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, numerous reports have shown both pro-inflammatory and anti-inflammatory functions of PMN-MPs⁵, suggesting a potential context-, disease-, species-, and organ-specific role of MPs.

Described protocols in this communication provide a cost-effective, innovative, and adaptable method to study the function of MPs in health and disease. They are applicable to many model organisms, organs, and stimulation conditions. They allow for the identification of several types of MPs and can be used in the future to address their pro-inflammatory and anti-inflammatory functions. As an example, described here is how to study the function of PMN-MPs in epithelial wound healing in vitro and in vivo. The presented protocol for isolation of mouse bone marrow-derived PMNs was adapted with some modifications from a previously described method⁶.

Furthermore, protocols described in this study allow for the detection and characterization of specific markers that can be found on PMN-MPs by two complementary methods: Western blot and flow cytometry. We find that immunoblotting of MPs using standard protocols⁵ is easy and reliable, however, recent advances in sensitivity of flow cytometry instruments, and improved noise-to-signal ratio now allow for further analysis of MPs using this method. The described protocols in this study incorporate recent advances and recommendations from original research articles, including modifications to centrifugation speed and time, the addition of sample filtering and freezing/storage conditions^7,8, and how to reduce the "background noise", improve the detection limit of PMN-MPs, and discriminate between different sizes of MPs.

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); (v) Characterization of MPs by Western Blotting; (vi) Characterization of MPs by Flow Cytometry; (viii) Application of isolated MPs to Study Wound Healing

1. Mouse Bone Marrow Cell Isolation

1. Dissection of the femur and tibia from the mouse hind legs
   1. Prepare a euthanasia box (e.g., an empty 1 mL tip box) for inhalation of anesthesia. Add a few drops of 100% isoflurane onto a sterile cloth taped to the inside of a plastic box lid. As per new IACUC guidelines, animals should not come in direct contact with isoflurane, thus place a tip holder between the mouse and the gauze containing the isoflurane.
      NOTE: Isoflurane is a powerful inhalational anesthetic and should be handled with extreme caution inside a fume hood.
   2. Euthanize mice as per IACUC recommendation. Place a mouse inside the euthanasia box described in step 1.1.1 for 1–5 min until it has ceased breathing. Ensure the animal's death by performing a cervical dislocation.
   3. Lift the abdominal skin using stainless steel, 12 cm curved, 0.17 mm X 0.1 mm tweezers and cut the skin half way between the upper and lower extremities. Peel the skin from here to the most distal section of the mouse body including the lower extremities. With 10 cm long, straight dissecting scissors, remove the muscles from the hind legs and dislocate the hip joint leaving the femur head intact.
   4. Use scissors to dissect the muscles from the femur and the tibia and separate the femur from the tibia. Make sure the ends of the bones remain intact as they contain significant amounts of bone marrow.
   5. Remove all remaining flesh by rolling the bones inside a paper towel. Place the clean bones in a Petri dish containing Serum-Free Medium (SFM, i.e., Dulbecco's Modified Eagle's medium (DMEM) without serum or antibiotics).
   6. Rinse the bones in 70% ethanol and then in SFM.
   7. Prepare 50 mL of SFM in a 50 mL conical tube. Load 10 mL of SFM into a 10 mL syringe and attach it to a 25 and 5/8 inch gauge needle.
   8. Trim the end of the bones with scissors until an opening to the bone marrow (reddish color) is visible.
   9. Flush the bone marrow cells into a new 50 mL tube. Use a 10 mL syringe with a 26 and 5/8 inch gauge needle with approximately 3 mL/bone of SFM. When finished flushing, resuspend the cells in the tube by pipetting (use disposable 1 mL plastic tips) to break cell aggregates.

