Why does quantification of intracellular amastigotes matter for target validation?

Quantifying intracellular amastigotes provides a direct measure of Leishmania virulence and replication competence within macrophages, enabling assessment of whether a compound disrupts the pathogen’s intracellular lifecycle. This phenotypic readout supports target validation by linking molecular inhibition to functional outcomes in a disease-relevant system.

How does isolation of the intracellular replication variable support the discovery pipeline?

By focusing on amastigote formation within parasitophorous vacuoles, the assay isolates the intracellular replication stage as a key virulence-dependent variable, allowing researchers to evaluate compounds that specifically block differentiation or survival inside host cells. This enables de-risking of targets involved in iron metabolism, phagosome maturation, or parasite persistence mechanisms.

What do quantitative measurements of infected macrophage nuclei enable?

Quantitative measurement of DAPI-stained amastigote nuclei clustered around macrophage nuclei enables objective comparison of infection rates across experimental conditions, supporting dose-response analysis and hit confirmation in screening campaigns. These data provide a scalable, microscopy-based readout for assessing compound effects on intracellular parasite burden.

Why do replication requirements matter for cross-functional collaboration?

Defined replication requirements—including standardized macrophage differentiation, infection timing, and fixation protocols—ensure assay consistency across laboratories and project teams, enabling reliable data sharing and comparison. This supports translational continuity by allowing discovery, screening, and preclinical groups to generate comparable virulence assessment data.

What statistical analysis capabilities are required before implementation?

Implementation requires the ability to quantify and compare infected cell frequencies across conditions using statistical methods such as t-tests or ANOVA to determine significant differences in amastigote load between treated and control groups. This enables data-driven hit selection and structure-activity relationship analysis in lead optimization efforts.

培养巨噬细胞中利什曼原虫毒力的评估

JoVE Encyclopedia of Experiments

Immunology

0 views • 4:46 min • July 8th, 2025

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从盖玻片上含有骨髓来源巨噬细胞的多孔板开始。

用利什曼原虫前鞭毛体（寄生虫的传染阶段）治疗巨噬细胞。

在

孵化过程中，寄生虫与巨噬细胞相互作用，促进 F-肌动蛋白在相互作用位点周围的组装。

巨噬细胞将寄生虫吞噬在 F-肌动蛋白包围的吞噬体中，形成寄生液泡或 PV——一个复制生态位。

在

PV 内，寄生虫释放生存因子，增加液泡周围的 F-肌动蛋白积累并抑制它们与溶酶体的融合。

此外，寄生虫上调铁调素（一种巨噬细胞肽激素）的产生，抑制铁释放并提高细胞内铁水平。

铁流入液泡促进前鞭毛体分化为致病性无鞭毛体并促进其繁殖，增强毒力。

孵育后，洗涤以去除游离寄生虫，然后固定并透化受感染的巨噬细胞。

引入核酸荧光染料对受感染的巨噬细胞和无鞭毛体的细胞核进行染色。

在

荧光显微镜下，聚集在巨噬细胞大核周围的小细胞核证实了细胞内寄生虫的存在，证明了利什曼原虫的毒力。

在培养后的第七天或第八天，使用安装在真空管线上的吸头将四个高压灭菌灭菌的 12 毫米玻璃盖玻片转移到六孔培养板的每个孔的底部，不要重叠。为实验的每个时间点准备相同的板。接下来，用新鲜的37摄氏度PBS和5毫升不含钙和镁的PBS洗涤骨髓来源的巨噬细胞培养物2次，每块板补充一毫摩尔EDTA。

在

37摄氏度下五分钟后，将分离的细胞汇集在50毫升锥形管中，用另外5毫升不含钙和镁的PBS冲洗每个板两次。离心后，将巨噬细胞重新悬浮在冰上的5至10ml新鲜骨髓来源的巨噬细胞培养基中。计数后，在新鲜的骨髓来源的巨噬细胞培养基中将细胞稀释至每毫升浓度5 x 10⁵，并将每孔2毫升细胞加入6孔板中。然后，将细胞在细胞培养箱中孵育过夜。

要用 L amazonensis 感染细胞，将寄生虫悬浮液稀释至适当的实验感染倍数，并在所有六孔板的每个孔中添加 50 至 100 微升寄生虫，包括实验的所有时间点。将骨髓来源的巨噬细胞孵育至适当的感染期和温度。

然后，每孔用两毫升不含钙或镁的新鲜37摄氏度PBS洗涤孔3次，并用1.5至2ml的2%多聚甲醛固定初始时间点样品10分钟。对于与较晚时间点相对应的板，向每个孔中加入2毫升新鲜的骨髓来源的巨噬细胞培养基，并将板放回适当的培养箱中。然后，如刚才所示，固定并洗涤每个剩余时间点的细胞，并将样品储存在新鲜的PBS中，温度为4摄氏度。

要用DAPI染色细胞，请用1.5ml不含钙和镁的新鲜PBS替换上清液，在室温下补充0.1%非离子洗涤剂10分钟，然后用不含钙或镁的PBS洗涤3次2毫升。接下来，在室温下用每毫升 2 微克 DAPI 在 1 毫升 PBS 中对细胞进行染色一小时。洗涤三次后，将盖玻片单元面朝下放在安装有市售防褪色封片试剂的单个玻璃显微镜载玻片上。

然后，为了量化感染细胞的数量，使用荧光显微镜的100倍浸油物镜和伊曼纽尔计数器来识别聚集在每个DAPI染色的大型巨噬细胞核周围的较小无鞭毛体细胞核的数量。