Abstract
BackgroundIn the poultry industry, quantitative analysis of chicken T cell proliferation is important in many biological applications such as drug screening, vaccine production, and cytotoxicity assessment. Several assays have been established to evaluate this immunological response in chicken cells. However, these assays have some disadvantages including use of radioactive labels ([3H]-Thymidine assay), necessity of DNA denaturation or digestion (BrdU incorporation assay), lack of sensitivity and underestimation of anti-proliferative effects (MTT assay), and modulation of activation molecules and cell viability reduction (CFSE assay). Overcoming these limitations, the EdU proliferation assay is sensitive and advantageous compared to [3H]-Thymidine radioactive labels in studies on cell proliferation in vitro and allows simultaneous identification of T cell populations. However, this assay has not been established using primary chicken cells to evaluate T cell proliferation by flow cytometry.ResultsHere, we established an assay to evaluate the proliferation of primary chicken splenocytes based on the incorporation of a thymidine analog (EdU) and a click reaction with a fluorescent azide, detected by a flow cytometer. We also established a protocol that combines EdU incorporation and immunostaining to detect CD4+ and CD8+ proliferating T cells. By inducing cell proliferation with increasing concentrations of a mitogen (Concanavalin A), we observed a linear increase in EdU positive cells, indicating that our protocol does not present any deficiency in the quantity and quality of reagents that were used to perform the click reaction.ConclusionsIn summary, we established a reliable protocol to evaluate the proliferation of CD4+ and CD8+ chicken T cells by flow cytometry. Moreover, as this is an in-house protocol, the cost per sample using this protocol is low, allowing its implementation in laboratories that process a large number of samples.
Highlights
In the poultry industry, quantitative analysis of chicken T cell proliferation is important in many biological applications such as drug screening, vaccine production, and cytotoxicity assessment
As rapidly proliferating cells must acquire more nutrients than non-proliferating cells [23], requiring high levels of glucose and amino acids [24], we evaluated the possibility of improving cell viability using a high-glucose medium like Dulbecco’s modified eagle medium (DMEM).F12 and a medium that was modified in our laboratory, supplemented with 0.5% chicken serum (ChS) (FARMEM medium, industrial secret)
Another study recommends the use of X-VIVOTM 15 medium (Lonza, MD, USA), a serum-free medium that reduces the background proliferation of primary chicken cells [19]
Summary
Quantitative analysis of chicken T cell proliferation is important in many biological applications such as drug screening, vaccine production, and cytotoxicity assessment. Several assays have been established to evaluate this immunological response in chicken cells These assays have some disadvantages including use of radioactive labels ([3H]-Thymidine assay), necessity of DNA denaturation or digestion (BrdU incorporation assay), lack of sensitivity and underestimation of anti-proliferative effects (MTT assay), and modulation of activation molecules and cell viability reduction (CFSE assay). Overcoming these limitations, the EdU proliferation assay is sensitive and advantageous compared to [3H]-Thymidine radioactive labels in studies on cell proliferation in vitro and allows simultaneous identification of T cell populations. An increase in cellular mitochondrial activity might not be just an indicator of proliferation as it can be affected by other factors like cell death and decreased or increased metabolic activity of non-proliferating cells [17, 18]
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