Guava easy cyte 8

To determine membrane binding of SLY to HEp-2 and NPTr cells, as well as undifferentiated PTEC and PBEC, 4 × 10⁵ trypsinized cells were incubated with 120 HU/mL rSLY in 1 mL cell culture medium for 2 h at 37 °C. Cells were washed with PBS (Sigma-Aldrich, Taufkirchen, Germany) with 2% (v/v) FCS (Biochrom, Berlin, Germany) and stained using polyclonal antiserum raised against rSLY [88 (link)] (diluted 1:1000 in PBS with 2% FCS, incubated for 1 h at RT) and Alexa Fluor^® 488 goat-anti-rabbit IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:1000 in PBS with 2% FCS, incubated for 30 min at RT). Additionally, cells were stained with the DNA-intercalating dye propidium iodide (PI, 2.5 µg/mL; Sigma-Aldrich, Taufkirchen, Germany) for 5 min at RT to discriminate viable and non-viable cells. SLY-cell association was measured using Guava^® EasyCyte8 (Merck Millipore, Darmstadt, Germany). The cell population was identified using forward- and side-scatter light, and green- and red-fluorescent cells were detected. In all the experiments, at least 5000 events were counted and analyzed with FlowJo software version 10.5.2 (Tree Star Inc., Ashland, OR, USA). The experiment was repeated at least three times. Results are expressed as percentage SLY-cell association and mean fluorescence intensity.

Suspensions of C. albicans SC5314 were prepared by taking one loop of pure culture growth from 24 h YPD agar plates into RPMI 1640 medium and adjusting optical density to 0.1 at 660 nm wavelength. Then, FLCpOH-TP10-NH2 and FLCpOH-TP10-7-NH2 were added at concentrations corresponding to 8 × MIC and 4 × MIC. FLC (8 × MIC) was used as a negative control. The cell suspensions were then treated for 0.5, 2, 4, 6, and 24 h at 37 °C with continuous agitation (180 rpm). The cells were then centrifuged and resuspended in 200 µL of PBS buffer (pH 7.4). Subsequently, cells were treated with propidium iodide (1 mg/mL, final concentration) and incubated for 30 min at room temperature in the dark. Cellular fluorescence was visualized using lens 20×, using Olympus Fluorescence Microscope BX60 (Olympus, Tokyo, Japan). The images were also post-processed utilizing cell Sens program. The fluorescence intensity emitted by DNA-bound propidium iodide was measured using a flow cytometer (Merck Millipore guava easyCyte 8, Darmstadt, Germany). Data were obtained from at least two independent experiments.

For mitochondrial staining, hESC-CMs cultured on coverslips were incubated with prewarmed medium containing 0.1 μM MitoTracker Red CMXRos (Thermo Fisher, United States) for 20 min. These cells were then stained with TNNT2 antibody, followed by incubation with Alexa Fluor 488-conjugated secondary antibody as shown in Supplementary Table 2. After nuclear staining with Hoechst 33342, cells were visualized under an LSM 880 confocal laser scanning microscope (Carl Zeiss, Germany). For the flow cytometric assay, the hESC-CMs incubated with 0.1 μM MitoTracker Red CMXRos were dissociated into single cells before subsequent TNNT2 and nuclear staining, and finally analyzed with Guava easyCyte 8 (EMD Millipore, Germany).

Cardiomyocytes were washed with D-PBS and then incubated with prewarmed medium supplemented with 0.1 μM MitoTracker Red CMXRos (M7512, ThermoFisher Scientific, USA) for 20 minutes. After 3 washes with D-PBS, the cells were dissociated into single cells using 0.25% trypsin-EDTA and fixed with 1% paraformaldehyde for 20 minutes. Then, the cells were washed with D-PBS and analyzed with Millipore Guava easyCyte 8 (EMD Millipore, USA).