2. PMN Isolation from Murine Bone Marrow

1. Red cell lysis
   1. Pellet the collected bone marrow by centrifuging at 350 x g for 8 min at 4 °C.
   2. Resuspend the cell pellet in 20 mL of 0.2% NaCl for approximately 20-30 s to lyse the erythrocyte fraction. Do not exceed 30 s as this will result in PMN lysis. Add 20 mL of 1.6% NaCl to restore osmolality.
      NOTE: This step may lead to very little PMN activation.
   3. Wash the cells with 2 mL of PBS and centrifuge as in step 2.1.1 above.Remove the supernatant.
   4. Proceed to isolating PMNs by density gradient centrifugation.
2. Isolation of neutrophils by density gradient centrifugation
   1. Warm polysucrose and sodium diatrizoate solutions, with densities of 1.119 g/mL and 1.077 g/mL (see Table of Materials), to room temperature (RT) before use.
   2. Prepare polysucrose and sodium diatrizoate gradients fresh, immediately prior to bone marrow cell application by slowly layering 3 mL of the 1.077 g/mL density solution over 3 mL of the 1.119 g/mL density solution in 15 mL conical tubes.
      NOTE: Preparation of the gradient solution too far in advance will result in mixing of the layers and poor neutrophil purity and recovery.
   3. Resuspend the bone marrow cell pellet in 1 mL of ice-cold PBS and overlay the resulting cell suspension on top of the polysucrose and sodium diatrizoate gradient (slowly, to avoid mixing of the layers).
   4. Centrifuge for 30 min at 850 x g at 25 °C at RT, without brake. Keep the reagents at RT for effective separation of the neutrophils from other cells present in the bone marrow. Set the centrifuge at slow deceleration to allow for the gradient to be efficiently formed and maintained after the centrifugation step.
   5. Visually locate a mononuclear cell layer at the interface of PBS and 1.077 g/mL density solution (upper layer). Remove this layer and the rest of the density solution without disturbing the PMN band.
   6. Collect an entire PMN layer and some of the underlying polysucrose and sodium diatrizoate 1.119 g/mL solution into 15 mL tubes using a transfer pipette.
      NOTE: The purity of the isolated PMNs will depend on a complete removal of the upper layer.
   7. Top the tubes with filtered PBS (filtered through a 0.1 µm pore size filter) and centrifuge at 300 x g for 5 min, at 4 °C.
   8. Wash the pelleted PMNs with 2 mL of PBS using centrifugation conditions as in step 2.2.7.
   9. Resuspend in 1 mL of filtered PBS and count the isolated PMNs by hemocytometer.
      NOTE: For this isolation method, the resulting PMN purity amounts to ~ 85-90%, as was assessed by flow cytometry. The remaining contaminating fractions consists primarily of mononuclear lymphocytes.
3. PMN stimulation to induce MP release
   NOTE: PMNs can be stimulated to release MPs using various activating conditions, including N-formyl-l-methionyl-leucyl-l-phenylalanine (fMLF), phorbol-12-myristate-13-acetate (PMA), or Interferon gamma (IFNγ). Importantly, prior to simulation, PMNs should be kept on ice at all times.
   1. Pellet PMNs (300 x g, 5 min, 4 °C) and resuspend in 0.1 µm filtered Hank's Balanced Salt solution (HBSS) solution, containing the stimulating agent of choice. For optimal stimulation, use 100 µL stimulation solution per 10 million PMNs for 20-30 min at 37 °C in 15 mL tubes.
      NOTE: The following concentrations of activators were successfully used in our previous work: fMLF(0.5-1 µM for human and 2-5 µM for mouse PMNs), PMA (200 nM), and IFNγ (100 ng/mL). If the release of pre-labeled MPs is desired, incubate unstimulated PMNs (1-5 x 10⁶) with N-(2-aminoethyl) maleimide-FITC (see Table of Materials) (1 µM in 100 µL HBSS, 15 min, 37 °C). Proceed with steps 2.3.1-2.3.3.
   2. Spin down at 850 x g for 5 min at 4 °C, to remove PMNs and transfer cell-free supernatants to new 1.5 mL microcentrifuge tubes.
   3. For MP isolation from cell-free supernatants, continue to step 4.2.

