Rapid Magnetic-microbead Method for Efficient Purification of Low-density Neutrophils

Summary

Low-density neutrophils (LDN) increase significantly in several diseases. LDN are usually isolated through cell sorting. We present a practical method to obtain pure and viable LDN. After density-gradient centrifugation of peripheral blood, cells are incubated with magnetic microbeads, and then LDN are separated through magnetic columns.

Abstract

Neutrophils, important leukocytes of innate immunity, have traditionally been considered a homogeneous cell population. Nevertheless, recent evidence has shown that neutrophils exist in several subpopulations. One such subpopulation is low-density neutrophils (LDN). LDN are found in small numbers in the blood of healthy individuals, but their numbers increase significantly in diseases such as systemic lupus erythematosus, autoimmune disorders, cancer, and infections. In these cases, LDN may participate in the pathogenesis of the disease. The only way to isolate LDN is through density-gradient centrifugation of peripheral blood. However, after centrifugation, LDN co-purify with mononuclear cells. Thus, studying this neutrophil subpopulation is challenging. There is no standard methodology to separate LDN from mononuclear cells. Typically, LDN are separated by cell sorting in a flow cytometer. However, this method requires long sorting times (hours) to obtain enough pure cells for further functional studies. This seriously affects the viability and function of cells. Here, we propose a practical method to obtain large numbers of pure and viable LDN in a short time. After density-gradient centrifugation, the mononuclear cell fraction is incubated with anti-CD66b magnetic microbeads, and then LDN are separated through magnetic columns in < 30 min. Purified LDN (CD66b⁺ cells) are labelled with monoclonal antibodies against CD10, CD11b, CD14, CD15, CD16b, CD33, CD62L, CD66b, and CD98, and are analyzed by flow cytometry for confirmation. Purified LDN are completely functional as indicated by their capacity to produce reactive oxygen species, and to form neutrophil extracellular traps. This new purification method results in LDN with high purity (more than 90%) and viability (more than 96%) in a short time period. This method can easily be scaled up to obtain large numbers of pure LDN to evaluate LDN functions in different diseases through biochemical or other omics analysis.

Introduction

Neutrophils, the predominant leukocytes in human blood¹, are key participants of the innate immune system. They arrive in large numbers to tissues with inflammation or infection². There, neutrophils activate several effector functions, such as phagocytosis^3,4, degranulation^5,6, and formation of neutrophil extracellular traps (NETs)⁷. Additionally, neutrophils also participate in the adaptive immune response⁸. The classical view of neutrophils considers them as homogeneous cells produced in the bone marrow with predetermined responses^9,10. However, recent studies reveal that neutrophils are heterogeneous cells with multiple phenotypic and functional states under both healthy and disease states^{11,12,13,14,15}.

Among several neutrophil subpopulations, the low-density neutrophils (LDN) have attracted much interest because of their intrinsic properties and because they increase in numbers in several diseases^16,17,18. LDN were found in the blood of systemic lupus erythematosus (SLE) patients in 1986¹⁹. They were detected following the process for separating leukocytes in a density gradient^20,21. After centrifugation of blood or leukocyte-rich plasma on a density medium (e.g., Ficoll-Paque), monocytes and lymphocytes (known as peripheral blood mononuclear cells [PBMC]) form a band in the upper (low-density) part of the tube. Neutrophils sediment at the bottom of the tube. Among the PBMC, a few neutrophils were also found; these are LDN (Figure 1). Small numbers of LDN are in the blood of healthy individuals²². However, LDN numbers increase significantly in multiple immunosuppression and chronic inflammation conditions, including SLE^23,24, sepsis²⁵, psoriasis²⁶, asthma²⁷, juvenile idiopathic arthritis²⁸, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis²⁹, HIV infection³⁰, malaria³¹ and tuberculosis³². Although LDN numbers increase in all the mentioned pathologies, LDN have been mostly studied in the context of SLE¹⁷. LDN from the blood of SLE patients seems to have a greater capacity to form NETs³³, to secrete large amounts of proinflammatory mediators³⁴, and to activate T cells²⁴. However, in other conditions, such as cancer, LDN are reported to consist of mature and immature neutrophils³⁵, with T-cell suppressive properties^36,37,38. Similarly, in COVID-19 patients, LDN are also reported to suppress T cell proliferation³⁹, but contrary to SLE, LDN seems to produce fewer NETs³⁹. Therefore, the origin, composition, and functional properties of LDN are still controversial.

