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Technical note: Flow cytometric identification of bovine milk neutrophils and simultaneous quantification of their viability

Sofie Piepers UGent, Sarne De Vliegher UGent, Kristel Demeyere UGent, Bart Lambrecht UGent, Aart de Kruif UGent, Evelyne Meyer UGent and Geert Opsomer UGent (2009) JOURNAL OF DAIRY SCIENCE. 92(2). p.626-631
abstract
The aim of the present study was to develop a flow cytometric procedure for the quantification of the proportion of viable, apoptotic, and necrotic polymorphonuclear neutrophilic leukocytes (PMNL) isolated from both low- and high-somatic-cell-count quarter milk samples. Milk PMNL were differentiated from other cells by indirect fluorescent labeling using a primary anti-bovine granulocyte monoclonal antibody (CH138A) and an Alexa 647-labeled secondary antibody. Polymorphonuclear neutrophilic leukocytes were identified flow cytometrically based on their cytoplasmic granularity and CH138A-positivity. Additional labeling with annexin-V-fluorescein isothiocyanate and propidium iodide was used to determine milk PMNL viability. Thirty milk samples were run in parallel to assess the repeatability of the immunoassay and 6 repeated measurements per sample were performed to assess the instrument stability. Fluorescence microscopic verification of the CH138A staining pattern showed both a high sensitivity (90.9%) and specificity (92.3%). The combination of the side-scatter properties of granulated PMNL and CH138A-Alexa 647 positivity allows the distinction of labeled PMNL from other milk cells and particles that may bind nonspecifically, and from autofluorescent particles present in milk. Quantification of the proportion of PMNL and viable, apoptotic, and necrotic subpopulations in parallel samples gave repeatable results with concordance correlation coefficients varying between 0.93 and 0.99. The average coefficient of variation for repeated measurements in identical samples ranged between 4.2 and 9.7%. In conclusion, this is the first flow cytometric method suited for the simultaneous quantification of viable, apoptotic, and necrotic bovine milk PMNL in a straightforward manner.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
MASTITIS, BLOOD, APOPTOSIS, DAIRY-COWS, POLYMORPHONUCLEAR LEUKOCYTES, MAMMARY-GLAND, milk, Bovine, neutrophil, flow cytometry, CELLS, SUBPOPULATIONS, EXPRESSION, LACTATION
journal title
JOURNAL OF DAIRY SCIENCE
J. Dairy Sci.
volume
92
issue
2
pages
626 - 631
publisher
Amer Dairy Science Assoc
place of publication
Savoy, USA
Web of Science type
Article
Web of Science id
000262654900022
JCR category
AGRICULTURE, DAIRY & ANIMAL SCIENCE
JCR impact factor
2.463 (2009)
JCR rank
2/49 (2009)
JCR quartile
1 (2009)
ISSN
0022-0302
DOI
10.3168/jds.2008-1393
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
537200
handle
http://hdl.handle.net/1854/LU-537200
date created
2009-04-02 14:29:27
date last changed
2009-12-11 15:01:51
@article{537200,
  abstract     = {The aim of the present study was to develop a flow cytometric procedure for the quantification of the proportion of viable, apoptotic, and necrotic polymorphonuclear neutrophilic leukocytes (PMNL) isolated from both low- and high-somatic-cell-count quarter milk samples. Milk PMNL were differentiated from other cells by indirect fluorescent labeling using a primary anti-bovine granulocyte monoclonal antibody (CH138A) and an Alexa 647-labeled secondary antibody. Polymorphonuclear neutrophilic leukocytes were identified flow cytometrically based on their cytoplasmic granularity and CH138A-positivity. Additional labeling with annexin-V-fluorescein isothiocyanate and propidium iodide was used to determine milk PMNL viability. Thirty milk samples were run in parallel to assess the repeatability of the immunoassay and 6 repeated measurements per sample were performed to assess the instrument stability. Fluorescence microscopic verification of the CH138A staining pattern showed both a high sensitivity (90.9\%) and specificity (92.3\%). The combination of the side-scatter properties of granulated PMNL and CH138A-Alexa 647 positivity allows the distinction of labeled PMNL from other milk cells and particles that may bind nonspecifically, and from autofluorescent particles present in milk. Quantification of the proportion of PMNL and viable, apoptotic, and necrotic subpopulations in parallel samples gave repeatable results with concordance correlation coefficients varying between 0.93 and 0.99. The average coefficient of variation for repeated measurements in identical samples ranged between 4.2 and 9.7\%. In conclusion, this is the first flow cytometric method suited for the simultaneous quantification of viable, apoptotic, and necrotic bovine milk PMNL in a straightforward manner.},
  author       = {Piepers, Sofie and De Vliegher, Sarne and Demeyere, Kristel and Lambrecht, Bart and de Kruif, Aart and Meyer, Evelyne and Opsomer, Geert},
  issn         = {0022-0302},
  journal      = {JOURNAL OF DAIRY SCIENCE},
  keyword      = {MASTITIS,BLOOD,APOPTOSIS,DAIRY-COWS,POLYMORPHONUCLEAR LEUKOCYTES,MAMMARY-GLAND,milk,Bovine,neutrophil,flow cytometry,CELLS,SUBPOPULATIONS,EXPRESSION,LACTATION},
  language     = {eng},
  number       = {2},
  pages        = {626--631},
  publisher    = {Amer Dairy Science Assoc},
  title        = {Technical note: Flow cytometric identification of bovine milk neutrophils and simultaneous quantification of their viability},
  url          = {http://dx.doi.org/10.3168/jds.2008-1393},
  volume       = {92},
  year         = {2009},
}

Chicago
Piepers, Sofie, Sarne De Vliegher, Kristel Demeyere, Bart Lambrecht, Aart de Kruif, Evelyne Meyer, and Geert Opsomer. 2009. “Technical Note: Flow Cytometric Identification of Bovine Milk Neutrophils and Simultaneous Quantification of Their Viability.” Journal of Dairy Science 92 (2): 626–631.
APA
Piepers, S., De Vliegher, S., Demeyere, K., Lambrecht, B., de Kruif, A., Meyer, E., & Opsomer, G. (2009). Technical note: Flow cytometric identification of bovine milk neutrophils and simultaneous quantification of their viability. JOURNAL OF DAIRY SCIENCE, 92(2), 626–631.
Vancouver
1.
Piepers S, De Vliegher S, Demeyere K, Lambrecht B, de Kruif A, Meyer E, et al. Technical note: Flow cytometric identification of bovine milk neutrophils and simultaneous quantification of their viability. JOURNAL OF DAIRY SCIENCE. Savoy, USA: Amer Dairy Science Assoc; 2009;92(2):626–31.
MLA
Piepers, Sofie, Sarne De Vliegher, Kristel Demeyere, et al. “Technical Note: Flow Cytometric Identification of Bovine Milk Neutrophils and Simultaneous Quantification of Their Viability.” JOURNAL OF DAIRY SCIENCE 92.2 (2009): 626–631. Print.