Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Summary

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

Abstract

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50–1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

Introduction

Recently, "microparticles/microvesicles" originating from the cell cytosol or plasma membrane have become of great scientific interest, as emerging data suggest that these structures, ranging from 50-1,000 nm in diameter, can carry biological information and serve as a non-canonical method of cellular communication. Immune cell-derived MPs and particularly those produced by polymorphonuclear neutrophils (PMNs) are of great interest given the important role of PMNs in host defense^1,2, inflammatory responses³, and wound-healing⁴. Intriguingly, thus far, num....

Protocol

All animal work was approved by the Northwestern IACUC. All experiments were completed in accordance and compliance with all relevant regulatory and institutional guidelines. For human subjects donating blood, an informed consent was presented and signed; in addition, all human subjects in this study were treated in accordance with the institutional and federal guidelines for human welfare.

NOTE: The protocol steps are listed under the following subsections: (i) Mouse Bone Marrow Cell Isolation; (ii) PMN Isolation from Murine Bone Marrow; (iii) PMN Isolation from Human Blood; (iv) MP Isolation from PMN Supernatants (by Ultracentrifugation);....

Results

Representative flow cytometric analysis of MPs that were isolated from human and mouse PMNs are shown in Figure 1. The size heterogeneity of PMN-MPs can be assessed by comparison to known sized beads as shown in Figure 1A, B for human MPs. Note, no significant differences in size heterogeneity were observed between mouse and human MPs. Similarly, using flow cytometry and fluorescence labeling, expression of prote.......

Discussion

Protocols for the isolation and characterization of PMN-derived MPs are described in this communication. Several key critical points must be taken into account for the success of the procedure. First, PMNs must be isolated fresh and used in experiments within 2 h of isolation to prevent spontaneous activation and degranulation. All handling of PMNs during isolation and up to the point of stimulation must be performed on ice to prevent activation and premature MP release¹². Second, ultracentrifugat.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Corning	10-041-CV
Fetal Bovine Serum (Heat inactivated)	Atlanta Biologics	S11150
Penicillin/Streptomycin (10,000 units penicillin / 10,000 mg/ml strep)	GIBCO	15140
0.5 M EDTA	Fisher	BP2482
Sodium chloride	Sigma	S9888	Sterile and filtered through a 0.1 micron filter unit
Sterile filtered Histopaque 1077	Sigma	10771
Sterile filtered Histopaque 1119	Sigma	11191	Density 1.119 g/ml.  Solution of polysucrose and  sodium diatrizoate. Bring to room temperature
Phosphate Buffered Saline (PBS) without Calcium and Magnesium	Cellgro	210-40-CV
Ethyl Alcohol (200 proof)	Decon labs	2701
HBBS	Corning	21-022-CV
Trypan Blue Solution, 0.4%	Sigma	T8154 SIGMA	Diluted 1:2 for cell viability counting
Isoflurane	Abbot	50033
Anti-human CD11b-APC conjugated	Biolegend	301350	Clone ICRD44 (Final concetration: 1 µg/mL)
BODIPY FL	Invitrogen	B10250	N-(2-aminoethyl) maleimide
Annexin V Binding Buffer	Biolegend	422201
Calibration Beads for Flow Cytometry	BioCytex	7803
FITC Annexin V	Biolegend	640906
Polymorphprep	Axis-Shield PoC AS		Sodium diatrizoate/Dextran 500, density 1.113 g/ml
C57BL/6 mice	Jackson Labs
15 ml centrifuge tubes	Corning	430053
50 ml centrifuge tubes	BD Falcon	352070
25 ml serological pipettes	Celltreat	229225B
10 ml serological pipettes	Celltreat	229210B
5 ml serological pipettes	Celltreat	229205B
Pasteur pipettes	BD Falcon	357575
25 G x 5/8 in. Needles (precision glide needles)	BD	305122
100 µm cell strainers	Celltreat	229485
Vacutainer tubes	BD	367251
Equipment
LSR Fortessa Special Order Research Product (SORP)	BD	(SORP)
Swinging bucket centrifuge	ThermoFisher Scientific	75007210
Ultracentrifuge	Beckman	(L8-80 M)
Micro-centrifuge	ThermoFisher Scientific	VV-17703-15 (Fresco 17)
Swinging bucket centrifuge	ThermoFisher Scientific	75004503 (Megafuge 40R)
Biopsy forceps, 28 cm	Storz	27071zj
Software
Image J	National Institute of Health	Open source