Abstract

Single cell immunophenotyping by flow cytometry has proven a useful and high sensitive method for the analysis of immune cell composition and phenotype in different lymphatic and non-lymphatic tissues. Fixation of stained cells is usually recommended when the cells need to be preserved for later analysis by flow cytometry to avoid changes in cell morphology and expression of the level of cellular antigens. In the present study, a stain-fix approach was used in combination with flow cytometry to investigate the impact of fixation of camel lymph node cell suspension (n = 5 camels) after labeling with monoclonal antibodies to some leukocyte antigens on their cellular composition and expression density of immune cell markers. The obtained results indicated that camel lymph node cell suspension stained with fluorochrome-conjugated mAbs to leukocyte antigens and fixed with paraformaldehyde (PFA) will keep stable values for their immune cell composition for at least six days when analyzed by flow cytometry. However, if cell subsets were to be identified, fixation may result in different values that were obtained when analyzing fresh stained unfixed cells. Especially the instability in the fluorescence intensity of CD14, CD172a, and MHCII will lead to significant changes in the frequency of monocyte subsets (classical versus intermediate or non-classical) and the identification of macrophage functional subtype (M1 versus M2). Similarly, the instability in CD44 expression may affect the identified phenotype of T cells with significantly lower frequency of activated T cells. In conclusion, flow cytometric data collected from stained and PFA-fixed cell suspension prepared from camel lymph nodes should be interpreted with care if the functional subtype of cells is to be identified based on surface molecule expression. Open Access: This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit https://creativecommons.org/licenses/by/4.0/. © The Author(s) 2024