Filtermat a

To determine cell proliferation in the stimulated cultures, at day 5 tritium thymidine (18 kBq/well) was added to the 96-well plates for overnight incubation at 37°C with 5% CO₂, to be incorporated in the cellular DNA with every cell division. Cells were harvested on a filter (Filtermat A, Perkin Elmer, Oss, The Netherlands) and incorporated label was determined as counts per minute (cpm) using a MicroBeta Counter (Perkin Elmer). Mean cpm from quadruplicate wells were used to calculate the Stimulation Indices (SI) by dividing the mean cpm of stimulated cultures by the mean medium control. Results are shown as mean SIs per group with standard deviations. SIs >2 were considered positive and in single peptide stimulated cultures indicative for the identification of an epitope. Immunodominance of epitopes was determined based on ranking of SIs in response to single synthetic peptides per mouse strain. The shared sequence between overlapping peptides with SIs >2 were considered to represent the epitope core.

Trophozoite stage parasites (1 % parasitaemia and 0.5 % haematocrit), different concentrations of test samples (≤100 μg/mL) and chloroquine as standard anti-malarial drug were used in the assays. After 24 h incubation period, 25 µL of medium containing [³H]-hypoxanthine (0.5 µCi/well) (PerkinElmer) was added per well, followed by another 18 h incubation at 37 °C [24 (link)]. The cells were harvested (Cell Harvester PerkinElmer) on glass fibre filters (Filtermat A, PerkinElmer) then placed onto sample bags (PerkinElmer) and immersed in scintillation fluid (UltimaGold, PerkinElmer). Radioactive emission was counted in a 1450 Microbeta TriLux Microplate Scintilation and Luminescence Counter (PerkinELmer). The inhibition of parasite growth was evaluated from the levels of [3H]-hypoxanthine incorporation, i.e., IC₅₀ values were evaluated by comparing the incorporation in drug-free control cultures and estimated by linear interpolation [25 (link)] using curve fitting software (Microcal Origin software 8.5). All experiments were performed three times, and each sample was tested in triplicate.

Approximately 16 h after ²¹³Bi-irradiation, cells were plated in quadruplicates at 4 × 10⁵ cells/mL in 100 μL in 96-well flat-bottom microtiter plates and incubated at 37°C. Forty-two hours after irradiation, 10 μL of ³H-thymidine (925 kBq/mL; Perkin Elmer) was added to each well and incubated at 37°C. Six hours later (i.e., 48 h after irradiation), cells were harvested (Harvester 96 – Tomtec) on glass fiber Filtermat A (Perkin Elmer). Radioactive emission was amplified with Betaplate Scint (Perkin Elmer) and read using 1450 Microbeta Plus counter (Wallac). Results are expressed as quadruplicate mean ± SD.

The amounts of cell-free synthesized proteins were quantified by measuring trichloroacetic acid (TCA)-insoluble radioactivity using a liquid scintillation counter (MicroBeta2, PerkinElmer, Waltham, MA). Radioactive L-[U-¹⁴C]-leucine added to the CFPS reaction mixture along with the 20 amino acids and carried out the standard CFPS reaction at 30°C for 20 h. The CFPS reaction was quenched by adding an equal volume of 0.5 M of potassium hydroxide and incubated at 37°C for 20 min. The same amounts of samples were spotted on two separate fiberglass paper sheets (Filtermat A, PerkinElmer, Waltham, MA) and dried at room temperature for 30 min. One of the fiberglass paper sheets was subjected to washing three times with 5% TCA solution at 4°C for 10 min. The sheet was rinsed in 100% ethanol at room temperature for 10 min to remove non-precipitated samples and was dried at room temperature for 40 min. The meltable cocktail (MeltiLex A, PerkinElmer, Waltham, MA) was heated to 95°C, and then radioactivity was measured using the liquid scintillation counter. The total and soluble yields were determined as described previously (Swartz et al., 2004 (link)).