Lsm 5 pascal scanning head

Assessment of apoptosis and necrosis in cultures was performed using DNA binding dyes—Hoechst 33342 and propidium iodide (PI). The analyses were performed under an Axiovert 200M confocal microscope with an LSM 5 PASCAL scanning head (Zeiss, Jena, Germany). Cell cultures for the analysis were carried out in a 35 × 10 mm tissue culture dish (SPL Life Sciences, Pocheon, South Korea) and inoculated with 2 mL of a cell suspension at a concentration of 1 × 10⁵ cells/mL. After 24 h incubation with the test fractions at a final concentration of 300 μg/mL and 2.5 mg/mL fibrinogen, 5 μL of Hoechst 33342 (0.4 mg/mL) and PI (0.5 mg/mL) mixed in a 2:3 ratio were added where applicable. The cultures were incubated for 5 min at 37 °C in the dark. After this time, the medium with the staining solution was removed and replaced with PBS buffer with Ca²⁺ and Mg²⁺ ions. The buffer exchange was followed by microscopic observations at an excitation wavelength of λ = 420 nm. The number of apoptotic and necrotic cells was calculated based on the observation of at least 1000 cells in randomly selected fields of view. Cells exhibiting blue fluorescence of fragmented nuclei were interpreted as apoptotic, whereas cells with pink fluorescence of whole nuclei were classified as necrotic. Each analysis was repeated three times [22 (link),35 (link)].

The LIVE/DEAD BacLight Bacterial Viability Kit allows quick determination of living and dead cells based on the integrity of their cell membranes [17 (link)]. Bacterial suspensions (10 µL) from the control and experimental groups were incubated with 3 µL of a dye mixture (5 µM Syto9 and 30 µM propidium iodide) for 15 min in the dark at room temperature. Then, 3 µL of the resulting suspension was applied to a microscope slide and observed under a Zeiss Axiovert confocal microscope with an LSM 5 Pascal scanning head.
The images of live and dead bacteria were collected as fluorescent green and red bacilli under a fluorescence microscope using appropriate single bandpass filter sets. The excitation/emission maxima were 480 nm/500 nm for Syto9 and 490 nm/635 nm for propidium iodide. The viability of the mycobacterial cells was estimated based on the percentage of Syto 9 fluorescence in the whole specimen. The images were analyzed using ImageJ 1.42q.
The minimum inhibitory concentration (MIC) for M. smegmatis was determined with the agar dilution method on solid Sauton’s medium (with 0.7% agar) [18 ]. The PSE with protein concentrations ranging from 6.25 to 150 μg/mL was analyzed. The MIC value corresponded to the lowest extract concentration at which no more than one colony developed. The experiment was repeated three times.