Cell trace far-red is a suitable erythrocyte dye for multi-color invasion phenotyping assays.

Unlabelled: 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.

Impact Statement: 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.