In cell analyzer 2000 microscope

The C. neoformans suspension and treatment preparation with the MK58911-NH₂ peptide were carried out as described above for the cell wall assay. After 4 h, fungal cells were centrifuged and washed with PBS. A sample from each group (control and MK) was suspended in India ink, and images were acquired in a Primovert inverted microscope (Carl Zeiss). Cellular and capsular sizes of parent cells were measured using ImageJ software. Another sample from each group was incubated with 10 μg/ml of capsule-binding mouse monoclonal antibody 18b7 at 37°C at 150 rpm for 1 h. Subsequently, 1 μl of anti-mouse IgG-FITC was added, and the samples were incubated at 37°C at 150 rpm for 30 min. The images were acquired with an In Cell Analyzer 2000 microscope (GE). The quantification of the average fluorescence intensity per cell to verify the action of the peptide on the capsule was performed with In Cell Investigator software. Three independent experiments were performed.

Images were acquired in an IN Cell Analyzer 2000 microscope (GE Healthcare) with a Nikon 20x/0.45 NA Plan Fluor objective (binning 1X1), using a large chip CCD Camera (CoolSNAP K4) with a pixel array of 2048x2048 (7.40 μm² pixel). Image field of view (FOV) x-y for this objective is 0.76x0.76 mm. With these settings and an approximate 70–80% cell confluence, 1500–2000 macrophages per well were imaged (per 4 FOV), a number we consider reasonable for the quantifications as it is 15 to 20-fold higher than that used with manual counting [15 (link)–17 (link)]. The excitation and emission filters used to detect DAPI, Alexa Fluor 568 and HCS CellMask^™ were DAPI, Texas Red, and Cy5, respectively.

C. neoformans colonies were dissolved in phosphate-buffered saline (PBS) and diluted in a solution containing RPMI 1640 culture medium, 0.165 M morpholinepropanesulfonic acid (MOPS) (pH 7.4), l-glutamine, and 2% glucose to obtain 1 × 10⁶ cells/ml. One hundred microliters of this fungal suspension was added to a 96-well plate. Subsequently, 100 μl of MK58911-NH₂ (15.625 μg/ml [1/2× MIC], 31.25 μg/ml [MIC], and 62.50 μg/ml [2× MIC]), amphotericin B (0.125 μg/ml [MIC]), and fluconazole (1 μg/ml [MIC]), all diluted in RPMI 1640 medium, was added to the plate. The fungal suspension and RPMI 1640 medium were used as the positive and negative controls, respectively. The plate was incubated at 37°C at 150 rpm for 4 h. After that, 10 μl of calcofluor white was added to reach a 50-μg/ml concentration, and the mixture was incubated for 5 min at room temperature. The samples were centrifuged at 3,500 rpm for 5 min (5810R centrifuge; Eppendorf) and washed with PBS. Next, 200 μl of PBS was added to each well, and the images were acquired with an In Cell Analyzer 2000 microscope (GE). The quantification of the average fluorescence intensity per cell to verify the action of the peptide on the chitin content present in the cell wall and the fungal cell count were performed by using In Cell Investigator software. Three independent experiments were performed.