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An EdU-based flow cytometry assay to evaluate chicken T lymphocyte proliferation.

BMC veterinary research (2020-07-08)
Karla Lucía F Alvarez, Astrid Poma-Acevedo, Manolo Fernández-Sánchez, Manolo Fernández-Díaz
ABSTRACT

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. 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. Here, 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. In 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Histopaque®-1077, sterile-filtered, density: 1.077 g/mL
Sigma-Aldrich
Dimethyl sulfoxide, ≥99.5% (GC), suitable for plant cell culture
Sigma-Aldrich
L-Ascorbic acid, BioXtra, ≥99.0%, crystalline
Sigma-Aldrich
Concanavalin A from Canavalia ensiformis (Jack bean), Type IV-S, lyophilized powder, aseptically processed, BioReagent, suitable for cell culture
Sigma-Aldrich
Trypan Blue solution, 0.4%, for microscopy
Millipore
EDTA, Disodium Salt, Dihydrate, Molecular Biology Grade - CAS 6381-92-6 - Calbiochem
Sigma-Aldrich
Saponin from quillaja bark, Sapogenin content ≥10 %
SAFC
RPMI-1640 Medium, AutoMod, without L-glutamine and sodium bicarbonate, powder, suitable for cell culture
Sigma-Aldrich
Copper(II) sulfate pentahydrate, suitable for plant cell culture, ≥98%
Sigma-Aldrich
Dulbecco′s Phosphate Buffered Saline, Without calcium chloride, powder, suitable for cell culture