in vivo. Apoptotic cells can be identified using the TUNEL method, which detects in situ DNA fragmentation.
The overall goal of this protocol is to illustrate the in vitro production of LMPs from a T lymphocyte cell line, and to demonstrate their proapoptotic effect on airway epithelial cells. In situ cell death detection demonstrated that LMPs strongly induce airway bronchial epithelial cell death, suggesting that LMPs-mediated injury to the airway epithelium may impact barrier function of the damaged epithelium.
NOTE: Male C57BL/6 mice (5–7 weeks old) are from Charles River Laboratories International, Inc. (St-Constant, Quebec, Canada.) and manipulated according to protocols approved by the CHU Sainte-Justine Animal Care Committee. Mouse bronchial tissue explants provide a good source of primary bronchial epithelial cells for investigating the proapoptotic effects of LMPs on epithelial cells. This protocol describes the in vitro generation of LMPs, as well as a method for detecting apoptotic epithelial cells on LMPs-treated bronchial tissue explants. This protocol consists of 3 sections.
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 condition, unless otherwise indicated.
1.1) Stimulation and Collection of MPs9
1.2) Characterization of MPs via FACS Analysis 4
1.3) Determination of MP Protein Concentration (Bradford Assay)
2. Bronchial Tissue Explants and LMPs Treatment
NOTE: Pay special attention to the sterile working environment, and aseptically prepare the solutions and medium used in following experiments. To prepare the Complete Healing Medium, add 1 ml of Tissue Healing Medium Supplements with Serum (thawed on ice) to 100 ml Tissue Healing Medium and mix well.
2.1) Preparation of Bronchial Tissue Explants
2.2) LMPs Treatment
3. Histopathological Examination
3.1) Prepare the Following Solutions Before Proceeding to the Next Steps
3.2) Explant Fixation and Tissue Section Deparaffinization
3.3) Hematoxylin and Eosin (H&E) Staining
3.4) In Situ Cell Death Detection: TUNEL Assay
LMPs were characterized with annexin V staining10 by fluorescence-activated cell sorting (FACS) analysis and gated using 1 µm beads in which 97% of MPs (≤1 µm) were annexin-V-Cy5 positive (Figure 1A and 1B). Typically, about 2.5 mg of LMPs were obtained following this protocol. Bronchial tissue explants from C57BL/6 mice were subjected to vehicle and LMPs treatment. Histopathological analysis of bronchial sections revealed the effect of LMPs on the structural integrity of the b...
MPs are active mediators of intercellular cross talk and their study is promising in many areas of science.11 This study presented a detailed protocol for in vitro large-scale generation of LMPs derived from an apoptotic T cell line. These MPs express a large repertoire of lymphocyte molecules and are biologically implicated in the regulation of cellular and tissue homeostasis. However, LMPs derived from different sources may be biologically different.4,9,12,13
L...
The authors have nothing to disclose.
This work is supported by grants from the Canadian Institutes of Health Research (178918), Fonds de recherche en santé du Québec – Vision Health Research Network.
Name | Company | Catalog Number | Comments |
LMPs production and characterization | |||
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 | |
0.22 µm filter | Sarstedt | 83.1826.001 | |
Annexin-VCy5 | BD Pharmagen | 559933 | |
FACS flow solution | BD Bio-sciences | 342003 | |
Fluorescent microbeads (1 µm) | Molecular Probes | T8880 | |
Polysterene counting beads (7 µm) | Bangs laboratories | PS06N/6994 | |
Polypropylene FACS tubes | Falcon | 352058 | |
1 ml pipet | Fisher | 13-678-11B | |
5 ml pipet | Falcon | 357543 | |
25 ml pipet | Ultident | DL-357551 | |
1.5 ml conical polypropylene micro tube | Sarstedt | 72.690 | |
15 ml conical polypropylene tube | Sarstedt | 62.554.205 | |
50 ml conical polypropylene tube | Sarstedt | 62.547.205 | |
50 ml high speed polypropylene copolymer tube | Nalgene | 3119-0050 | |
250 ml high speed polypropylene bottle | Beckman | 356011 | |
Protein assay (Bradford assay) | Bio-Rad Laboratories | 500-0006 | |
Protein assay standard II | Bio-Rad Laboratories | 500-0007 | |
Test tube 16 x 100 | VWR | 47729-576 | |
Test tube 12 x 75 | Ultident | 170-14100005B | |
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 | |
Fow cytometry | BD Bio-sciences | FACS Calibur | |
Spectrophotometer | Beckman | Series 600 | |
Bronchial tissue explants and sections | |||
C57BL/6 mice (5-7 weeks old) | Charles River Laboratories, Inc. | ||
Mouse Airway PrimaCell™ System: | CHI Scientific, Inc. | 2-82001 | |
Rib-Back Carbon Steel Scalpel Blades | Becton Dickinson AcuteCare | 371310 | #10 |
Scalpel Handle | Fine Science Tools Inc. | 10003-12 | #7 |
phase-contrast inverted microscope | Olympus Optical CO., LTD. | CK2 | |
high O2 gas mixture | VitalAire Canada Inc. | ||
modular incubator chamber | Billups-Rothenberg Inc. | MIC-101 | |
MaxQ 4000 incubated orbital shaker | Barnstead Lab-Line, | SHKA4000-7 | |
12-well tissue culture plate | Becton Dickinson and Company | 353043 | |
Plastic tissue culture dishes (100 mm) | Sarstedt, Inc. | 83.1802 | |
Surgical scissors | Fine Science Tools Inc. | 14060-09 | Straight, sharp, 9cm longth |
Half-curved Graefe forceps | Fine Science Tools Inc. | 11052-10 | |
humidified CO2 incubator | Mandel Scientific Company Inc. | SVH-51023421 | |
Histopathological examination | |||
formalin formaldehyde | Sigma-Aldrich, Inc. | HT5011 | |
paraffin | Fisher scientific International, Inc. | T555 | |
ethyl alcohol | Merck KGaA, Darmstadt | EX0278-1 | |
glutaraldehyde | Sigma-Aldrich, Inc. | G6403 | |
Cacodylate | Sigma-Aldrich, Inc. | 31533 | |
microscope slides | VWR Scientific Inc. | 48300-025 | 25x75 mm |
Xylene | Fisher scientific International, Inc. | X5-4 | |
Mayer's hematoxylin | Sigma-Aldrich, Inc. | MHS16 | Funnel with filter paper |
HCl | Fisher scientific International, Inc. | A144s-500 | |
eosin | Sigma-Aldrich, Inc. | HT110116 | Funnel with filter paper |
Permount™ Mounting Medium | Thermo Fisher Scientific Inc. | SP15-100 | |
glass coverslip | surgipath medical industries, Inc. | 84503 | 24×24 #1 |
TUNEL detection kit | In Situ Cell Death Detection, POD | 11 684 817 910 | |
oven | Despatch Industries Inc. | LEB-1-20 | |
rotary Microtome | Leica Microsystems Inc. | RM2145 | |
filter paper | Whatman International Ltd. | 1003150 | #3 |
Microscope | Nikon Imaging Japan Inc. | E800 | |
staining dish complete | Wheaton Industries, Inc. | 900200 | including dish, rack, cover |
1.5 ml eppendorf tube | Sarstedt Inc. | 72.69 | 39x10 mm |
Orbital and Reciprocating Water Bath | ExpotechUSA | ORS200 | |
phosphate buffered saline | GIBCO | 14190-144 | |
fume hood | Nicram RD Service | 3707E |
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