Measurement of canine Th17 cells by flow cytometry

Area of Science:

• Immunology
• Veterinary Medicine
• Cell Biology

Background:

• T helper 17 (Th17) cells are critical immune cells involved in inflammation and autoimmune diseases.
• Investigating Th17 cell roles in canine diseases requires efficient and reliable identification methods.
• Th17 cells are a rare population, necessitating optimized techniques for their detection and analysis.

Purpose of the Study:

• To establish fast and accurate methods for identifying Th17 cells in dogs for clinical research.
• To evaluate different preselection and enrichment strategies for isolating CD3+CD4+ cells.
• To assess the impact of sample storage and cell stimulation on Th17 cell measurement.

Main Methods:

• Comparison of two density gradients for mononuclear cell isolation, with Lymph24+ Spin Medium showing superior reduction of polymorphonuclear cells.
• Enrichment of CD3+CD4+ cells using autoMACS Pro Separator by depleting CD8alpha+, CD11b+, and CD21+ cells.
• Evaluation of cell stimulation and overnight blood storage effects on Th17 cell quantification via flow cytometry.

Main Results:

• Lymph24+ Spin Medium significantly decreased polymorphonuclear cells, yielding a purer cell population (p=0.01).
• Cell sorting significantly increased CD3+CD4+ cell purity and decreased unwanted cell populations (p<0.001).
• Cell stimulation significantly impacted Th17 cell percentages (p<0.001), while overnight storage did not affect results for either stimulated or unstimulated samples.

Conclusions:

• A density gradient (Lymph24+ Spin Medium) and cell sorting effectively enrich for CD3+CD4+ cells, improving purity for Th17 cell analysis.
• Stimulation is crucial for accurate Th17 cell measurement, and overnight blood storage is a viable option without compromising results.
• A modified protocol enables efficient overnight storage and flow cytometry analysis of canine blood samples for Th17 cell research in various disease states.