158 To cite this paper: Al-Sukruwah MA, Althagafi H, Al-Abdulsalam NK, and Hussen J (2024). Impact of Fixation of Camel Lymph Node Cells on Marker Expression Stability in Flow Cytometry. World Vet. J., 14(2): 158-168. DOI: https://dx.doi.org/10.54203/scil.2024.wvj20 2024, Scienceline Publication World ’s Veterinary Journal World Vet J, 14(2): 158-168. ISSN 2322-4568 Impact of Fixation of Camel Lymph Node Cells on Marker Expression Stability in Flow Cytometry Mohammed Ali Al-Sukruwah 1 , Hind Althagafi 2* , Najla K Al Abdulsalam 3 , and Jamal Hussen 1* 1 Department of Microbiology, College of Veterinary Medicine, King Faisal University, Al-Ahsa, Saudi Arabia 2 Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh 11671, Saudi Arabia 3 Department of Biological Sciences, College of Science, King Faisal University, P.O. Box 380, Al-Ahsa 31982, Saudi Arabia *Corresponding author's Email: hjalthagafi@pnu.edu.sa; jhussen@kfu.edu.sa 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. Keywords: Camel, Fixation, Flow cytometry, Immune cell, Lymph node ORIGINAL ARTICLE Received: March 20, 2024 Revised: April 22, 2024 Accepted: May 15, 2024 Published: June 25, 2024 INTRODUCTION Lymph nodes are secondary organs with specialized structures that filter the lymph throughout the body (Yang et al., 2019). Lymph nodes provide sites for cross-talk between antigen-specific adaptive immune cells and antigen-presenting cells for the development of adaptive immune responses (Liao and von der Weid, 2015; Rehfeld et al., 2017). The cellular composition of the node lymphatic tissue is dominated by lymphoid cells with lower numbers of neutrophils and monocytes and their derivatives, such as macrophages and dendritic cells. Alterations in the composition and phenotype of immune cells in the lymph node reflect important events of the immune response, including migration and homing of immune cells, antigen trapping, presentation, stimulation of adaptive immunity, and generation of effector cells (Lun et al., 2007). Therefore, the analysis of these changes provides an effective method to evaluate the immune response against infection and vaccination, or for the diagnosis of immunopathology (Koets et al., 2002; Caucheteux et al., 2013). Single-cell immunophenotyping by flow cytometry has proven a useful and highly sensitive method for the analysis of immune cell composition and phenotype in different lymphatic (Aalbers et al., 2015) and non-lymphatic tissues (Dong et al., 2016; Hagberg et al., 2018; Tighe et al., 2019). Immunophenotyping steps usually include cell separation and the preparation of cell suspension followed by labeling cellular antigens with monoclonal antibodies conjugated with fluorescent dyes (Maecker et al., 2012). One of the big challenges during immunophenotyping studies, especially in veterinary medicine, where flow cytometers are still not available in every laboratory, is extending the time between cell staining and flow cytometric analysis without affecting cell composition or phenotype (Laurin et al., 2015). Fixation of stained cells represents a way to avoid protein denaturation and loss of antigenic structure leading to extending the time between sampling and analysis (Ng et al., 2012; Qin et al., 2021). In addition, the detection of intracellular epitopes, such as cytokines or signaling molecules, requires cell fixation and permeabilization to access the target epitope by antibodies (Paavilainen et al., 2010; Cheng et al., 2019). For the selection of a fixative agent, the fixative must not impact parameters related to cell structure, including cell size and granularity, as well as the reactivity of cellular epitopes with DOI: https://dx.doi.org/10.54203/scil.2024.wvj20 PII: S232245682400020-14