West African Centre for Cell Biology of Infectious Pathogens, College of Basic and Applied Sciences, University of Ghana, P. O. Box LG54, Legon, Accra.
Department of Biochemistry, Cell and Molecular Biology, College of Basic and Applied Sciences, University of Ghana, P. O. Box LG54, Legon, Accra.
Exp Biol Med (Maywood). 2020 Jan;245(1):11-20. doi: 10.1177/1535370219897393. Epub 2020 Jan 5.
erythrocyte invasion phenotyping assays are a very useful tool for assessing parasite diversity and virulence, and for characterizing the formation of ligand–receptor interactions. However, such assays need to be highly sensitive and reproducible, and the selection of labeling dyes for differentiating donor and acceptor erythrocytes is a critical factor. We investigated the suitability of cell trace far-red (CTFR) as a dye for invasion phenotyping assays. Using the dyes carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and dichloro dimethyl acridin one succinimidyl ester (DDAO-SE) as comparators, we used a dye-dilution approach to assess the limitations and specific staining procedures for the applicability of CTFR in invasion phenotyping assays. Our data show that CTFR effectively labels acceptor erythrocytes and provides a stable fluorescent intensity at relatively low concentrations. CTFR also yielded a higher fluorescence intensity relative to DDAO-SE and with a more stable fluorescence intensity over time. Furthermore, CTFR did not affect merozoites invasion of erythrocytes and was not toxic to the parasite’s intraerythrocytic development. Additionally, CTFR offers flexibility in the choice of combinations with several other DNA dyes, which broaden its usage for erythrocyte invasion assays, considering a wider range of flow cytometers with various laser settings.
In recent years, flow cytometry has become a cornerstone in investigating phenotypic diversity using multiple dyes to discriminate between donor and acceptor erythrocytes. To broaden the applicability of such assays, we optimized the staining conditions of a newly developed cytoplasmic dye, cell trace far-red (CTFR), and assessed its suitability for use in invasion phenotyping assays. We showed that CTFR has a very narrow emission peak excited by red lasers. Furthermore, CTFR labeling of target erythrocytes, achieved even at low concentrations, is stable over time and did not impair parasite development. erythrocyte invasion phenotyping assays revealed that CTFR is suitable for use in combination with several DNA dyes in multiplex assays. This will allow for high throughput phenotyping of parasites as well as facilitate the evaluation of preference of erythrocytes by merozoites. Altogether, these make screening for potential invasion-blocking interventions possible.
红细胞入侵表型分析是评估寄生虫多样性和毒力，以及描述配体-受体相互作用形成的非常有用的工具。然而，此类分析需要具有高度的灵敏度和重现性，并且区分供体和受体红细胞的标记染料的选择是一个关键因素。我们研究了细胞示踪远红（CTFR）作为一种染料用于入侵表型分析的适用性。我们使用羧基荧光素二乙酸琥珀酰亚胺酯（CFDA-SE）和二氯二甲基吖啶酮琥珀酰亚胺酯（DDAO-SE）作为比较染料，使用染料稀释方法评估 CTFR 在入侵表型分析中的适用性的局限性和特定染色程序。我们的数据表明，CTFR 有效地标记受体红细胞，并在相对较低的浓度下提供稳定的荧光强度。CTFR 产生的荧光强度也高于 DDAO-SE，并且随着时间的推移具有更稳定的荧光强度。此外，CTFR 不影响裂殖子入侵红细胞，并且对寄生虫的红细胞内发育没有毒性。此外，CTFR 提供了与几种其他 DNA 染料结合的灵活性，这拓宽了其在红细胞入侵测定中的用途，因为可以考虑使用具有各种激光设置的更广泛范围的流式细胞仪。
近年来，流式细胞术已成为使用多种染料区分供体和受体红细胞以研究表型多样性的基石。为了扩大此类分析的适用性，我们优化了新开发的细胞质染料细胞示踪远红（CTFR）的染色条件，并评估了其在入侵表型分析中的适用性。我们表明，CTFR 的发射峰非常狭窄，仅被红色激光激发。此外，CTFR 对靶红细胞的标记，即使在低浓度下，也能在很长一段时间内保持稳定，并且不会损害寄生虫的发育。红细胞入侵表型分析表明，CTFR 适合与几种 DNA 染料组合用于多重测定。这将允许高通量表型分析寄生虫，以及便于评估裂殖子对红细胞的偏好。总之，这些使筛选潜在的入侵阻断干预措施成为可能。