Because LDN are found together with PBMC, studying them is technically complicated. To fully explore LDN properties in different pathologies, it is necessary to purify them. A common procedure of LDN separation is fluorescent cell sorting^{22,40,41,42,43,44,45}. However, cell sorting requires a long time for cell separation. This may lead to cell loss or low recovery if the sorting time is not sufficient. In addition, cells are subjected to excessive stress, causing variable results while studying the function of these cells. Long sorting time also increases the cost of experimental procedures and requires specialized personnel.

Here, we present a rapid and efficient method for obtaining large numbers of pure and viable human LDN in a very short time. After density-gradient centrifugation, LDN are separated by magnetic cell sorting (MACS; Figure 1). The main advantage of this purification method is that LDN are separated with high purity (more than 90%) and viability (more than 96%). Also, purified LDN are completely functional as indicated by their capacity to generate reactive oxygen species (ROS) and to release NETs. This protocol can be easily implemented in any laboratory interested in neutrophil biology to quickly separate LDN from the blood of people with multiple diseases in order to further study these cells.

Protocol

All procedures in this protocol follow the guidelines of the Human Research Bioethics Committee at Instituto de Investigaciones Biomédicas - Universidad Nacional Autónoma de México (UNAM). All participants provided informed consent.

1. Isolation of peripheral blood mononuclear cells (PBMC) and neutrophils from human blood

1. Obtain about 10 mL of blood from a healthy adult volunteer by venipuncture. Add 10 U/mL of heparin as an anticoagulant.
   ​CAUTION: Dispose of the needle into a biohazard needle disposal container. Syringes and other materials in touch with blood should be placed in a bag and autoclaved before disposal.
2. In a 15 mL conical centrifuge tube, add 2 mL of 6% dextran T500 in PBS. Then add 10 mL of blood by draining it down the side of the tube. Invert the tube a couple of times to mix the blood and dextran.
3. Let the tube rest for 45 min, while erythrocytes sediment. The leukocyte-rich plasma appears above the erythrocyte layer.
4. In another 15 mL centrifuge tube, add 5 mL of density gradient medium. Carefully pipette the plasma, without touching the erythrocytes, and layer it on top of the medium. Two separate phases must be formed.
5. Centrifuge the tube at 516 x g for 20 min at 4 °C. Mononuclear cells (PBMC) form a band between plasma and the medium layers. Neutrophils form a pellet at the bottom (Figure 1).
6. Isolation of PBMC
   1. Eliminate by aspiration the plasma on top of the PBMC without touching the cells. Collect the cells from the band between the plasma and the medium. Make sure to aspirate as little medium as possible.
   2. Place cells into a 50 mL conical centrifuge tube. Add 20 mL of PBS. Centrifuge the tube at 400 x g for 5 min at 4 °C.
   3. Carefully aspirate the supernatant and scrape the tube to separate the cells. Add 10 mL of cold PBS to resuspend the cells. Count the PBMC using a Neubauer chamber.
      NOTE: Do not use a pipette to resuspend the cells, because this may damage the cells.
7. Isolation of neutrophils
   1. Remove the density gradient medium. Separate the cells by scraping the tube and add 10 mL of cold PBS.
   2. Put the cells into a fresh 50 mL centrifuge tube and centrifuge at 400 x g for 5 min at 4 °C. Aspirate the supernatant and separate the cells as described in step 1.6.3.
8. To eliminate residual erythrocytes, add 10 mL of cold hypotonic solution (0.2% NaCl, 1% BSA, 20 mM Hepes, pH = 7.4), and mix gently for exactly 1 min.
9. Quickly add 10 mL of cold hypertonic solution (1.6% NaCl, 1% BSA, 20 mM HEPES, pH = 7.4) to make the solution isotonic.
10. Count the neutrophils using a Neubauer chamber (purity > 95%). Pellet the cells by centrifuging as in step 1.6.2. and resuspend them in cold PBS. Maintain the tube on ice.
    NOTE: It is reported that the half-life of neutrophils in vitro is 8 - 12 h^46,47,48. In our experience with this protocol, neutrophils maintain their functions for approximately 6 to 8 h.

