Materials and Reagents

Pluronic acid F-127 (Thermo Fisher Scientific, Invitrogen^TM, catalog number: P3000MP )
20% (w/v) stock in DMSO. An amphiphilic mild detergent that facilitates Fura-2 AM solubilization
Probenecid (Thermo Fisher Scientific, Invitrogen^TM, catalog number: P36400 )
An inhibitor of the anionic transporters improving intracellular accumulation of the fluorophore
Fura-2 acetoxymethyl ester (Fura-2 AM) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: F1225 )
Digitonin (Merck, Sigma-Aldrich^TM, catalog number: 300410 )
EGTA (Ethylene-bis(oxyethylenenitrilo)tetraacetic acid) (Merck, Sigma-Aldrich^TM, catalog number: E3889 )
Calcium chloride (CaCl₂) (Merck, Sigma-Aldrich^TM, catalog number: C1016 )
Potassium chloride (KCl) (Merck, Sigma-Aldrich^TM, catalog number: P9333 )
Potassium phosphate monobasic (KH₂PO₄) (Merck, Sigma-Aldrich^TM, catalog number: P0662 )
Sodium phosphate dibasic (Na₂HPO₄) (Merck, Sigma-Aldrich^TM, catalog number: S0876 )
Magnesium sulfate (MgSO₄) (Merck, Sigma-Aldrich^TM, catalog number: M7506 )
Sodium chloride (NaCl) (Merck, Sigma-Aldrich^TM, catalog number: S7653 )
HEPES (Merck, Sigma-Aldrich^TM, catalog number: H0887 )
Glucose (Merck, Sigma-Aldrich^TM, catalog number: G8270 )
Modified Tyrode’s loading buffer (MTLB) (pH 7.4) (see Recipes)
MTLB without Ca²⁺(pH 7.4) (see Recipes)

Equipment

Neubauer chamber
Spectrofluorometer with Fast Filter Accessory (PerkinElmer, model: LS-55 ) or Spectrofluorometer HITACHI DW 7000
Benchtop Centrifuge (Eppendorf, model: 5415D )
Quartz SUPRASIL Macro/Semi-micro Cell (PerkinElmer, catalog number: B0631132 )

Software

FL WinLab (PerkinElmer)
Excel^® (Microsoft)
OriginPro 6 (OriginLab Corporation, https://www.originlab.com/)

Procedure

Stock solutions/Buffers
1. Digitonin (MW: 1229.3 g/mol) stock at 5 mM in distilled water.
2. EGTA (MW: 380.35 g/mol) stock at 500 mM (pH 8) in distilled water.
3. Fura-2 AM (MW: 1001.86 g/mol) stock at 1 mM in DMSO.
4. Modified Tyrode’s loading buffer (MTLB) (see Recipes).
5. MTLB without Ca²⁺(see Recipes).
6. Probenecid (MW: 285.36 g/mol) stock at 1 M in DMSO.
Parasite loading with Fura-2 AM
1. Count parasites using a Neubauer chamber and calculate the title.
2. Collect 1 x 10⁸ parasites at exponential phase by centrifugation at 600 x g for 2.5 min and discard the supernatant.
3. Wash twice with 500 µl of MTLB at 29 °C and discard supernatant.
4. Resuspend the parasites in 500 µl MTLB and add 1 µl Fura-2 AM (1:1,000 from stock to final concentration 1 µM), 1.2 µl probenecid (3:1,250) from stock to final concentration 2.4 mM), and 1.25 µlpluronic acid (1:400 from stock to final concentration 0.05%).
5. Incubate the parasites at 29 °C at darkness with continuous stirring for 2 h.
6. Wash Fura-2 loaded parasites with 500 µl of loading buffer, in either the presence or absence of Ca²⁺.
7. Transfer 500 µl of the parasite suspension to a quartz cuvette and place it in a spectrofluorometer with gentle stirring at 29 °C.
8. Adjust the spectrofluorometer at 340 and 380 nm for excitation (Ex) and at 510 nm for emission (Em).
9. Start measurement using FL WinLab software.
Controls
To end the experiment, add the followings reagents (the data they generated are needed for analysis of results):
1. When the monitor trace is stable (constant slope), meaning that Ca²⁺ is no changing further add digitonin at 30 µM to obtain the maximal ratio of fluorescence (R_max).
2. Wait until the monitor trace is stable again and add EGTA at 10 mM to obtain the minimal ratio of fluorescence (R_min).

