Trypanosoma cruzi Intracellular Amastigotes Isolated by Nitrogen Decompression Are Capable of Endocytosis and Cargo Storage in Reservosomes

Epimastigote forms of Trypanosoma cruzi (the etiologic agent of Chagas disease) internalize and store extracellular macromolecules in lysosome-related organelles (LROs) called reservosomes, which are positive for the cysteine protease cruzipain. Despite the importance of endocytosis for cell proliferation, macromolecule internalization remains poorly understood in the most clinically relevant proliferative form, the intracellular amastigotes found in mammalian hosts. The main obstacle was the lack of a simple method to isolate viable intracellular amastigotes from host cells. In this work we describe the fast and efficient isolation of viable intracellular amastigotes by nitrogen decompression (cavitation), which allowed the analysis of amastigote endocytosis, with direct visualization of internalized cargo inside the cells. The method routinely yielded 5x107 amastigotes—with typical shape and positive for the amastigote marker Ssp4—from 5x106 infected Vero cells (48h post-infection). We could visualize the endocytosis of fluorescently-labeled transferrin and albumin by isolated intracellular amastigotes using immunofluorescence microscopy; however, only transferrin endocytosis was detected by flow cytometry (and was also analyzed by western blotting), suggesting that amastigotes internalized relatively low levels of albumin. Transferrin binding to the surface of amastigotes (at 4°C) and its uptake (at 37°C) were confirmed by binding dissociation assays using acetic acid. Importantly, both transferrin and albumin co-localized with cruzipain in amastigote LROs. Our data show that isolated T. cruzi intracellular amastigotes actively ingest macromolecules from the environment and store them in cruzipain-positive LROs functionally related to epimastigote reservosomes.


Introduction
Intracellular amastigotes can be isolated directly from infected spleen and liver [25] or from infected tissue culture cells [26,27] by density gradient centrifugation (using metrizamide and/or Percoll). Density gradient-based protocols are, however, very laborious and involve several medium changes, which can lead to a considerable loss of parasite viability. A different protocol, adapted from a method to purify T. cruzi metacyclic trypomastigotes [28,29], used anion-exchange chromatography to separate T. cruzi intracellular amastigotes from cell debris and trypomastigotes, after needle-based disruption of infected cells [30]. Although this is a robust option to purify amastigotes in a larger scale, chromatography is also time consuming, and the positively-charged resin may induce changes in the parasite's surface glycoconjugates.
In the present work, we introduce a rapid cavitation procedure to isolate T. cruzi amastigotes from Vero cells. In this protocol, cavitation by nitrogen decompression is followed by a few centrifugation steps (but no density gradients), allowing the rapid purification of viable intracellular amastigotes that are competent for endocytosis. Flow cytometry, fluorescence microscopy and western blotting analyses demonstrated that the T. cruzi amastigotes isolated using this new methodology were capable of internalizing transferrin and albumin from the extracellular milieu, and that these molecules were directed efficiently to LROs. Importantly, we detected co-localization of ingested transferrin and albumin with cruzipain, in LROs. Our data also suggest that the LROs of T. cruzi intracellular amastigotes correspond to reservosomes.
In vitro-derived T. cruzi metacyclic trypomastigotes were obtained by incubating epimastigotes in TAU3AAG medium, according to a previously described metacyclogenesis (i.e., epimastigote-to-trypomastigote differentiation) protocol [33]. After 72 h of cultivation in this medium,~80% of the cells in the supernatant were trypomastigotes.
Vero cells (ATCC CCL-81) were maintained in 75-cm 2 cell culture flasks (Corning Incorporated, Corning, NY, USA), in RPMI medium supplemented with 5% FBS, at 37°C (in a humidified 5% CO 2 atmosphere). Cells were infected with in vitro-derived metacyclic trypomastigotes (at a ratio of 10 parasites/cell), 24h after passage. After 4 h of interaction, host cell monolayers were washed with PBS (to remove non-adherent parasites) and then incubated in the same conditions with 7-10 ml DMEM medium supplemented with 10% FBS for four days, when trypomastigote production peaked. Then, culture supernatants were collected, and cell-derived trypomastigotes that had been released into the supernatant were harvested by centrifugation at 3,000g, for 15 min.
