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Combined Boyden-Flow Cytometry Assay Improves Quantification and Provides Phenotypification of Leukocyte Chemotaxis
Chemotaxis has been studied by classical methods that measure chemotactic and random motility responses in vitro, but these methods do not evaluate the total number and phenotype of migrating leukocytes simultaneously. Our objective was to develop and validate a novel assay, combined Boyden-flow cyt...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3235166/ https://www.ncbi.nlm.nih.gov/pubmed/22174892 http://dx.doi.org/10.1371/journal.pone.0028771 |
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author | Gomez-Lopez, Nardhy Vadillo-Ortega, Felipe Estrada-Gutierrez, Guadalupe |
author_facet | Gomez-Lopez, Nardhy Vadillo-Ortega, Felipe Estrada-Gutierrez, Guadalupe |
author_sort | Gomez-Lopez, Nardhy |
collection | PubMed |
description | Chemotaxis has been studied by classical methods that measure chemotactic and random motility responses in vitro, but these methods do not evaluate the total number and phenotype of migrating leukocytes simultaneously. Our objective was to develop and validate a novel assay, combined Boyden-flow cytometry chemotaxis assay (CBFCA), for simultaneous quantification and phenotypification of migrating leukocytes. CBFCA exhibited several important advantages in comparison to the classic Boyden chemotaxis assay (CBCA): 1) improved precision (intra-assay coefficients of variation (CVs): CBFCA-4.7 and 4.8% vs. CBCA-30.1 and 17.3%; inter-observer CVs: CBFCA-3.6% vs. CBCA 30.1%); 2) increased recovery of cells, which increased assay to provide increased sensitivity; 3) high specificity for determining the phenotype of migrating/attracted leukocytes; and 4) reduced performance time (CBFCA 120 min vs. CBCA 265 min). Other advantages of CBFCA are: 5) robustness, 6) linearity, 7) eliminated requirement for albumin and, importantly, 8) enabled recovery of migrating leukocytes for subsequent studies. This latter feature is of great benefit in the study of migrating leukocyte subsets. We conclude that the CBFCA is a novel and improved technique for experiments focused on understanding leukocyte trafficking during the inflammatory response. |
format | Online Article Text |
id | pubmed-3235166 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-32351662011-12-15 Combined Boyden-Flow Cytometry Assay Improves Quantification and Provides Phenotypification of Leukocyte Chemotaxis Gomez-Lopez, Nardhy Vadillo-Ortega, Felipe Estrada-Gutierrez, Guadalupe PLoS One Research Article Chemotaxis has been studied by classical methods that measure chemotactic and random motility responses in vitro, but these methods do not evaluate the total number and phenotype of migrating leukocytes simultaneously. Our objective was to develop and validate a novel assay, combined Boyden-flow cytometry chemotaxis assay (CBFCA), for simultaneous quantification and phenotypification of migrating leukocytes. CBFCA exhibited several important advantages in comparison to the classic Boyden chemotaxis assay (CBCA): 1) improved precision (intra-assay coefficients of variation (CVs): CBFCA-4.7 and 4.8% vs. CBCA-30.1 and 17.3%; inter-observer CVs: CBFCA-3.6% vs. CBCA 30.1%); 2) increased recovery of cells, which increased assay to provide increased sensitivity; 3) high specificity for determining the phenotype of migrating/attracted leukocytes; and 4) reduced performance time (CBFCA 120 min vs. CBCA 265 min). Other advantages of CBFCA are: 5) robustness, 6) linearity, 7) eliminated requirement for albumin and, importantly, 8) enabled recovery of migrating leukocytes for subsequent studies. This latter feature is of great benefit in the study of migrating leukocyte subsets. We conclude that the CBFCA is a novel and improved technique for experiments focused on understanding leukocyte trafficking during the inflammatory response. Public Library of Science 2011-12-09 /pmc/articles/PMC3235166/ /pubmed/22174892 http://dx.doi.org/10.1371/journal.pone.0028771 Text en Gomez-Lopez, et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Gomez-Lopez, Nardhy Vadillo-Ortega, Felipe Estrada-Gutierrez, Guadalupe Combined Boyden-Flow Cytometry Assay Improves Quantification and Provides Phenotypification of Leukocyte Chemotaxis |
title | Combined Boyden-Flow Cytometry Assay Improves Quantification and Provides Phenotypification of Leukocyte Chemotaxis |
title_full | Combined Boyden-Flow Cytometry Assay Improves Quantification and Provides Phenotypification of Leukocyte Chemotaxis |
title_fullStr | Combined Boyden-Flow Cytometry Assay Improves Quantification and Provides Phenotypification of Leukocyte Chemotaxis |
title_full_unstemmed | Combined Boyden-Flow Cytometry Assay Improves Quantification and Provides Phenotypification of Leukocyte Chemotaxis |
title_short | Combined Boyden-Flow Cytometry Assay Improves Quantification and Provides Phenotypification of Leukocyte Chemotaxis |
title_sort | combined boyden-flow cytometry assay improves quantification and provides phenotypification of leukocyte chemotaxis |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3235166/ https://www.ncbi.nlm.nih.gov/pubmed/22174892 http://dx.doi.org/10.1371/journal.pone.0028771 |
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