An alternative flow cytometry strategy for peripheral blood dendritic cell enumeration in the setting of repetitive GM-C
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BioMed Central
Open Access
Methodology
An alternative flow cytometry strategy for peripheral blood dendritic cell enumeration in the setting of repetitive GM-CSF dosing Kehui Wang1, Kevin P Nishimoto2, Rita S Mehta1 and Edward L Nelson*1,2,3 Address: 1Department of Medicine, Division of Hematology/Oncology, School of Medicine, University of California, Irvine, USA, 2Department of Molecular Biology & Biochemistry, School of Biological Sciences, University of California, Irvine, USA and 3Center for Immunology, University of California, Irvine, USA Email: Kehui Wang - [email protected]; Kevin P Nishimoto - [email protected]; Rita S Mehta - [email protected]; Edward L Nelson* - [email protected] * Corresponding author
Published: 24 April 2006 Journal of Translational Medicine2006, 4:18
doi:10.1186/1479-5876-4-18
Received: 08 January 2006 Accepted: 24 April 2006
This article is available from: http://www.translational-medicine.com/content/4/1/18 © 2006Wang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract Background: Enumeration of circulating peripheral blood dendritic cells (DCs) is complicated by the absence of a unique cell surface marker expressed on all DC subsets and by the use of various biological adjuvants to modulate the DC compartment, including granulocyte macrophage colony stimulating factor (GM-CSF). Common methods employ a cocktail of antibodies, typically including anti-CD14, to define a lineage negative, MHC class II positive, putative DC population. Reported flow cytometry protocols include highly variable gating strategies and DC identification criteria. Increasing appreciation of DC pleiomorphism, GM-CSF biology, and recognition of CD14 expression in some DC subsets led us to consider an alternative lineage cocktail to improve identification of the circulating DC pool. Methods: Standard whole blood staining with appropriate fluorochrome conjugated antibodies to MHC class II and either standard CD14 containing, or an alternate CD66acde containing, lineage cocktail was performed on samples obtained from normal donors and breast cancer patients before and after administration of dose-dense, cytotoxic chemotherapy with daily GM-CSF hematopoetic growth factor support. Putative DCs were enumerated by standard flow cytometry. Data set differences were evaluated using two tailed Mann-Whitney or Wilcoxon signed rank tests. Cellular morphology was examined in cellsorted populations from post GM-CSF samples. Results: Use of either antibody cocktail defined comparably sized lineage negative, MHC class II positive populations in normal donors and at baseline in cancer patients. However, selection of lineage negative subsets with increasing MHC class II expression levels yielded larger putative DC populations identified with the alternate cocktail. Bo
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