Identification of B cells through negative gating—An example of the MIFlowCyt standard applied
Open Access
- 3 February 2010
- journal article
- research article
- Published by Wiley in Cytometry Part A
- Vol. 77A (6), 546-551
- https://doi.org/10.1002/cyto.a.20862
Abstract
Polychromatic flow cytometric analysis takes advantage of the increasing number of available fluorophores to positively identify and simultaneously assess multiple parameters in the same cell ( 1 ). Additional parameters may be analyzed through negative identification (i.e., through exclusion of particular stains or antibodies employed). In this report, we tested whether such negative‐gating strategy would identify human B lymphocytes in innate immune phenotyping studies. To this end, B cells were identified as the negatively‐stained subpopulation from the CD123 vs. CD11c plot of the CD14neg‐low, MHC IIhigh human peripheral blood mononuclear cells. To test the specificity of this negative gating approach, we confirmed that negatively gated B cells indeed expressed CD19, the bona fide marker for human B cells. However, a small number of unidentified cells were contained in the negatively‐gated B cells. Furthermore, a small percentage cells expressing markers used to identify monocytes and myeloid dendritic cells (mDC) coexpressed CD19. This identifies such negative B‐cell gating approach as potentially problematic. When applied to the analysis of Toll‐like receptors (TLR) stimulation experiments, we were however able to interpret the results, as B‐cells respond to TLR stimulation in a qualitative different pattern as compared to monocytes and DC. This report is presented in a manner that is fully compliant with the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard, which was recently adopted by the International Society for Advancement of Cytometry (ISAC) ( 2 ) and incorporated in the publishing policies of Cytometry and other journals. We demonstrate how a MIFlowCyt‐compliant report can be prepared with minimal effort, and yet provide the reader with a much clearer picture of the portrayed FCM experiment and data. © 2010 International Society for Advancement of CytometryKeywords
This publication has 14 references indexed in Scilit:
- Publishing flow cytometry dataAmerican Journal of Physiology-Lung Cellular and Molecular Physiology, 2010
- A chromatic explosion: the development and future of multiparameter flow cytometryImmunology, 2008
- Promoting coherent minimum reporting guidelines for biological and biomedical investigations: the MIBBI projectNature Biotechnology, 2008
- Intracellular cytokine optimization and standard operating procedureNature Protocols, 2006
- Quality assurance for polychromatic flow cytometryNature Protocols, 2006
- Interpreting flow cytometry data: a guide for the perplexedNature Immunology, 2006
- Toll‐like receptor stimulation as a third signal required for activation of human naive B cellsEuropean Journal of Immunology, 2006
- Standardizing the standardsMolecular Systems Biology, 2006
- The toll-like receptor repertoire of human B lymphocytes: inducible and selective expression of TLR9 and TLR10 in normal and transformed cellsBlood, 2003
- A role for Toll-like receptors in acquired immunity: up-regulation of TLR9 by BCR triggering in naive B cells and constitutive expression in memory B cellsBlood, 2003