Dibac4 3

Studies regarding the effect of the P-EO and HAp-P samples against the microbial membrane were conducted by flow cytometry using a sensitive fluorescent dye, DiBAC4(3). The microbial strains were cultivated in the presence of the tested samples for 24 h at 37 °C. After 24 h, 0.5 μg/mL of DiBAC4(3) (Invitrogen/Life technologies, Carlsbad, CA, USA) was used to stain the microbial strains. The positive control was represented by an untreated microbial cell suspension. After 30 min of incubation with the DiBAC4(3), the intensity of fluorescence (IF) was measured using an Accuri C6 plus flow cytometer (Becton Dickinson, Biosciences, (San Jose, CA, USA)) in channel of fluorescence fluorescein isothiocyanate (FITC) (filters 530/30 nm).

DiBAC₄ (3) (Molecular Probes, Eugene, OR, USA) was used to measure plasma membrane potential by flow cytometry and microscopy. 0.5 mL of 0.5 μM (in DMEM with 10% FCS) dye was added into the slides and incubated for 30 min in dark at 37 °C. Cells were gently dislodged from surface using 1 mL of non-enzymatic cell dissociation solution (Sigma Aldrich, Schnelldorf, Germany) and around 10,000 cells were added into flow cytometry tubes. Later, cells were centrifuged and suspended in 0.25 mL of PBS and immediately analyzed on flow cytometer at 488 nm. 5000 cells in triplicates per sample were counted. An aliquot of cell suspension was separated to register baseline value. The sampling interval of DiBAC₄ (3) fluorescence measurements were in the range of 4 and 5 s. Data analysis was performed using CellQuest software (Becton Dickinson, Heidelberg, Germany).
For microscopy, cells were incubated with 0.5 mL of 0.1 μM DiBAC₄ (3) dye (in DMEM with 10% FCS) for 5 min at 37 °C. Cells were washed once with 1 mL PBS and immediately imaged under Olympus IX81 inverted fluorescence microscope with excitation maxima of 490 nm. Cells were imaged at 6–8 random places from the slides. Mean fluorescence intensity of 40 cells in triplicates was analyzed using ImageJ.