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Published on: December 17, 2013
An EdU-based flow cytometry assay to evaluate chicken T lymphocyte proliferation
Karla Lucía F Alvarez1, Astrid Poma-Acevedo2, Manolo Fernández-Sánchez2
1Research and Development Laboratories, FARVET, Carretera Panamericana Sur N°766 Km 198.5, Ica, Peru. karla.alvarez@farvet.com.
BMC Veterinary Research
|July 8, 2020
Summary
A new assay effectively measures chicken T cell proliferation using EdU incorporation and flow cytometry. This method overcomes limitations of older assays and is cost-effective for large-scale studies.
Area of Science:
- Immunology
- Poultry Science
- Cell Biology
Background:
- Quantitative analysis of chicken T cell proliferation is crucial for drug screening, vaccine development, and cytotoxicity assessment in the poultry industry.
- Existing assays for chicken T cell proliferation have limitations, including radioactivity, DNA denaturation requirements, low sensitivity, and effects on cell viability.
- The EdU proliferation assay offers advantages over traditional methods but had not been established for primary chicken cells using flow cytometry.
Purpose of the Study:
- To establish a reliable and sensitive assay for evaluating T cell proliferation in primary chicken cells.
- To adapt the EdU incorporation assay for use with chicken splenocytes and flow cytometry detection.
- To enable simultaneous detection of proliferating CD4+ and CD8+ T cells.
Main Methods:
- Developed a protocol utilizing 5-ethynyl-2'-deoxyuridine (EdU) incorporation into newly synthesized DNA of proliferating chicken splenocytes.
- Employed a click reaction with a fluorescent azide to detect incorporated EdU, analyzed via flow cytometry.
- Combined EdU detection with immunostaining for CD4 and CD8 surface markers to identify specific T cell populations.
Main Results:
- Successfully established a flow cytometry-based assay for measuring chicken T cell proliferation using EdU incorporation.
- Demonstrated a linear correlation between mitogen (Concanavalin A) concentration and the percentage of EdU-positive cells, validating assay sensitivity.
- The protocol allows for the simultaneous identification and quantification of proliferating CD4+ and CD8+ T cells.
Conclusions:
- A reliable and cost-effective protocol for evaluating CD4+ and CD8+ chicken T cell proliferation by flow cytometry has been established.
- This in-house developed assay is suitable for laboratories handling numerous samples due to its low cost per sample.
- The assay provides a valuable tool for immunological studies in poultry, overcoming limitations of previous methods.
