Microtitre plate wells

We assessed the ability of amoebae to internalize cryptococcal cells: (1) obtained from strains C. neoformans UOFS Y-1378, (2) C. gattii R265, (3) C. neoformans LMPE 046 in the absence or presence of 3-hydroxy C9:0, i.e., 0 mM, 0.2 and 1 mM, using the phagocytosis stain, pHrodo^TM Green Zymosan A BioParticles (Life Technologies, US). The stain only fluoresces when excited at acidic pH, such as inside a food vacuole or phagosome. Cryptococcal cells were standardized to 1 × 10⁶ cells/ml in PBS (which has a neutral pH) and stained (1 μl of stain: 999 μl of cells) for 1 h at room temperature while slowly agitating. Next, cryptococcal cells were washed with PBS, spun down and suspended in sterile 1000 μl of PBS. A 100-μl suspension of cells was then transferred to microtitre plate wells (Greiner Bio-One, Germany) and allowed to interact with amoebae (100 μl; 1 × 10⁵ cells/ml) for 2 or 6 h at 30°C. The amoebae were standardized in fresh PYG broth (pH 7). At the end of the incubation period, the induced fluorescence was measured (492 nm; ex/538 nm; em) using a Fluoroskan Ascent FL (Thermo-Scientific, USA) microplate reader, which converts logarithmic signals to relative fluorescence units. The fluorescence was also measured for fungal cells alone in order to normalize the readings.

The ability of rMHJ_0461 to influence the conversion of plasminogen to plasmin in the presence of tPA was determined using a method described previously [7 (link)]. Purified porcine plasminogen (50 μg ml⁻¹) [11 ] was incubated for 1 h at 37°C with 1 : 0.5, 1 : 1, 1 : 2, 1 : 4 and 1 : 8 molar ratios of rMHJ_0461 in microtitre plate wells (Greiner Bio One, Germany) before the addition of tPA and Spectrozyme-PL. Controls included plasminogen alone, plasminogen and rMHJ_0461 in the absence of tPA, plasminogen and tPA in the absence of rMHJ_0461. Protein controls using substance P and insulin as substitutes for rMHJ_0461 at 1 : 1 molar ratios were also tested. Absorbance at 405 nm was read every 5 min for 90 min. This experiment was performed twice, each time in triplicate.