Cytexpert flow cytometer

Bacterial PMF was determined using DiOC₂(3) [71 (link)]. E. coli G92 was cocultured with PS (0, 5, 10, and 20 mM) or fumarate (0, 25, 50, and 100 mM) at 37 °C for 4 h. Cells were harvested and washed, and resuspended with M9CA to OD₆₀₀ = 0.3. DiOC₂(3) was dissolved in dimethyl sulfoxide and prepared as a sterile stock solution of 3 mM. During oscillation at 37 °C for 15 min, the bacteria were incubated with 20 μl of DiOC₂(3). Dye-free samples were used to control for autofluorescence and carbonyl cyanide 3-chlorophenylhydrazone (5 μM) was added as the depolarized control sample. The detection of green fluorescence was performed using a band-pass filter with a bandwidth of 488 to 530 nm, whereas red fluorescence was detected using a band-pass filter with a bandwidth of 488 to 610 nm. The CytExpert Flow Cytometer (Beckman, USA) determined a total of 100,000 ungated events in each sample. Then, they were analyzed by FlowJo 10.8.1 software (Becton, Dickinson and Company, USA). The calculation of PMF was based on the intensity ratio of red/green fluorescence.

Serum inflammatory cytokines were detected using a commercial mouse inflammation panel kit (BioLegend, San Diego, USA) and the CytExpert Flow Cytometer (Beckman, USA) according to the instructions of manufacturer. Data were analyzed by the LEGENDplex™ software (BioLegend, San Diego, USA).

RAW264.7 cells (1 × 10⁶⋅mL^–1) were cultured in 6 well plates at 37°C for 12 h, and then exposed to ZnONPs, AMP, AMP-ZnONPs (1.95 μg⋅mL^–1) or LPS (0.5 μg⋅mL^–1) for 24 h, respectively. The cells were incubated with 1 mg⋅mL^–1 FITC-dextran for 1 h, then the reaction was stopped by cold PBS. The FITC-dextran intensity of cell samples was analyzed by CytExpert flow cytometer (Beckman Coulter, CA, United States).

The 2′,7′-dichlorofluorescein diacetate (DCF-DA) dye was used to determine intracellular ROS generation. Briefly, DCF-DA (100 μM) was added to the donor E. coli DH5α and recipient E. coli J53 suspensions (in PBS at approximately 10⁶ CFU/mL). After shaking at 100 rpm for 30 min in the dark, the cells were washed twice using PBS to remove excess probes. Subsequently, antibiotics at subinhibitory concentrations (0, 1/8 MIC and 1/4 MIC) were added to the bacterial cells, which were incubated for 2 h at room temperature in the dark. Bacterial cell fluorescence was measured using a CytExpert Flow Cytometer (Beckman, USA) at an excitation wavelength of 488 nm and an emission wavelength of 630 nm.

BacLight bacterial membrane potential kit (Thermo Fisher Scientific) was used to measure the PMF. Briefly, 1 mL bacterial culture obtained from the log‐phase was directly diluted to 10⁶ CFUs per mL in the culture medium without washing and co‐cultured with 10 µL of 3 × 10⁻³m DiOC₂(3) (3,3’‐diethyloxa‐carbocyanine iodide) as treated samples. Another two tubes should be prepared for a depolarized control and an unstrained control. 10 µL of 500 × 10⁻⁶m CCCP was added to the depolarized control sample and mixed. Samples were incubated at 37 °C for 30 min. Signal intensity was assessed by CytExpert Flow Cytometer (Beckman, USA) and analyzed by FlowJo V10.8.1. The green fluorescence was detected through a 488 to 530 nm bandwidth band‐pass filter, and the red fluorescence was detected through a 488 to 610 nm bandwidth band‐pass filter. The forward scatter, side scatter, and fluorescence were collected with logarithmic signal amplification. The PMF was determined and normalized as the intensity ratio of the red/green fluorescence. The membrane potential was calculated with the following formula: PMF = lg(10^3/2×(redfluorescencegreenfluorescence)).

The bladder cancer cells treated with shGFP or shNAT10 were cultured in 96‐well plates, whereas untreated cells were used as blank controls. Cell viability was evaluated and quantified by measuring the absorbance using a Tecan Infinite 200 PRO (Tecan, Switzerland) with the CCK‐8 assay. Seventy‐two hours after transient transfection, approximately 1 × 10⁶ cells were collected from 6‐well tissue culture plates. The proportion of apoptotic cells was determined with the Annexin V‐FITC Apoptosis Kit (eBioscience, USA) using a CytExpert flow cytometer (Beckman Coulter, Inc.).

The fluorescent dye propidium iodide (PI) (30 μM) was added to the same cells to perform the outer membrane permeability assay. After shaking at 100 rpm for 30 min in the dark, fluorescence was measured using a CytExpert Flow Cytometer (Beckman, USA) at an excitation wavelength of 488 nm and an emission wavelength of 630 nm. The heat-treated (2 h at 80°C) cells were used as the control for the inner membrane damaged cells. All tests were performed using biological triplicates. The results were analyzed using the CytExpert 2.0 software (Beckman, USA).