Characterization of Chicken Leukocyte Subsets from Lymphatic Tissue by Flow Cytometry

Abstract

Determining the abundance of leukocyte subtypes, including lymphocyte subpopulations, not only in blood but also in lymphatic tissues, is inevitable to assess the immune status of an organism for research purposes. However, nucleated thrombocytes and erythrocytes exacerbate many hematological techniques in avian species. In order to enable a rapid discrimination of leukocyte subsets from lymphatic tissues of chicken, we adapted existing flow cytometric methods for counting leukocytes in chicken blood. We established staining and gating strategies allowing the flow cytometric characterization and enumeration of total leukocytes, thrombocytes, monocytes/macrophages, CD8α+ lymphocytes, CD4+ T cells, γδ T cells, and B cells in chicken spleen and CD8α+ lymphocytes, CD4+ T cells, γδ T cells, and B cells among intraepithelial lymphocytes in chicken cecal tonsils. For this, we prepared single‐cell suspensions of spleen and isolated intraepithelial lymphocytes from cecal tonsils without density centrifugation, and performed antibody staining of cells without subsequent washing steps to prevent cell loss and falsification of obtained cell counts. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.

Development of lymphocyte subpopulations in local breed chickens

“…The data showed similarity to those in genetically divergent breeds of chicken ( Table-1 ). In addition, the weights and weight percentage of the primary and secondary lymphoid organs were within the normal range when compared to those of their commercial counterparts (light breeds in particular) ( Table-2 ) [ 27 ]. Like other vertebrates, avian lymphocytes are distributed in the primary lymphoid organs; they migrate to the secondary lymphoid organs where they encounter the antigen.…”

Short- and long-term consequences of stocking density during rearing on the immune system and welfare of laying hens

“…Lymphatic tissue was processed according to Hofmann and Schmucker (2021) . In brief, the spleen was cut into pieces under sterile conditions, transferred into gentleMACS C-Tube (#130-096-334, Miltenyi Biotec, Bergisch Gladbach, Germany) containing PBS + 0.05 mg/mL Gentamycin (Biochrom, Berlin, Germany) and dissociated with a gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany).…”

“…Specific immune cell populations were characterized using flow cytometric analysis, followed by the no-lyse-no-wash method described by Seliger et al (2012) with some modifications and amplifications described in detail in Hofmann and Schmucker (2021) . The following antibodies were used for the discrimination of blood or tissues leukocytes: Anti-CD45-APC (# 8270-11, clone LT40), anti-Monocyte/Macrophage (# 8420-09, clone Kul01), anti-CD4-Pacblu (# 8210-26, CT-4), or -PE (# 8210-09,), anti-CD8α-FITC (# 8220-02, clone CT-8) and anti-Bu-1-FITC (# 8395-02, clone AV20) were obtained from SouthernBiotech (Birmingham, USA).…”

“…Heterophils were identified based on their FSC/SSC characteristics. The detailed staining and gating strategy are described in Hofmann and Schmucker (2021) . In short, 20 µL of stabilized EDTA-blood were diluted with 980 µL staining buffer (PBS pH 7.4, 2% BSA, 0.1% NaN 3 , 5mg/mL EDTA).…”

“…Flow cytometric analysis of splenocytes and intraepithelial lymphocytes of cecal tonsils including gating strategy is described in detail in Hofmann and Schmucker (2021) . In brief, single-cell suspensions were stained with respective antibody mixtures to discriminate all leukocytes and leukocyte subsets according to a no-lyse no-wash single-step two-tube protocol.…”