2. Purification of low-density neutrophils (LDN)

1. Centrifuge PBMC at 400 x g for 5 min at 4 °C. Remove supernatant and resuspend cells in 120 µL of cold wash buffer (1% BSA in PBS).
2. Add 35 µL of CD66b magnetic microbeads. Incubate the mixture in the dark at 4 °C for 30 min. Mix gently every 10 min to prevent cell aggregation.
3. Add 1 mL of cold wash buffer. Place cells in a microcentrifuge and spin at 400 x g for 3 min.
4. Remove supernatant, separate the cell pellet by scraping the tube, and resuspend cells in 1 mL of wash buffer.
5. Place a magnetic separation column onto a magnet. Add 0.5 mL of wash buffer to the column and let it pass through the column.
6. Transfer cells (1 mL) onto the column. Let the buffer pass through the column drop by drop.
7. Add 0.5 mL of wash buffer into the column and let it pass through. Add another 0.5 mL of wash buffer into the column.
8. Remove the column from the magnet and put it in a microcentrifuge tube. Add 1 mL of wash buffer to the column.
9. Insert the plunger on top of the column and gently apply pressure to elute the cells. Remove the plunger and put the column on top of a new microcentrifuge tube.
10. Add another 1 mL of wash buffer. Insert the plunger on top of the column and gently apply pressure to elute the cells.
11. Place both tubes in a microcentrifuge and spin them at 800 x g for 3 min. Finally, resuspend the cells (LDN) from both tubes into 1 mL of cold PBS. Keep on ice.
    NOTE: The first 2 mL of flow-through contains the rest of the PBMC. These cells can be used for other studies.

3. Multicolor flow cytometry

1. Resuspend purified cells at 1 x 10⁶ cell/mL in labeling buffer (1% FBS in PBS). Add 250 µL of cells into a 1.5 mL microcentrifuge tube.
2. Add the corresponding antibodies against neutrophil membrane molecules (for details, see Table of Materials). Incubate the cells for 30 min at 4 °C, protecting them from light.
3. Add 1 mL of PBS. Place tubes in a microcentrifuge and spin them at 800 x g for 3 min.
4. Aspirate the supernatant, break the cell pellet by tapping the tube, and resuspend cells in 0.5 mL of 1% paraformaldehyde.
5. Keep the cells at 4 °C protected from light, until analyzed by flow cytometry. Analyze cells by flow cytometry, capturing 10,000 events per sample. Perform cell sorting as previously described²².
   CAUTION: Paraformaldehyde is an irritant to the skin and the respiratory tract. Make sure you do not breathe the vapors.