Axenic amastigotes were obtained by in vitro amastigogenesis [34]. Briefly, trypomastigotes were collected from the supernatant of infected Vero cell cultures (4-days post-infection), washed with PBS and incubated in high glucose DMEM medium, at pH 5, and 37°C (in a humidified 5% CO 2 atmosphere). After 24 hours, almost 100% of the cells had an amastigote shape.

Isolation of T. cruzi intracellular amastigotes by nitrogen cavitation
To isolate intracellular amastigotes from host cells, five culture flasks with 1x10 6 infected Vero cells each were washed three times with PBS (under gentle agitation), 48 hours post-infection (i.e., at the peak of intracellular amastigote replication), to eliminate contamination with extracellular amastigotes. Then, cultures were trypsinized for 10 min at 37°C, and the trypsinization was halted by the addition of cold FBS 1:1 (v/v). The infected cells in suspension were lysed by nitrogen decompression (cavitation), in a 4639 cell disruption vessel (Parr Instrument Company, Moline, IL, USA), using 180 psi pressure, for 5 min. Intact Vero cells were removed by centrifugation at low speed (10 min, 800g), and intracellular amastigotes were recovered from the supernatant. Cell debris were removed from the amastigote fraction by three centrifugation steps at 2,000g for 5 minutes, to produce a supernatant of isolated intracellular amastigotes.

Endocytosis assay
Isolated intracellular amastigotes, axenic amastigotes and culture epimastigotes (positive control) were subjected to a previously described endocytosis assay [5], using 2x10 6 parasites for flow cytometry analyzes, 5x10 6 for fluorescence microscopy studies and 10 7 for western blotting. Briefly, after 15 min under stress in PBS at 25°C, parasites were incubated with 50 μg/ml transferrin-AlexaFluor 633 or albumin-AlexaFluor 488, for 30 min, at 37°C (amastigotes) or 28°C (epimastigotes). Alternatively, parasites were incubated at 4°C to allow transferrin binding without internalization [6], or the retention of albumin in the flagellar pocket [10]. Negative control cells were incubated in the absence of labeled transferrin or albumin.

Transferrin dissociation assay
Membrane-bound transferrin was dissociated using a previously described method [17], with modifications. Parasites were subjected to endocytosis assays as described above (using 50 μg/ml transferrin-AlexaFluor 633) and then incubated for 5s at 4°C with 0.25 M acetic acid/ 0.5 M sodium chloride, pH 3.5, after which the pH was quickly neutralized with 1 M sodium acetate. Transferrin dissociation from the parasite's surface was analyzed by western blotting and flow cytometry, as described below.
Transferrin band densitometry was performed using the ImageJ 1.45s software (National Institute of Mental Health-NIMH, Bethesda, Maryland, USA). Integrated band densities estimated by ImageJ were used to calculate the ratios between the signal at 28°C (epimastigotes) or 37°C (amastigotes) and that at 4°C, for both untreated and acetic acid-treated parasites.

Flow cytometry
Live parasites that had internalized transferrin-AlexaFluor 633 or albumin-AlexaFluor 488 were analyzed in a FACSAria II flow cytometer (Becton-Dickinson, San Jose, USA), using 660/ 20 and 530/30 nm band-pass filters, respectively. A total of 20,000 events were acquired in the regions of the FSC x SSC plot previously shown to correspond to parasites [37]. Data analysis was performed using the FlowJo software (FlowJo, Ashland, OR, USA), and the normalized median fluorescence intensities of transferrin and albumin were calculated as the ratios between the median fluorescence intensities of treated and untreated cells. Statistical analysis was performed using the software GraphPad Instat (GraphPad Software Inc, La Jolla, CA, USA), by ANOVA followed by Tukey-Kramer multiple comparisons test.
