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

Abstract

Plasmodium falciparum 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 P. falciparum 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 P. falciparum 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 P. falciparum 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 P. falciparum 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 P. falciparum 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. P. falciparum 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.

Keywords: Cell trace far-red; Plasmodium falciparum; erythrocyte invasion; flow cytometry.

Figure 1.

Gating strategy of data generated by CTFR staining. (a): Pseudocolor plot of all events gated in the forward scatter area (FSC-A) by side scatter area (SSC-A). (b): Selection of single erythrocytes using forward scatter hight (FSC-H) by the FSC-A. (c–d): Dot plot and histogram plot showing CTFR fluorescent intensity by the SSC-A and number of erythrocytes count, respectively. PMT voltages were adjusted so that CTFR-stained erythrocytes register between 10³ and 10⁵ on the logarithmic scale. Black dots represent outliers.

Figure 2.

Labeling conditions of acceptor erythrocytes with CTFR. Erythrocytes were labeled using different concentrations of CTFR diluted in different solvents – 1× PBS (a–b) or RPMI 1640 (c–d) – at different hematocrits – 4% (a and c) or 8% (b and d) – and incubated at 37°C for 2 h in a shaking incubator. Labeled erythrocytes were further mixed with unlabeled erythrocytes and analyzed by flow cytometry.

Figure 3.

Graph showing the variation of CTFR stain index at two-time points, day 0 and day 15 post-staining. To monitor the stability of CTFR, labeled erythrocytes were co-incubated with unlabeled erythrocytes for at least 24 h prior to data acquisition by flow cytometry. CTFR SI was monitored for 15 days and compared to that of CFDA-SE and DDAO-SE.

Figure 4. CTFR labeling does not impair P. falciparum erythrocyte invasion and normal parasite growth. Erythrocytes labeled with either CFDA-SE (20 µM), DDAO-SE (10 µM), or CTFR (2 µM) were individually incubated with equal volumes of schizont-infected erythrocytes from 3D7 and Dd2 parasite cultures under normal P. falciparum culturing conditions. (a) Graphs showing the parasite growth patterns for two successive asexual replication cycles in labeled erythrocytes relative to the unlabeled control erythrocytes. (b) Giemsa-stained parasites showing the parasite morphology in unstained (black squares), CFDA-SE-stained (blue squares), DDAO-SE-stained (green squares), and CTFR-stained erythrocytes (red squares). Culture aliquots harvested at regular time intervals were fixed with 100% methanol and stained with 10% Giemsa on glass microscope slides. Images were taken with a Cole Palmer light microscope (Vernon Hills, Illinois 60061, USA) coupled with a MoticamBTW8 camera (Motic China Group Co., Ltd).

Figure 5.

Invasion phenotypes of two P. falciparum laboratory strains: 3D7 and Dd2 into enzyme-treated erythrocytes (define abbreviations here). Schizont-infected unlabeled erythrocytes were co-incubated in a 1:1 ratio with either DDAO-SE or CTFR-labeled erythrocytes, at 37°C for 18–24 h. Following incubation, parasites were stained with either Hoechst 33342 or SYBR Green I and final parasitemias in the target populations were determined by gating on the fluorescently labeled erythrocyte population using flow cytometry. Invasion efficiencies were determined as a percentage of the final parasitemia of a mock-treated and labeled positive control erythrocyte population. The data are presented as the mean ± standard error of the mean of two independent experiments conducted in triplicates. UT: untreated; NT: neuraminidase treatment; TT: trypsin treatment; CT: chymotrypsin treatment.