Performance of plate-based cytokine flow cytometry with automated data analysis

Area of Science:

• Immunology
• Cellular Immunology
• Flow Cytometry

Background:

• Cytokine flow cytometry (CFC) is a multiparameter method for quantifying antigen-specific T cells.
• CFC offers an alternative to ELISPOT assays, with potential for higher throughput.
• Optimizing sample processing is key to increasing the efficiency of CFC assays.

Purpose of the Study:

• To develop and optimize high-throughput methods for cytokine flow cytometry (CFC).
• To adapt CFC protocols for use with 96-well and 24-well plates for whole blood and PBMC samples.
• To evaluate the performance of plate-based CFC compared to traditional tube-based methods.

Main Methods:

• Developed protocols for stimulating and processing whole blood and peripheral blood mononuclear cells (PBMCs) in deep-well and round-bottom plates.
• Tested samples from both HIV-1-seronegative and HIV-1-seropositive donors.
• Compared plate-based methods with traditional tube-based stimulation and processing, assessing percent response, staining intensity, and cell recovery.
• Evaluated automated gating templates against manual gating for data analysis.

Main Results:

• Established protocols for plate-based stimulation and processing of whole blood and PBMCs.
• Demonstrated comparable percent response, staining intensity, and cell recovery between plate-based and tube-based CFC methods.
• Showed equivalence between automated gating templates and manual gating for CFC data analysis.
• Confirmed successful testing with samples from both HIV-1-seronegative and HIV-1-seropositive donors.

Conclusions:

• Plate-based CFC methods, combined with automated analysis, offer a higher throughput platform.
• These optimized methods reduce operator-induced variability, crucial for large-scale studies.
• The developed platform is suitable for processing large sample numbers in clinical trials and for epitope mapping.