To detect the amastigote-specific surface protein Ssp4 [38], in vitro-derived metacyclic trypomatigotes and amastigotes (intracellular and axenic) were washed twice in PBS and fixed for 15 min in 4% formaldehyde. Cells were then incubated for 30 min at 28°C with an anti-Ssp4 monoclonal antibody [38] diluted to 1:3200, in PBS (pH 7.2). Samples were washed with PBS and incubated for 30 min at 28°C with a goat anti-mouse secondary antibody coupled to Alexa-Fluor 488 (1:1000 in PBS). Cells were washed with PBS and immediately analyzed by flow cytometry (as described above), using a 530/30 nm band pass filter. In parallel, anti-Ssp4 stained cells were adhered to poly-L-lysine coated slides and observed under a Nikon E600 epifluorescence microscope (Nikon Instruments, Tokyo, Japan).

Immunofluorescence microscopy
To detect native cruzipain, isolated amastigotes and culture epimastigotes were washed twice in PBS, fixed for 30 min with 4% paraformaldehyde, permeabilized for 5 min in 0.5% Triton in PBS, and incubated for 2 h at 37°C with 50 μg/ml anti-cruzipain monoclonal antibody (CZP-315.D9; [39]), in PBS. Samples were then washed three times in PBS and incubated, for 2 h at 37°C, with a goat anti-mouse secondary antibody coupled to AlexaFluor 488 diluted to 1:600 in 'incubation buffer' (PBS with 1.5% BSA).
To co-localize transferrin-AlexaFluor 633 or albumin-AlexaFluor 488 with cruzipain, parasites that had been subjected to endocytosis assays were fixed for 30 min in 4% paraformaldehyde and permeabilized for 5 min with 0.5% Triton in PBS. For co-localization with transferrin-AlexaFluor 633, samples were incubated with both CZP-315.D9 (50 μg/ml) and the anti-transferrin goat antiserum (to enhance the AlexaFluor 633 signal; diluted to 1:150 in incubation buffer), washed three times in PBS and then incubated with goat anti-mouse-AlexaFluor 488 (to detect cruzipain) and rabbit anti-goat-AlexaFluor 594 (to detect transferrin) secondary antibodies (diluted to 1:600 in incubation buffer). For co-localization with albumin-Alexa-Fluor 488, samples were incubated with CZP-315.D9 and then with a goat anti-mouse-AlexaFluor 594 secondary antibody (using the same dilution conditions mentioned above).
After antibody incubations, all samples were washed three times in PBS, incubated for 5 min with 1.3 nM Hoechst 33342, mounted with Prolong Gold antifade reagent and examined under a Nikon Eclipse E600 epifluorescence microscope.

Results and Discussion
Rapid and efficient isolation of viable T. cruzi intracellular amastigotes by nitrogen decompression T. cruzi epimastigote forms internalize albumin and transferrin and store these molecules in reservosomes, the late endocytic organelles in this parasite stage [5,8]. In contrast, little is known about the endocytic activity of the proliferative intracellular amastigote form, which prevails in chronic chagasic patients and represents the most clinically relevant form of this parasite [40]. Thus, the aim of our study was to analyze the endocytic activity of amastigotes by using transferrin and albumin as probes.
Given that the study of endocytosis by intracellular amastigotes is hindered by the concomitant endocytic activity of host cells, we first attempted to make fluorescent-labeled endocytic tracers available to amastigotes directly in the host cell's cytoplasm, using mild detergent treatments (with saponin and lysolecithin) of infected cells; however, host cells were killed by the lowest detergent concentrations required for transferrin entry into the cytoplasm (data not shown).
