Characterization of Chicken Leukocyte Subsets from Lymphatic Tissue by Flow Cytometry

Area of Science:

• Immunology
• Avian biology
• Flow cytometry

Background:

• Assessing immune status requires quantifying leukocyte subtypes in blood and lymphatic tissues.
• Nucleated thrombocytes and erythrocytes complicate avian hematological analysis.
• Accurate enumeration of immune cells is crucial for understanding avian immune responses.

Purpose of the Study:

• To adapt flow cytometry for rapid discrimination and enumeration of leukocyte subsets in chicken lymphatic tissues.
• To establish reliable staining and gating strategies for avian immune cell analysis.
• To overcome challenges posed by nucleated erythrocytes and thrombocytes in avian samples.

Main Methods:

• Adapted existing flow cytometry methods for chicken blood to analyze spleen and cecal tonsil cells.
• Developed specific antibody staining and gating strategies for immune cell identification.
• Prepared single-cell suspensions and isolated intraepithelial lymphocytes without density centrifugation.
• Performed antibody staining without washing steps to minimize cell loss and ensure accurate counts.

Main Results:

• Successfully characterized and enumerated total leukocytes, thrombocytes, monocytes/macrophages, CD8α+ lymphocytes, CD4+ T cells, γδ T cells, and B cells in chicken spleen.
• Quantified CD8α+ lymphocytes, CD4+ T cells, γδ T cells, and B cells among intraepithelial lymphocytes in chicken cecal tonsils.
• Established a streamlined protocol minimizing cell loss and falsification of counts.

Conclusions:

• The developed flow cytometry protocol enables efficient and accurate characterization of immune cell populations in chicken spleen and cecal tonsils.
• This method provides a valuable tool for avian immunology research, facilitating immune status assessment.
• The protocol overcomes common challenges in avian hematology, offering a robust approach for immune cell enumeration.