Calcein am

Rhodamine123 (Rh123) drug efflux was carried out using a Multidrug-Resistance Direct Dye Efflux Activity Kit (Millipore, Darmstadt, Germany), with some modifications. 2.5 × 10⁵ cells were collected and incubated at 4 °C for 1 hour in cold efflux buffer supplemented with 10 µg/mL of Rh123 and 22 µmol/L of drug (CTR-20, colchicine) or a vehicle control. Cell pellets were washed with cold efflux media containing sham control or 22 µmol/L of drug, and resuspended in pre-warmed efflux media containing 22 µmol/L of drug (or vehicle) and incubated for 1 hour in 37 °C water bath. Cells were then placed on ice and fluorescence levels were quantified by flow cytometry using a Beckman Coulter FC500 flow cytometer, 488 nm excitation, FL1 (525 nm/40) emission. Calcein-AM (Santa Cruz, Dallas) efflux was carried out on 1 × 10⁷ cells, which were co-treated with sham control or 22 µmol/L of CTR-20 (or colchicine/verapamil) and 0.5 µmol/L Calcein-AM for 15 min at 37 °C on 10 cm plates, followed by washing/pelleting with ice-cold media. Reading was carried out with a Beckman Coulter FC500 flow cytometer.

To assess cell viability, the LIVE/DEAD test was used using an InCell Analyzer 2000 automated microscope (GE Healthcare, Chicago, IL, USA). Cells of examined cell line were seeded on the surface of tested materials at constant amount of about 12 × 10⁴ per well in 2 mL of medium suitable for a given cell line and then cultured for 48 h under standard conditions. As a control, a well with no sample was used. Before observations under a fluorescent microscope, the medium was removed and the cells were washed with PBS solution. The samples were then incubated for 15 min at room temperature in a balanced Hank salt solution containing a mixture of fluorescent dyes: Hoechst 33342 (Molecular Probes, Eugene, OR, USA), calcein AM (Santa Cruz Biotechnology, Dallas, TX, USA), and propidium iodide (Molecular Probes).
The obtained images were analyzed using InCell Analyzer (GE Healthcare) software. All cells were divided into two subpopulations, i.e., live (stained with calcein-AM—gives green color) and dead (stained with propidium iodide—red color). The total number of cells was determined based on blue fluorescence of Hoechst 33342.

A total of 1 × 10⁴ A549 cells were seeded in each well of a 96-well culture plate until they reached 80% confluence. The cells were infected with bacteria (MOI = 50) or bacterial culture media for 2 h. Cell viability was detected with a CCK-8 (Beyotime) according to the manufacturer’s instructions. In addition, the cell viability was determined by calcein-AM (Santa Cruz) staining54 (link). A total of 1 × 10⁵ cells were cultured in cell culture dishes (15 mm) and infected with the indicated PAO1 strains (MOI = 50) for 2 h. The cells were loaded with 4 μm calcein-AM for 30 min in D-Hanks buffer at room temperature (RT), followed by 30 min incubation at RT. Image acquisition was performed with a Leica TCS SP5 confocal laser scanning microscope.