Therefore, we developed a protocol to isolate viable, endocytosis-competent intracellular amastigotes from infected host cells, using nitrogen decompression (also known as nitrogen cavitation). Nitrogen decompression is an effective method for lysis and homogenization of cell suspensions [41], and is based on oxygen-free nitrogen dissolution under high pressure. After a sudden release of the pressure, nitrogen 'bubbles out' from the solution, including the cell interior, culminating in plasma membrane disruption. Depending on the cell type, higher pressures (leading to higher nitrogen dissolution) are needed for effective lysis. Mammalian cells are successfully disrupted after the release of 250-500 psi of pressure, while smaller cells with a resistant cell wall, such as bacteria, need around 1,500 psi [41]. Trypanosomatids have a sub-pellicular corset of microtubules that maintains parasite shape and provides resistance to mechanical pressure [42]. Hence, higher pressures are required to disrupt T. cruzi cells by cavitation, when compared with those needed to lyse mammalian cells, and we explored this difference efficiently to release intracellular amastigotes from infected cells.
Cell disruption by nitrogen decompression allowed the release of~5x10 7 amastigotes per 5x10 6 infected Vero cells (NIH 3T3 fibroblasts and H9C2 myoblasts were also used successfully for intracellular amastigote isolation; data not shown). Morphological analysis by light microscopy showed that the parasites obtained by nitrogen cavitation had the typical amastigote shape (Fig 1A). Flow cytometry analysis of parasites labeled for the amastigote-specific surface protein Ssp4 showed that isolated intracellular amastigotes had strong Ssp4 expression, similar to that observed in axenic amastigotes (Fig 1B), while trypomastigotes and epimastigotes did not express Ssp4. These data confirmed that the isolated parasites were indeed amastigotes. Interestingly, isolated intracellular amastigotes had a narrower profile of Ssp4 expression than axenic amastigotes, indicating that axenic amastigotes constitute a more heterogeneous population formed by parasites in a variety of differentiation stages.
Infected Vero cells containing intracellular amastigotes were more susceptible to cavitation than uninfected ones, and could be lysed effectively with as little as 180 psi, a condition that did not affect intracellular amastigotes (S1 Fig). Flow cytometry using a GFP-fluorescent T. cruzi clone [43] showed that, after cavitation, almost all infected Vero cells were disrupted, releasing GFP-positive viable intracellular amastigotes (S1 Fig). In contrast, uninfected Vero cells could only be lysed efficiently after the release of 350-400 psi, which also disrupted~25% of intracellular amastigotes (not shown). Hence, by performing nitrogen cavitation at 180 psi, only infected cells were disrupted, resulting in increased recovery of intact amastigotes and a reduction in the amount of cell debris, which could be removed easily by successive washes  Fig 1A). We used a 5-min cavitation period routinely, and longer cavitation periods (10 min) did not increase parasite yield (data not shown).
We confirmed the expression of Ssp4 by indirect immunofluorescence, and also to analyzed the localization of the reservosome/LRO marker cruzipain in intracellular amastigotes ( Fig  1C). We observed a strong Ssp4 signal on the cell surface of isolated intracellular amastigotes (Fig 1C), as previously described for extracellular amastigotes obtained from the transformation of blood or cell culture trypomastigotes [38]. The anti-cruzipain monoclonal antibody CZP-315.D9 recognized an LRO-like structure posterior to the nucleus (Fig 1C), a localization compatible with that of LROs [13].
Previously described methods for the isolation of T. cruzi amastigotes are laborious and involve several medium changes and/or anion-exchange chromatography [25][26][27][28][29], with considerable loss of parasite viability and likely changes in the amastigote surface glycoconjugates (due to the interaction with the positively-charged resin). The nitrogen decompression method for intracellular amastigote isolation described here is easier and faster than previously described protocols, and yields viable amastigotes efficiently, and is likely to represent an excellent tool for studies on different aspects of amastigote biology.

