Solving technical issues in flow cytometry to characterize porcine CD8α/β expressing lymphocytes

Area of Science:

• Immunology
• Cell Biology
• Flow Cytometry

Background:

• CD8 is a T cell co-receptor binding MHC class I, crucial for immune responses.
• In pigs, CD8 exists as αα homodimers on some lymphocytes and αβ heterodimers on cytolytic T cells.
• Monoclonal antibodies (mAbs) targeting CD8α and CD8β chains are vital tools for flow cytometry analysis.

Purpose of the Study:

• To investigate potential interferences between different CD8α and CD8β monoclonal antibody (mAb) clones during multi-color flow cytometry staining in pigs.
• To identify optimal mAb combinations for reliable detection of porcine CD8αβ heterodimers on cytolytic T cells.
• To provide guidance for improved flow cytometry panel design in porcine immunology research.

Main Methods:

• Evaluation of four specific mAb clones for porcine CD8α (11/295/33, 76-2-11) and CD8β (PPT23, PG164A).
• Assessment of co-staining compatibility by incubating different mAb combinations.
• Analysis of mAb binding efficiency and signal intensity using flow cytometry.

Main Results:

• The CD8α mAb clone 11/295/33 significantly inhibited the binding of both tested CD8β mAb clones (PPT23, PG164A).
• This inhibition by clone 11/295/33 led to reduced CD8β signal and potential misidentification of CD8αβ cytolytic T cells.
• The CD8α mAb clone 76-2-11 did not show any binding inhibition with either CD8β mAb clone.

Conclusions:

• The combination of CD8α clone 11/295/33 with CD8β clones is not suitable for multi-color flow cytometry in pigs.
• CD8α clone 76-2-11 is the preferred antibody for CD8α detection in multi-color panels due to its compatibility with CD8β staining.
• These findings will enhance the accuracy of flow cytometry studies involving porcine immune cells, particularly cytolytic T cells.