4. Detection of reactive oxygen species (ROS)

1. In a 1.5 mL microcentrifuge tube, add 2.5 x 10⁵ cells. Place tubes in a microcentrifuge and spin at 800 x g for 3 min.
2. Aspirate the supernatant, scrape the tube to separate the cells, and resuspend the cells in 100 µL of 15 µM dihydrorhodamine 123 in PBS.
3. Incubate the cells protected from light at 37 °C for 20 min. Add 1 mL of PBS.
4. Place tubes in a microcentrifuge and spin at 800 x g for 3 min. Aspirate the supernatant, scrape the tube to separate the cells, and resuspend them in 100 µL of 50 nM phorbol 12-myristate 13-acetate (PMA) in PBS.
5. Incubate the cells protected from light at 37 °C for 45 min. Add 1 mL of PBS.
6. Place tubes in a microcentrifuge and spin them at 800 x g for 3 min. Aspirate the supernatant, scrape the tube to separate the cells, and resuspend them in 0.5 mL of 1% paraformaldehyde in PBS.
7. Keep the cells at 4 °C protected from light until analyzed by flow cytometry. Analyze cells by flow cytometry, capturing 10,000 events per sample.

5. Visualization of neutrophil extracellular traps (NETs)

1. Place coverslips in wells of a 12-well plate and cover them with 10 µg/mL poly-L-Lysine. Incubate the plate overnight at 4 °C with gentle agitation.
2. Remove the poly-L-Lysine, and wash the coverslips with PBS, incubating the plate for 5 min at room temperature with gentle agitation. Wash the coverslips with PBS two more times.
3. Remove the PBS and let the coverslips dry by placing the plate inside a laminar-flow hood for 2 h.
4. Resuspend LDN at 1 x 10⁶ cell/mL in RPMI-1640 medium with 5% FBS. Take 350 µL (3.5 x 10⁵ cells) of the cell suspension and put them into a well of the 12-well plate containing the coverslips.
5. Keep the plate for 30 min in a 5% CO₂ incubator at 37 °C. Add 3.5 µL of 5 µM PMA diluted in PBS to each well (final concentration of PMA is 50 nM).
6. Keep the plate for 4 h in a 5% CO₂ incubator at 37 °C. Add 350 µL of 8% paraformaldehyde in PBS, and keep the plate overnight in a 5% CO₂ incubator at 37 °C.
7. Place a transparent film over a test tube stand as previously described⁴⁹, so that wells are formed on the tube holes.
8. Fill each well with the various solutions to wash or stain the coverslips, forming a big drop. Remove the coverslips and put them upside down on a drop of water for 5 min. In the same manner, wash the coverslips three more times with water.
9. Place the coverslips upside down on a drop of blocking buffer (5% BSA in PBS) and incubate for 20 min at room temperature.
10. Transfer the coverslips to a drop of the corresponding primary antibody (e.g., anti-elastase or anti-citrulline) in blocking buffer and incubate for 60 min at room temperature.
11. Wash coverslips 2x in 0.01% Tween-20 in PBS. Transfer the coverslips to a drop of the corresponding secondary antibody in blocking buffer containing 150 nM DAPI, and incubate for 60 min at room temperature, protected from light.
12. Wash coverslips 2x in 0.01% Tween-20 in PBS. Place a drop of antifade mounting medium onto a glass slide and put coverslips upside down.
13. Seal coverslips around the perimeter with nail polish. Store mounted slides at 4 °C, protected from light. Visualize NETs using a fluorescence microscope.

6. Detection of neutrophil extracellular traps (NETs)

1. Resuspend cells at 5 x 10⁵ cell/mL in 500 nM Sytox Green, diluted in RPMI-1640 medium with 5% FBS.
2. Take 100 µL (5 x 10⁴ cells) of the cell suspension and put them into a well of a 96-well tissue culture plate.
3. Keep the plate for 20 min in a 5% CO₂ incubator at 37 °C. Add to each well 20 µL of 300 nM PMA diluted in RPMI-1640 medium (final concentration of PMA is 50 nM).
4. Transfer the plate to a pre-warmed microplate reader. Incubate the plate at 35 °C for 4 h, taking fluorescence measurements from the bottom of the plate every 5 min (using the 485 nm excitation and 528 nm emission filters).

Results

Low-density neutrophils (LDN) in healthy individuals represent around 5% of the PBMC (Figure 2A). The protocol described here typically delivers pure (> 90%) LDN. Cells are also efficiently recovered (yield around 98%) with high viability (> 95%; Figure 2B). For comparison, LDN were also purified by fluorescent-activated cell sorting as previously described²². Briefly, PBMC were labelled with anti-CD...