Isolated intracellular amastigotes internalize fluorescently-labeled transferrin
Most data suggestive of endocytosis by amastigotes is indirect [19][20][21][22][23]. Soares and De Souza (1991) showed direct evidence of transferrin binding to the surface of cell-derived amastigotes, but not of transferrin internalization, suggesting a low level of endocytic activity by amastigotes [5]. In our work we have also tried to visualize transferrin endocytosis directly by transmission electron microscopy (TEM) in isolated intracellular amastigotes, but no intracellular transferrin-gold complexes were observed (data not shown). These results were likely due to the relatively low endocytic activity of amastigotes, compared with that of epimastigotes, where cargogold complexes can be readily detected by TEM. Thus, we attempted to detect endocytosis in isolated intracellular amastigotes by more sensitive techniques than TEM, such as western blotting and flow cytometry.
Using western blotting, we confirmed the presence of holo-transferrin (MW 76-81 kDa) in protein extracts of amastigotes and epimastigotes (positive control cells) subjected to a transferrin uptake assay (Fig 2). The anti-TcGAPDH antiserum used as a loading control recognized a polypeptide of the correct molecular weight (~40 kDa) in all T. cruzi developmental forms (Fig 2A).
Whole cell extracts of isolated intracellular amastigotes incubated at the endocytosis-permissive temperature of 37°C (likely to include both surface bound and internalized transferrin) contained more transferrin than extracts of parasites incubated at 4°C, when endocytosis does not occur and, thus, only surface-bound transferrin should be detected (Fig 2A). After a rapid (5s) acetic acid treatment, which leads to the dissociation of surface-bound transferrin [20], holo-transferrin levels in amastigotes incubated at 37°C remained similar to those observed without acetic acid treatment. In epimastigotes, transferrin localizes inside the flagellar pocket and the cytostome/cytopharynx complex, and then accumulates in the reservosomes [5,8]. While the intracellular pool of labeled transferrin is not accessible to acetic acid treatment, this treatment is expected to remove most surface-bound transferrin, in parasites incubated at 4°C. In this study, we observed a residual signal after acetic acid treatment in parasites incubated at 4°C (Fig 2A), probably due to surface-bound transferrin that could not be readily removed from to the interior of the cytostome/cytopharynx or from the flagellar pocket (Fig 2A). Densitometry analysis showed that the ratio between the transferrin signal at 28°C and that at 4°C was higher for parasites treated with acetic acid compared with untreated parasites (Fig 2B), in agreement with the hypothesis that acid treatment removed the predominantly surface-bound transferrin at 4°C, while the internalized transferrin prevalent in parasites incubated at 37°C was resistant to acid treatment.
Overall, the western blotting data suggest that transferrin binds to the surface of amastigotes (at 4 and 37°C) and is internalized by parasites (at 37°C). A positive control for endocytosis using epimastigotes (at an endocytosis-permissive temperature of 28°C, instead of 37°C) showed a very similar pattern to that observed for amastigotes, supporting our interpretation of the amastigote data.

Isolated intracellular amastigotes have higher intracellular levels of labeled transferrin than axenic amastigotes
Flow cytometry analysis provided further support to the notion that transferrin is internalized by isolated intracellular amastigotes, since the transferrin-AlexaFluor 633 signal in these parasites increased significantly (p<0.001) when endocytic assays were performed at the endocytosis-permissive temperature 37°C, compared with samples incubated at 4°C (Fig 3A). The fluorescence signal increased in cells incubated at 4°C, compared with controls without transferrin, in line with the western blotting data that suggest that binding of transferrin to the surface of isolated intracellular amastigotes occurs at this temperature. The transferrin-AlexaFluor 633 signal decreased after acetic acid treatment in isolated intracellular amastigotes incubated Endocytosis in Isolated T. cruzi Amastigotes at 4°C, and also in those incubated at 37°C. However, the fluorescence intensity of cells incubated at 37°C and treated with acetic acid remained higher than that of cells incubated at 4°C, but not subjected to acid treatment (Fig 3A). These results suggest that a considerable proportion of the labeled transferrin in cells incubated at 37°C was found in internal organelles and, thus, was resistant to acid treatment (Fig 3A).
