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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present three methods to assess neutrophil migration and infiltration both in vivo and in vitro. These methods can be used to discover promising therapeutics targeting neutrophil migration.

Abstract

Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. Neutrophils can be recruited to inflammation sites via the guidance of cytokines and chemokines. Overwhelming infiltration of neutrophils can lead to indiscriminate tissue damage, such as in rheumatoid arthritis (RA). Neutrophils isolated from peritoneal exudate respond to a defined chemoattractant, N-formyl-Met-Leu-Phe (fMLP), in vitro in Transwell or Zigmond chamber assays. The air pouch experiment can be used to evaluate the chemotaxis of neutrophils towards lipopolysaccharide (LPS) in vivo. The adjuvant-induced arthritis (AA) mouse model is frequently used in RA research, and immunohistochemical staining of joint sections with anti-myeloperoxidase (MPO) or anti-neutrophil elastase (NE) antibodies is a well-established method to measure neutrophil infiltration. These methods can be used to discover promising therapies targeting neutrophil migration.

Introduction

Neutrophils are the most abundant white blood cell and account for 50−70% of the whole white blood cell population in humans1. Neutrophils are one of the primary responders during acute inflammation. Neutrophils can be recruited to inflammation sites via the guidance of cytokines and chemokines released by tissue-resident cells2,3,4, which is mediated by the interactions between cell adhesion molecules on the surface of neutrophils and vascular endothelium cells5. Neutrophils are fundamental to host defense and play a role....

Protocol

All experimental procedures were reviewed and approved by the Beijing University of Chinese Medicine Animal Care and Use Committee.

NOTE: C57BL/6 mice (7-8 weeks old) were used.

1. Neutrophil isolation

  1. Acquisition of peritoneal exudate cells
    1. Prepare fresh 10% proteose peptone solution in ddH2O. Calculate the volume needed according to the number of mice.
      NOTE: Set the number of mice as N, (2N+1) mL of solution must be disso.......

Representative Results

Peritoneal exudate cells were collected from lavage fluid of mice. Cells were resuspended in 1 mL of RPMI-1640 complete medium, layered onto a two-step (54.8%/70.2%) discontinuous density gradient (Figure 1A), and centrifuged at 1,500 x g for 30 min. Neutrophils (≥95%, ~1 x 107 neutrophils/mouse) were recovered from the lower interface (Figure 1B).

Discussion

Detailed protocols of highly-purified neutrophils from peripheral blood7, bone marrow and tissues18 have been available for a long time. Here we adopt a method of isolating neutrophils from peritoneal fluid19 in which mature neutrophils remain inactivated for further anti-inflammatory and antioxidant studies.

We used the air pouch experiment to explore the LPS-induced infiltration of neutrophils in vivo. This method has be.......

Acknowledgements

This work was supported by the National Natural Science Foundation of China (grant numbers 81430099 and 31500704), International Cooperation and Exchange Projects (grant number 2014DFA32950), and the research program of Beijing University of Chinese Medicine (grant numbers BUCM-2019-JCRC006 and 2019-JYB-TD013).

....

