Research and Development Laboratories, FARVET, Carretera Panamericana Sur N°766 Km 198.5, Ica, Peru.
BMC Vet Res. 2020 Jul 6;16(1):230. doi: 10.1186/s12917-020-02433-0.
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.
在禽类养殖业中，定量分析鸡 T 细胞增殖在药物筛选、疫苗生产和细胞毒性评估等许多生物学应用中都很重要。已经建立了几种测定法来评估鸡细胞中的这种免疫反应。然而，这些测定法有一些缺点，包括使用放射性标记物([3H]-胸苷测定法)、需要 DNA 变性或消化（BrdU 掺入测定法）、缺乏敏感性和对增殖抑制作用的低估（MTT 测定法），以及激活分子的调节和细胞活力的降低（CFSE 测定法）。为了克服这些限制，与[3H]-胸苷放射性标记物相比，EdU 增殖测定法在体外细胞增殖研究中更为敏感和有利，并且允许同时鉴定 T 细胞群。然而，尚未建立使用原代鸡细胞通过流式细胞术评估 T 细胞增殖的该测定法。
在这里，我们建立了一种测定法，通过流式细胞术基于胸苷类似物（EdU）的掺入和与荧光叠氮化物的点击反应来评估原代鸡脾细胞的增殖。我们还建立了一种方案，将 EdU 掺入和免疫染色相结合来检测 CD4 和 CD8 增殖性 T 细胞。通过用浓度递增的有丝分裂原（刀豆蛋白 A）诱导细胞增殖，我们观察到 EdU 阳性细胞的线性增加，表明我们的方案在用于进行点击反应的试剂的数量和质量方面没有任何缺陷。
总之，我们建立了一种可靠的通过流式细胞术评估 CD4 和 CD8 鸡 T 细胞增殖的方案。此外，由于这是一种内部方案，因此使用该方案的每个样本的成本很低，允许在处理大量样本的实验室中实施。