Similar results were obtained for a positive control for endocytosis using epimastigotes (at an endocytosis-permissive temperature of 28°C, instead of 37°C): the fluorescence signal was higher in cells incubated at 28°C, when compared with those incubated at 4°C (Fig 3A). Also, acetic acid treatment decreased the fluorescence intensity in both cases, although a higher signal remained in cells incubated at 37°C, indicating transferrin internalization.
Interestingly, we observed a relatively low fluorescence signal in axenic amastigotes incubated at 37°C, similar to that seen in cells incubated at 4°C (Fig 3A). These data indicate that axenic amastigotes ingest only a limited amount of transferrin and, therefore, do not represent a reliable model for studies on T. cruzi endocytosis. Normalized medians of the fluorescence peaks showed that the endocytic activity is higher in epimastigotes than in isolated intracellular amastigotes, while axenic amastigotes showed only minimal endocytic activity (Fig 3B).

Labeled transferrin co-localizes with cruzipain in lysosomal-related organelles (LROs) of isolated intracellular amastigotes
To examine the destination of the internalized transferrin, isolated intracellular amastigotes were subjected to endocytosis assays and then processed for immunofluorescence for the detection of transferrin and also of cruzipain, a marker of LROs (i.e., the reservosomes), the final destination of internalized transferrin in epimastigotes. In positive control cells (epimastigotes) incubated at 4°C, only cruzipain was detected in the reservosomes, located at the posterior end of the cell (Fig 4A and 4D), while transferrin was found at the anterior end of the cell (likely Endocytosis in Isolated T. cruzi Amastigotes corresponding to the cytostome) and in dense spots close to the nucleus, likely corresponding to the bottom of the cytopharynx [9,17,44]. In epimastigotes incubated at 28°C there was co-localization of transferrin with cruzipain at the reservosomes (Fig 4E and 4H), as previously shown [11,12,39].
As expected, transferrin labeling in isolated intracellular amastigotes (4 or 37°C) was not intense, but was clearly detectable in most cells. No co-localization with cruzipain occurred at 4°C: transferrin labeling was prominent at a discrete spot close to the nucleus and the kinetoplast (corresponding to the location of the bottom of the cytopharynx), while cruzipain localized to the posterior region of the cell (Fig 5A and 5B). In contrast, we detected co-localization of transferrin and cruzipain in LROs at the posterior region of the cell, in amastigotes incubated at 37°C, although co-localization was not clear in all cells (Fig 5C and 5D). A similar result was obtained when amastigotes were subjected to endocytosis assays and then treated with acetic acid (Fig 5E and 5F).These data indicate a low level of protein uptake; nevertheless, internalized transferrin was delivered to (and stored in) cruzipain-positive LROs at the posterior of the cell.
Ingested albumin co-localizes with cruzipain in isolated intracellular amastigotes, confirming a storage function for amastigote LROs To examine the internalization of a different endocytic marker, we analyzed the uptake of albumin-AlexaFluor 488 complexes by isolated intracellular amastigotes, using flow cytometry. As expected, we observed an intense fluorescence signal indicative of albumin internalization in epimastigotes (i.e., positive control cells) incubated with Albumin-AlexaFluor 488 at 28°C; this signal was significantly higher (p<0.001) than that observed in controls cells incubated at 4°C or not given the labeled tracer (Fig 6). However, only a weak albumin-AlexaFluor 488 fluorescence signal was detected in isolated intracellular amastigotes, regardless the experimental condition (Fig 6), indicating minimal endocytosis of albumin by this developmental form.