3. PMN Isolation from Human Blood

1. Recruit healthy volunteers for blood extraction according to Institutional IRB guidelines.
   1. Collect blood from the forearm of a healthy volunteer into commercially available 5 mL sodium citrate-containing tubes (see Table of Materials).
   2. Pipette 5 mL of dextran and sodium diatrizoate solution (density of 1.113 g/mL, see Table of Materials) into sterile 15 mL tubes and slowly layer 5 mL of freshly isolated human peripheral blood on top of it.
   3. Centrifuge the tubes for 50 min at 400 x g at RT (25 °C) with low acceleration and no brake.
   4. At the end of the centrifugation, remove the plasma and mononuclear cells (upper layer) using a sterile plastic suction pipette.
   5. Transfer the bottom layer (PMNs) into a new sterile 50 mL conical tube.
   6. Add an equal volume of 0.45% NaCl to PMNs (mix well and gently by rotating the tube).
   7. Centrifuge for 10 min at 400 x g at 25 °C.
2. Red cell lysis
   1. Add 10 mL of ice-cold, sterile water to pelleted cells for 45 s followed by 10 mL of ice-cold sterile 1.8% NaCl to restore osmolality.
      NOTE: All following steps for PMN isolation should be performed on ice to avoid PMN activation and premature MP release. The lysis step itself results in minor but not significant PMN activation, however, it is essential for isolating a pure PMN population for functional assays.
   2. Centrifuge the cells for 10 min at 250 x g at 4 °C with soft deceleration.
   3. Repeat steps 3.2.1 and 3.2.2.
   4. Resuspend PMNs in 5 mL of ice-cold HBSS without calcium or magnesium. Count total live cells (use a hemocytometer and viability staining at 1:2 dilution, see Table of Materials) before stimulation.
      NOTE: Isolated PMNs should be used for stimulation or other experiments within 2 h of isolation to avoid functional and cellular changes and cell death.

4. MP Isolation from Activated PMN Supernatants

NOTE: A similar protocol is used for the isolation of MPs from murine and human PMNs.

1. Pellet PMNs (300 x g, 5 min, 4 °C) and resuspend in 0.1 µm filtered HBSS solution, containing the stimulating agent of choice (for example 1 µM fMLF, see Note: 2.3.1). For optimal stimulation use 100 µL stimulation solution per 10 million PMNs for 20-30 min at 37 °C in 15 mL tubes.
2. Following stimulation, spin down activated PMNs at 600 x g for 5 min at 4 °C and transfer cell-free supernatants to new 1.5-mL microcentrifuge tubes.
3. Centrifuge the PMN-cleared supernatant at 13,000 x g, 10 min, 4 °C to remove cell debris and transfer the cleared supernatant into a new ultracentrifuge tube.
4. Centrifuge for 1 h at 100,000 x g, at 4 °C. Remove supernatants before MP storage. As needed, store pelleted MPs in paraffin sealed ultracentrifuge tubes at -80 °C until use in experiments.

5. Examination of PMN-MPs by Western Blotting

1. Add 1% SDS buffer (50-200 µL with 100 mM Tris pH: 7.4) to MPs pellet (from step 4.4), transfer to new 1.5 mL microcentrifuge tubes and boil the lysed MPs for 5 min.
   NOTE: The number of MPs and the lysis buffer volume must be adjusted and optimized for each protein of interest.
2. Load equal amounts of PMN-MPs per lane in loading buffer containing 10% β-mercapto-ethanol.
   ​NOTE: With our method we load MPs that were isolated from the same number of stimulated PMNs.
   1. Separate the lysates by SDS-PAGE on 10% polyacrylamide gel (use 50-100 V for 1-1.5 h) and transfer total proteins onto nitrocellulose membranes as described⁹.
3. Block membranes for 1 h with 5% non-fat milk in 0.05% Tween-20 Tris-buffered saline, and incubate it with an appropriate primary (overnight at 4 °C), followed by secondary HRP-conjugated antibodies.
4. Visualize the bands by adding chemoluminescence solution to the membrane and exposure to an X-ray film inside a light-proof cassette (1-24 h).