Discussion

Generally, neutrophils were thought of as homogeneous cells. However, recent evidence has shown that neutrophils could exist as cells with different activation states and/or multiple phenotypes. Thus, various neutrophil subpopulations may exist^13,14,15. One neutrophil subpopulation, the low-density neutrophils (LDN), has acquired great interest due to their particular intrinsic properties and also because they increase in multip...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-Citrulline rabbit polyclonal antibody	AbCam	ab100932	1/50 dilution
96-well tissue culture plate	Costar; Corning, Inc.	3596
Alexa Fluor 488 anti-human CD11b mouse IgG1 antibody	BioLegend	301317	0.25 μg/mL
Alexa Fluor 647 anti-human CD66b mouse IgM antibody	BioLegend	305109	0.05 μg/mL
Anti-Mouse IgG (H+L) goat antibody, Alexa Fluor 488	Invitrogen - Thermo Fisher Scientific	A-11001	1/50 dilution
Anti-Neutrophil Elastase mouse monoclonal IgG1 antibody	Santa Cruz Biotechnology	sc-55549	10 µg/mL
Anti-Rabbit IgG (H+L) donkey antibody, Alexa Fluor 555	Invitrogen - Thermo Fisher Scientific	A-31572	1/50 dilution
APC anti-human CD62L mouse IgG1 antibody	BioLegend	304809	0.03 μg/mL
APC/Cyanine7 anti-human CD14 mouse IgG1 antibody	BioLegend	367107	0.25 μg/mL
Attune NxT flow cytometer	Thermo Fisher Scientific, Inc.	(blue/red lasers)
Bovine serum albumin (BSA) Fraction V	MP Biomedicals	810025
Brilliant Violet 510 anti-human CD33 mouse IgG1 antibody	BioLegend	366609	0.30 μg/mL
DAPI	Calbiochem/EMD Millipore	268298
Dextran T500	Pharmacosmos A/C	5.51005E+11
Dihydrorhodamine-123	Anaspec, Inc.	AS-85711
FACS cell sorter	Becton Dickinson	Model FACSAria
Fetal bovine serum (FBS)	Gibco	16000-044
Ficoll-Paque	Merck KG	GE 17-1440-03
FITC anti-human CD98 mouse IgG1 antibody	BioLegend	315603	0.30 μg/mL
FlowJo Software	Becton Dickinson	Version 10, 2019
Fluorescence inverted microscope	Olympus	Model IX-70
Heparin	Inhepar - PiSA Farmaceutica	3660149	5000 UI/mL
Hepes	Sigma Aldrich	H7006
MACSprep Chimerism CD66b Microbeads	Miltenyi Biotec	130-111-552
Magnet	Miltenyi Biotec	130-042-102
Magnetic separation column	Miltenyi Biotec	130-042-201
Microcentrifuge	Eppendorf	Model 5415C
Microplate reader	BioTek Instruments	Model Synergy HT
Paraformaldehyde	Sigma Aldrich	158127
PE anti-human CD10 mouse IgG1 antibody	BioLegend	312203	0.05 μg/mL
PE anti-human CD16b mouse IgG2a antibody	BD Pharmingen	550868	1/40 dilution
PE/Cyanine5 anti-human CD15 mouse IgG1 antibody	BioLegend	323013	0.25 μg/mL
Phorbol 12-myristate 13-acetate (PMA)	Sigma Aldrich	P8139
poly-L-Lysine	Sigma Aldrich	P2658
Refrigerated centrifuge	Eppendorf	Model 5702R
RPMI-1640 tissue culture medium	Gibco	23400-021
SYTOX Green	Molecular Probes, Inc.	S-7020
Tween-20	Sigma Aldrich	P2287
Vectashield	Vector Laboratories	H-1000