Axioskop upright microscope

Sorted cells were mounted on superfrost slides using a Cytospin centrifuge (Cytospin 4; Thermo Fisher Scientific) operating for 5 min at 500 rpm. Cells were fixed with ice-cold methanol and stored at room temperature. Cells were subsequently stained with hematoxylin and acidic eosin and mounted with DPX. Images were collected on an Axioskop upright microscope (ZEISS) using a 100× objective and captured using a CoolSNAP ES camera (Photometrics) through MetaVue software (Molecular Devices). Images were then analyzed and processed using ImageJ (National Institutes of Health) and Image-Pro Premier software (Media Cybernetics).

Germination and appressorium formation were assayed on polystyrene and on onion epidermis. Ten-μL droplets of washed macroconidia (see above) containing 100 and 1000 spores were inoculated onto 90-mm polystyrene Petri dishes (Greiner Bio One) and the hydrophobic face of onion epidermal strips, respectively. After incubation for 24 h in a humid chamber at 23°C in darkness, infection structures were counted by phase contrast microscopy using an Axiovert 40 CFL inverted microscope (Carl Zeiss, Jena, Germany) equipped with a 10 x / 0.25 Ph1 objective for germination assays on polystyrene and an Axioskop upright microscope (Zeiss) equipped with a 20 x / 0.45 Ph 2 objective for germination assays on onion epidermis.

In vitro cultures of human cells may deliver improved understanding of the reactions of a particular cell type to specific drugs. Following the procedure mentioned above, confluent HeLa cell cultures were obtained.
Nanoformulations of (coated and uncoated) cubosomes (CIS, PAX, and DUAL) were added to the cell culture at the ratio of 1:1000 as was demonstrated by Murgia et al.^{17 (link)} (2 µl of cubosomal solutions were added to 2 ml of media containing cells). Cell Explorer™ fluorescence imaging kits (Biochem Life Sciences, India) was used for staining the HeLa cells. This specific kit is intended to consistently stain live HeLa cells in green fluorescence for comparatively extended time. The cells were fixed to preserve the pattern of imaging. The kit utilizes a nonfluorescent dye that becomes intensely fluorescent upon entering into live cells. This gives the live HeLa cells a stable fluorescence signal for moderately lengthier time period. This dye consists of hydrophobic molecules which easily permeates live cells. Observations were made using a Zeiss (Axioskop) upright microscope (Zeiss, Germany). Cellular fluorescence intensity was determined by ImageJ software. The fluorescent intensity of ten cells in the image were measured, and mean was calculated to determine mean fluorescent intensity.