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 ....

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 autoclave....

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-.......

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