PMID- 30199030
OWN - NLM
STAT- MEDLINE
DCOM- 20181211
LR  - 20200824
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 138
DP  - 2018 Aug 24
TI  - In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative 
      Analysis by Fluorescence Microscopy.
LID - 10.3791/58083 [doi]
LID - 58083
AB  - In response to invading pathogens, neutrophils release neutrophil extracellular 
      traps (NETs), which are extracellular networks of DNA decorated with histones and 
      antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release 
      during sepsis is associated with multiple organ dysfunction and mortality in mice 
      and humans but its implications in dogs are unknown. Herein, we describe a 
      technique to isolate canine neutrophils from whole blood for observation and 
      quantification of NETosis. Leukocyte-rich plasma, generated by dextran 
      sedimentation, is separated by commercially available density gradient separation 
      media and granulocytes collected for cell count and viability testing. To observe 
      real-time NETosis in live neutrophils, cell permeant and cell impermeant 
      fluorescent nucleic acid stains are added to neutrophils activated either by 
      lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in 
      nuclear morphology and NET formation are observed over time by fluorescence 
      microscopy. In vitro NETosis is further characterized by co-colocalization of 
      cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) 
      using a modified double-immunolabelling protocol. To objectively quantify NET 
      formation and citH3 expression using fluorescence microscopy, NETs and 
      citH3-positive cells are quantified in a blinded manner using available software. 
      This technique is a specific assay to evaluate the in vitro capacity of canine 
      neutrophils to undergo NETosis.
FAU - Li, Ronald H L
AU  - Li RHL
AD  - Department of Veterinary Surgical and Radiological Sciences, School of Veterinary 
      Medicine, University of California, Davis; rhli@ucdavis.edu.
FAU - Tablin, Fern
AU  - Tablin F
AD  - Department of Anatomy, Physiology and Cell Biology, School of Veterinary 
      Medicine, University of California, Davis.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Video-Audio Media
DEP - 20180824
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
SB  - IM
MH  - Animals
MH  - Dogs
MH  - Extracellular Traps/*genetics
MH  - Humans
MH  - Microscopy, Fluorescence/*methods
MH  - Neutrophils/*metabolism
PMC - PMC6231810
EDAT- 2018/09/11 06:00
MHDA- 2018/12/12 06:00
PMCR- 2020/08/24
CRDT- 2018/09/11 06:00
PHST- 2018/09/11 06:00 [entrez]
PHST- 2018/09/11 06:00 [pubmed]
PHST- 2018/12/12 06:00 [medline]
PHST- 2020/08/24 00:00 [pmc-release]
AID - 58083 [pii]
AID - 10.3791/58083 [doi]
PST - epublish
SO  - J Vis Exp. 2018 Aug 24;(138):58083. doi: 10.3791/58083.