Despite the failure to detect albumin-AlexaFluor 488 internalization by flow cytometry, we could detect endocytosis of this labeled tracer by isolated intracellular amastigotes using fluorescence microscopy (Fig 7); however, labeling was relatively weak and not observed in all cells. Control experiments with epimastigotes showed that, at 4°C, albumin-AlexaFluor 488 complexes were occasionally found at the flagellar pocket region, but did not co-localize with cruzipain in the reservosomes (Fig 7A). However, at 28°C albumin and cruzipain co-localized in reservosomes, as shown here for the first time (Fig 7B). Similar results were obtained with isolated intracellular amastigotes: at 4°C no co-localization was observed (Fig 7C), while at 37°C albumin and cruzipain occasionally co-localized at LROs (Fig 7D). At both temperatures cruzipain was also detected in the anterior region of cell, maybe en route to LROs and/or to the secretory pathway. These results, combined with the flow cytometry data, indicate that amastigotes are capable of albumin uptake, albeit only at low levels.
Thus, contrary to the suggestion that amastigote LROs are not bona fide components of the endocytic pathway [13], our findings show that T. cruzi amastigote forms internalize Cells were allowed to ingest transferrin-AlexaFluor 633 and were then incubated with anti-transferrin antibody (in red) and anti-cruzipain mAb (CZP-315.D9; in green). (A-B) Isolated intracellular amastigotes incubated without transferrin at 4°C or 37°C, showed no transferrin labeling (negative control), while cruzipain was located at the posterior region of the cell. (C) Isolated intracellular amastigotes incubated with transferrin at 4°C. No co-localization with cruzipain was observed. (D) Isolated intracellular amastigotes incubated with transferrin at 37°C. Co-localization with cruzipain was observed at the posterior region of the cell. (E-F) Isolated intracellular amastigotes incubated with transferrin at 4°C and 37°C and then subjected to acetic acid treatment. No colocalization of transferrin with cruzipain was observed at 4°C (E), while co-localization with cruzipain occurred at the posterior region at 37°C (F). The nucleus (n) and the kinetoplast (k) were stained with Hoechst 33342 (in blue). DIC, differential interference contrast. Scale bars, 2.5 μm. doi:10.1371/journal.pone.0130165.g005 Endocytosis in Isolated T. cruzi Amastigotes macromolecules and target ingested cargo to cruzipain-positive LROs. To our knowledge, these data represent the first direct evidence that amastigote LROs correspond to the reservosomes observed in epimastigote forms (Fig 8).

Conclusions
Host cell lysis by nitrogen decompression (cavitation) allows rapid and efficient isolation of viable T. cruzi intracellular amastigotes that have the typical amastigote shape and are positive for the amastigote-specific antigen Ssp4. Our data show that isolated intracellular amastigotes of T. cruzi display endocytic activity, although at lower levels than epimastigotes. Also, we show that internalized cargo (transferrin and albumin) accumulates in amastigote LROs, a clear indication that these organelles are functionally related to the reservosomes observed in epimastigotes. Fluorescence microscopy analysis of albumin-AlexaFluor 488 endocytosis by isolated intracellular amastigotes of T. cruzi. Cells were allowed to ingest albumin-AlexaFluor 488 (in green) and were then labeled with the anti-cruzipain mAb CZP-315.D9 (in red). In epimastigotes (A) and isolated intracellular amastigotes (C) incubated at 4°C, we did not observe co-localization of albumin (at the anterior end of the cell, nearest to the kinetoplast -k) and cruzipain (at the posterior end of the cell, nearest to the nucleus-n). In contrast, in epimastigotes (B) and isolated intracellular amastigotes (D) incubated at endocytosis-permissive temperatures (28°C or 37°C, respectively), we detected co-localization of albumin and cruzipain at the posterior region of the cell. The nucleus (n) and kinetoplast (k) are stained with Hoechst 33342 (in blue). DIC, differential interference contrast. Scale bars, 5 μm.