6. Examination of PMN-MPs by Flow Cytometry

1. For antibody staining, dilute antibodies appropriately in FACS buffer (PBS with 0.1 mM EDTA, 0.1% sodium azide). Resuspend pelleted PMN-MPs (derived from 1-3 x 10⁶ PMNs/condition) in 100 µL antibody solution.
   1. If staining for Annexin V, resuspend pelleted PMN-MPs in Annexin V buffer containing 5 µL of FITC-conjugated Annexin V. If co-staining with antibodies, add the desired antibodies (for an example see Table of Materials) directly to the Annexin V solution. Add fluorescent lipid dye, N-(2-aminoethyl) maleimide (see Table of Materials), at 1 µM concentration in 100 µL of FACS buffer to label and identify MPs.
2. Incubate the MPs in staining solution for at least 20 min at 4 °C.
3. Wash the MPs by ultracentrifugation (1 h at 100,000 x g, at 4 °C); discard the supernatant.
4. Resuspend in FACS buffer and run the samples on a designated flow cytometer equipped with an upgraded electronic unit for increased sensitivity (see Table of Materials).

7. Applications for Isolated PMN-MPs to Study PMN Function in Wound Healing

1. In vivo administration of PMN-MPs to colonic wounds
   1. Anesthetize mice by an intraperitoneal injection of a mixture of ketamine (100 mg/kg) and xylazine (5 mg/kg). Confirm anesthesia by pedal reflex (firm toe pinch) and adjust as necessary.
   2. Place the mouse on its stomach and using 28-cm biopsy forceps and an endoscope equipped with a high-resolution camera (such as a veterinary endoscope for small animal use, see Table of Materials), generate 3-5 superficial wounds along the dorsal side of the colon. Upon completion of wounding, return mice to a cage placed on a temperature-controlled (37 °C) mat until recovered.
   3. Anesthetize mice (as in step 7.1.1). Use an endoscope to acquire images of inflicted wounds 24 h (day 1) post-wounding. Let mice recover as in step 7.1.2.
   4. At the same time (24 h post-wounding), administer murine PMN-MPs derived from 2 x 10⁶ PMNs in 100 µL of HBSS+ directly into each wound site using a colonoscopy-based microinjection system (use a 29 G needle)⁹. Three days later (day 4 post-wounding) anesthetize mice (as in step 7.1.1) and use an endoscope to reacquire images of healing wounds. Let mice recover as in step 7.1.2.
   5. Using preferred image analysis software (see Table of Materials), outline visible wound regions in acquired images, measure the area of the same wound at days 1 and 4 post-wounding, and calculate the percentage of wound closure.
2. Human epithelial cells in vitro wound healing
   1. Count Caco-2 BBe or T84, human intestinal epithelial cells by hemocytometer and seed 5 x 10⁵ cells/well in 24-well culture plates. Incubate the cells in a standard incubation chamber at 37 °C and 5% CO₂. Caco-2 BBe or T84 reach confluency within 48 h⁹. To culture epithelial cells, use DMEM and DMEM-F12 (50:50) with supplements as previously described⁹.
   2. Generate mechanical scratch wounds in the monolayer using a pipette tip and low suction as previously described^4,9. Acquire images of wound areas immediately after scratching using an inverted phase-contrast differential interference microscope (use 5X or 10X objectives), to be used as a time = 0 reference point.
   3. Add MPs (derived from 2-4 X 10⁶ PMNs) to scratched-wounded epithelial cell monolayers, and incubate for an additional 24 h (48 h post-wounding, at 37 °C with 5% CO₂). At this time re-acquire images of the scratch-wounds.
   4. Use preferred image analysis software (see Table of Materials) to measure wound areas at t = 0 and t = 48 h. Calculate the percentage of wound closure by determining the difference in wound area at the selected time points.

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

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