Cold Stress Modulates the Phenotype of Lymphocyte Subsets and the Function of Blood Phagocytes in Goats
Cold Stress Modulates the Phenotype of Lymphocyte Subsets and the Function of Blood Phagocytes in Goats
Mohammed Ali Al-Sukruwah, Mohammed Ali Al Hejji, Baraa Falemban, Jamal Hussen*
Gating strategy for the flow cytometric analysis of lymphocyte subsets. Leukocytes were separated from goat blood, labeled with antibodies to cell surface markers, and analyzed by flow cytometry. After gating on signle cells (for the exclusion of cell doublets) in a forward scatter aria (FSC-A) against forward scatter height (FSC-H) density plot (A), lymphocytes (L) were gated based on their FSC and side scatter (SSC) characteristics (B). The lymphocyte subsets CD4+ ab T cells, CD8+ ab T cells (C), BAQ44A+ B cells (D), NKp46+ (CD335) natural killer (NK) cells (E), and WC1+ gd T cells (F) were identified based on positive staining with the corresponding mAb. Isotype control staining with secondary anti-mouse IgG2, anti-mouse IgM (G), and anti-mouse IgG1 (H) was performed in parallel.
Cell counts of goat lymphocytes subsets in blood. Leukocytes were separated from goat blood, counted under microscope, labeled with fluorescent antibodies, and analyzed by flow cytometry. The absolute numbers of CD4+ ab T cells, CD8+ ab T cells, the NKp46+ natural killer (NK) cells, B cells, and WC1+ Ƴδ T cells were calculated by multiplication of their percentages with the absolute number of lymphocytes. As the absolute leukocyte count was missing for the time point 2, no absolute count of lymphocyte subsets could be calculated for this time point. (n = 5 goats; * indicates significant differences (One-Way Anova; p < 0.05)).
Flow cytometric analysis of the abundance of CD44 on goat CD4+ and CD8+ T cells. Leukocytes were separated from goat blood and labeled with fluorescent antibodies to CD4, CD8, and CD44 and analyzed by flow cytometry. After the identification of CD4+ (A) and CD8+ (B) cell subsets within the lymphocyte population, overlapping histrograms were prepared to show the abundance of CD44 on CD4+ (C) and CD8+ T cells (D). Staining with anti-CD44 antibody was shown as black line, while control antibody staining was shown as red line. The expression levels of CD44 were calculated as mean fluorescence intensity (MFI) for CD4+ T cells (E) and CD8+ T cells (F). (n = 5 goats; * indicates significant differences (One-Way Anova; p < 0.05)).
Phagocytosis activity. Blood leukocytes were incubated in vitro with FITC-labeled and inactivated Staphylococcus aureus bacteria and analyzed by flow cytometry. A) Cell duplets were excluded based on forward scatter (FSC) and side scatter (SSC) properties. After gating on neutrophils (N) and monocytes (M), phagocytic cells were identified based on the increased fluorescence in the FITC channel. B) the percentage of phagocytosis-positive cells within neutrophils and monocytes was calculated and presented. (n = 5 goats; * indicates significant differences (One-Way Anova; p < 0.05)).