Data analysis

Each experiment must be performed at least in triplicates.
[(Ca²⁺)_i] is evaluated as described by Grynkiewicz et al. (1985) by applying the following equation:

[Ca²⁺]_i= K_d× (R – R_min/R_max – R) × F_min/F_max

K_d: Dissociation constant of Fura-2 (244 nM).
R: Ratio of the fluorescence emission at 510 nm obtained by alternating the excitation between 340 nm and at 380 nm by mean of the fast filter accessory at a given experimental condition.
R_max: Ratio of the fluorescence emission at 510 nm obtained by alternating the excitation between 340 nm and at 380 nm under saturated Ca²⁺ concentrations (in the presence of digitonin to permeabilize the parasite plasma membrane).
R_min: Ratio of the fluorescence emission at 510 nm obtained after alternating the excitation between 340 nm and at 380 nm in the absence of Ca²⁺ (in the presence of digitonin and presence of EGTA to chelate any remanent Ca²⁺).
F_max: Fluorescence of Fura-2 at 380 nm under saturated Ca²⁺ concentrations
F_min: Fluorescence of Fura-2 at 380 nm in the absence of Ca²⁺

Maximum and minimum values are obtained after the addition of 30 µM digitonin, which allows Ca²⁺ flow into the interior of the cell and thus, Fura-2 will display maximal fluorescence. Then, 10 mMEGTis added to chelate all Ca²⁺ to nominal 0 level (minimal fluorescence).
Post acquisition data must be converted to a suitable format for Excel^®, and then those rows and columns should be copied on the OriginPro software and be plotted as a XY graph, where the X is the time values and Y 340/380 relation values or intracellular Ca²⁺ concentration (nM).

Notes

It is strongly recommended the use of recently (maximum 1 month) isolated parasites from mice, instead of parasites after many culture passages. Fura-2 AM must be cleaved to Fura-2 and this cleavage is carried out by unspecified intracellular esterases. Parasites recently obtained from infected mice (or infected macrophages) have much higher levels of esterases, when compared to parasites after many passages, since these enzymes are lost by selection in frequently passed parasite culture.
The incubation time for a successful parasite load can vary extremely, depending on the amount of esterases present in the parasite population at a given moment, which is very variable. In some cases a 2 h incubation is enough, but in others, loading time can take even 6 h. Even more, in some cases loadingwithFura 2 may not be achieved, because of total lack of esterases in that particular batch.
Since Fura-2 AM is photosensitive, it is strongly recommended to cover aliquots with aluminum foil.
It is well known that Fura-2 AM and other acetoximethyl esters from other fluorescent Ca²⁺ indicators (as Rhod 2-AM and Fluo 3-AM) can be accumulated and transformed to the fluorescent species inside organelles like endoplasmic reticulum, mitochondrion and nucleus. In this regard, combinations of incubation time and temperature for loading the fluorescence probes must be taken in consideration to avoid this phenomenon. On the other hand, this could nevertheless constitute a large advantage, because this can be rationally used to load these organelles with the fluorescent probes, by manipulating incubation time and temperature, and thus performing difficult tasks as study Ca²⁺ movements inside mitochondria and endoplasmic reticulum, otherwise very difficult to measure (for example, see Benaim et al., 2006).

Recipes

1.Modified Tyrode’s loading buffer (MTLB) (pH 7.4)
137 mM NaCl
4 mM KCl
1.5 mM KH₂PO₄
8.5 mM Na₂HPO
11 mM glucose
1 mM CaCl₂
0.8 mM MgSO₄
20 mM HEPES-NaOH
MTLB without Ca²⁺ (pH 7.4)
137 mM NaCl
4 mM KCl
1.5 mM KH₂PO₄
8.5 mM Na₂HPO₄
11 mM glucose
0.8 mM MgSO₄
100 µM EGTA
20 mM HEPES-NaOH

Acknowledgments

Supported by Fondo Nacional de Ciencia, Tecnología e Investigación, Venezuela (FONACIT) (grants 2017000274 and 2018000010) and the Consejo de Desarrollo Científico y Humanístico-Universidad Central de Venezuela (CDCH-UCV) grant PG-03-8728-2013/2 to G.B.This protocol was essentially extracted from Benaim et al. (2014) and Rodriguez-Duran et al. (2019)

Competing interests

There are no conflicts of interest or competing interest.

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