Evos fl cell imaging system

A commercial LIVE/DEAD yeast viability kit (L-7009; Molecular Probes, Leiden, The Netherlands) was used to analyze yeast metabolic activity after treatment with LMM11 (for 24 hours) at the concentrations 16 μg/mL, 32 μg/mL and 64 μg/mL. In the dead control, the yeast were treated with 70% alcohol for 15 minutes. Untreated yeast cells were used as live control. Yeast cells were suspended in MOPS buffer containing 2% glucose. FUN-1 (10 μM) and Calcofluor White M2R (12.5 μM) cell dyes were added to the yeast cell suspensions. After incubation in the dark at 30°C for 30 min, the stained yeast was analyzed with an inverted fluorescence microscope (EVOS FL Cell Imaging System, Life Technologies, CA, USA), using appropriate filter sets, at x 400 magnification. The viability of fungal cells was determined by fluorescence analysis in at least 20 fields. Staining and the interpretation of fluorescence were performed according to the manufacturer’s instructions. Metabolically active cells showed red fluoresce in their structures while dead cells or cells with little or no metabolic activity exhibited diffuse bright green cytoplasmic fluorescence with no discernable red structures [33 (link)].

Microspores viability and the level of intensity after treatment with CPP-Cys-mCherry proteins were measured using BioTek Synergy Mx microplate reader (BioTek Instruments, Inc., USA). For this, the microspore solution was resuspended and 12,500 cells in 50 μl volume were pipetted into every well of the black 96-well plate (Costar, Fisher Scientific). For FDA and mCherry measurements, the fluorescent reading was done at excitation/emission wavelengths of 492/517 and 587/610 nm, respectively. The data normalization was done by setting the fluorescent reading from the FDA measurement in the untreated control sample to 100%. For the mCherry signal detection, the reading from untreated control was set to 1 and the fold difference as compared to control was calculated. No cross-reading was detected between two fluorescent signals under aforementioned conditions. All measurements were done in three biological and two technical repeats.
The visual examination of treated microspores was done using both EVOS FL cell imaging system (Life Technologies) and confocal laser scanning microscope Olympus, FV1000 (Olympus).

2500 Vybrant CM-Dil-labeled HSC-3 cells and 5000 Vybrant DiO-labeled Mfs were co-cultured in TC Lab-Tek Chamber slides (Thermo Fisher Scientific, MA, USA). The cells were allowed to attach o/n in normal growth medium. Thereafter cells were washed with PBS, and medium changed to SF Optimem (Life Technologies, CA, USA). The following experimental groups were created (n = 4): co-cultures of HSC-3 cells and Mfs (M1 Mf, M2 Mf, R848 Mf) were incubated with either DMSO (1:20 000), 0.3 mM Amiloride (Sigma-Aldrich Co.LLC, MO, USA), 3 mM Amiloride, 5 μM NF-κB inhibitor BAY 11-7082 (MerckMillipore, MA, USA) or 3 mM Amiloride plus 5 μM BAY 11-7082. Also monocultures of HSC-3 cells and Mfs were incubated with the same experimental molecules. Incubation was continued for up to 7 days and monitored once every day with the Evos FL Cell Imaging System (Life Technologies, CA, USA).

For the enumeration of echinocytes, samples were gently mixed before a drop (~10 μL) was transferred to a glass slide, smeared, and imaged immediately using phase-contrast microscopy at 40 × using an EVOS FL Cell Imaging System (Life Technologies). About 100 random RBCs were counted per sample and echinocytes were identified by their distinct spiculations.
Wright-Giemsa staining was performed according to standard procedures. Briefly, a drop (~10 μL) of sample was smeared on a glass slide, air-dried, fixed in 100% methanol, and dipped in Wright-Giemsa stain (Sigma) for 30 seconds before rinsing in deionized water. Images were captured with a Nikon DS-Ri1 color camera (12-bit; 1280 × 1024 resolution) using a Nikon 100 × Apo VC 100 × /1.40 oil objective on a Nikon Eclipse 90i microscope.