Materials

NameCompanyCatalog NumberComments
0.1% Fast Green SolutionSolarbio8348bTransfer 20 mg of fast grene FCF in one vial into another 100 mL beaker. Add 20 mL of H2O into the beaker and dissolve the stain by stirring to make 0.1% fast green solution, and filter it using a Nalgene PES 75mm filter
0.1% Safranin O Staining SolutionSolarbio8348aTransfer 20 mg of safranin O stain in one vial into a 100 ml beaker. Add 20 mL of H2O into the beaker and dissolve the stain by stirring to make 0.1% safranin O staining solution, and filter it using a Nalgene PES 75mm filter
0.5 M Ethylenediaminetetraacetic acid solution (EDTA), pH 8.0Sigma324506Sterile
100% EthanolBeijing Chemical Works
100% MethanolBeijing Chemical Works
15 mL Conical Polypropylene Centrifuge TubeFalcon14-959-53A
23 G x 1 1/4" NeedleBD305120
26 G x 3/8" NeedleBD305110
3% bovine serum albumin (BSA)Dissolve 0.3 g BSA in 10 mL PBS
3% H2O2Mix 1 mL 30% H2O2 with methanol with 9 mL methanol
3,3'-diaminobenzidine (DAB) kitZSGB-BIOZLI-9018
30 G x 1/2" NeedleBD305106
30% H2O2Beijing Chemical Works
5 mL SyringeBDZ683574
50 mL Conical Polypropylene Centrifuge TubeFalcon14-432-22
50% EthanolMix 500 mL 100% ethanol with 500 mL dH2O
54.8% PercollMix 2.74 mL SIP with 2.26 mL 1×PBS, stand still
70% EthanolMix 700 mL 100% ethanol with 300 mL dH2O
70.2% PercollMix 3.51 mL SIP with 1.49 mL 1×PBS, stand still
80% EthanolMix 800 mL 100% ethanol with 200 mL dH2O
95% EthanolMix 950 mL 100% ethanol with 50 mL dH2O
Acid Alcohol Superfast Differentiation SolutionBeyotimeC0165S
ANTIBODIES
Anti-Myeloperoxidase AntibodyAbcamab208670
Anti-Neutrophil Elastase AntibodyAbcamab21595
Automatic Hematology AnalyzerSysmexXS-800i
Bovine Serum Albumin (BSA)VWR0332-100G
Complete Freund's Adjuvant, 10 mg/mlsigma1002036152
Cover SlipCITOGLAS10212432C
Dial Thickness GaugeMitutoyo7301
Eppendorf Microtubes, 1.5 mLSigmaZ606340
Foetal Bovine Serum (FBS) PremiumPANP30-1302
Gas Anesthesia SystemZS DichuangZS-MV-IV
Goat Anti-Rabbit IgG H&L (HRP)PPLYGENC1309This is the secondary antibody used in the immunohistochemical staining.
Hank's Balanced Salt Solution (HBSS)Biological Industries, Beth HaEmek, Israel02-016-1ASterile
Hematoxylin Staining SolutionZSGB-BIOZLI-9609
Lipopolysaccharide (LPS)SigmaL3012
MEDIA AND SUPPLEMENTS
Modified Safranin O-fast Green FCF Cartilage Stain KitSolarbioG1371
N-formyl-Met-Leu-Phe (fMLP)Sigma47729
Penicillin Streptomycin Solution, 100×Invitrogen1514022
PercollGE Healthcare10245207Density gradient medium
Permeabilization BufferMix 100 μL Triton X-100 with 1 L dH2O to get 0.01% Triton X-100
Phosphate Buffer Saline (PBS), 1×Mix 90% ddH2O with 10% (v/v) 10×PBS, autoclaved
Phosphate Buffer Saline (PBS), 10×Dissolve 16 g NaCl, 0.4 g KCl, 2.88 g Na2HPO4·2H2O, 0.48 g KH2PO4 (anhydrous) in 200 mL ddH2O, adjust pH 7.4, autoclaved
PLASTIC WARES AND EQUIPMENTS
POWDER
Proteose PeptoneOxoid1865317
Retrieval BufferMix 18 mL retrieval buffer A with 82 mL retrieval buffer B, add dH2O to 1000 mL, adjust pH to 6.0
Retrieval Buffer A Stock for IHCDissolve 4.2 g citric acid (C6H5O7·H2O) in 200 mL dH2O
Retrieval Buffer B Stock for IHCDissolve 5.88 g trisodium citrate dihydrate (C6H5Na3O7·2H20) in 200 mL dH2O
Roswell Park Memorial Institute (RPMI)-1640 mediumSigmaR8758
RPMI-1640 Complete MediumRPMI-1640 medium is supplemented with 10% FBS and 1% penicillin/streptomycin.
Shu Rui U40 Disposable Sterile Insulin Injection Needle 1 mLBD328421
SlideCITOGLAS10127105P-G
SOLUTION
Stock Isotonic Percoll (SIP)Mix 90% (v/v) of percoll with 10% (v/v) 10×PBS, stand still for 20 min
Wash Buffer in Air Pouch AssayDilute 0.5M EDTA to 10mM with HBSS
XyleneBeijing Chemical Works

References

  1. Klein, C. Genetic defects in severe congenital neutropenia: emerging insights into life and death of human neutrophil granulocytes. Annual Review of Immunology. 29, 399-413 (2011).
  2. Nuzzi, P. A., Lokuta, M. A., Huttenlocher, A., Coutts, A. S.

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