96 multiwell plate

The parasite load was determined by limiting dilution assay [21 (link),52 (link)]. In VL, parasitism was quantified in the spleen and liver, while in cutaneous leishmaniasis, it was analyzed in the skin at the point of parasite inoculation. Briefly, the skin, spleen, and liver were individually weighed, homogenized in S10, and then diluted 1:2000 (skin) or 1:500 (spleen and liver). An initial homogenized suspension was placed into the first well (200 μL) and serial dilutions (1:4) were distributed in a 96-multiwell plate (Nunc, Germany) and subjected to 12 serial dilutions with four replicate wells. After 10 days at 25 °C, each well was examined by optical microscopy and the final titer was set as the highest dilution for which the well contained at least one parasite. The viable parasitic load per gram of homogenized organ was calculated as follows: (reciprocal titer of the last positive well per total volume of homogenized tissue x dilution factor) divided by the weight (gram) of the homogenized tissue. The parasite load was expressed as the number of parasites per gram of homogenized organ.

BMMs as prepared previously were seeded in droplets of 5 μl (5 × 10³ cells) in 96-multiwell plates (Nunc, Roskilde, Denmark). The cells were left to adhere for 10 min whereafter 200 μl of complete α-MEM supplemented with M-CSF (30 ng/ml) and with or without RANKL (4 ng/ml) (R&D Systems/Biotechne) and CCL5 (1 μg/ml) (R&D Systems/Biotechne) was added. The cells were incubated at 37 °C in 5% CO₂ for various times (designated in Results section), whereafter the culture media were harvested and stored in aliquots at −20 °C until analysis of the active isoform 5b of TRAP5b (Immunodiagnostic systems [IDS], Copenhagen, Denmark). Adherent cells were fixed at the bottom of the 96-well plate and stained for TRAP using the leukocyte acid phosphatase kit (Sigma–Aldrich, St Louis, MO, USA), following the manufacturer's instruction. TRAP-positive cells with three or more nuclei were considered osteoclasts. Images were acquired using an Olympus BX41 light microscope. For gene expression analyses, BMMs were seeded in droplets of 40 μl (4 × 10⁴ cells) in 12-multiwell plates (Nunc) and cultured as mentioned previously.

Subsamples of ca. 200 diapausing eggs from each sample were individually placed in 96-multiwell plates (NUNC) and induced to hatch under constant light, 6 g L⁻¹ salinity and 25 °C (standard hatching conditions^{64 (link)}). These well plates were monitored daily for 28 days in order to estimate the diapausing egg hatching fraction and the timing of hatching.