Rh123

The cells were cultured in a 6-well plate for 24 h, then incubated with different ratios of CIK (10:1 or 20:1) for 72 h. The cells were then digested, resuspended, incubated with P-gp antibodies for 30 min at 4°C and washed twice in PBS. The fluorescence intensity of fluorescein isothiocyanate (FITC)-P-gp (Abcam, Burlingame, CA, USA) was analyzed by flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) at 488 nm.
The cells were cultured in a 6-well plate for 24 h, then incubated with different ratios of CIK for 72 h. The cells were then digested, resuspended, incubated with 10 μM Rh-123 (Sigma-Aldrich, San Francisco, CA, USA) for 60 min and washed twice in PBS. The fluorescence intensity of Rh-123 was analyzed by flow cytometry (FACSCalibur; BD Biosciences) at 488 nm.
The cells were cultured in a 6-well plate for 24 h, then incubated with different ratios of CIK (10:1 or 20:1) for 72 h. A total of 10 μg/ml ADR was added and co-cultured for 24 h, then the cells were digested, resuspended, incubated with Annexin V-FITC and propidium iodide (PI) for 15 min at 37°C and washed twice in PBS. The apoptosis rate was analyzed by flow cytometry (FACSCalibur; BD Biosciences) at 488 nm.

MMP in HFS and LFS was measured using rhodamine 123 (Rh123; Sigma-Aldrich, St. Louis, MO, USA). Briefly, spermatozoa were mixed with 1 μM Rh123 diluted in PBS and the concentration of spermatozoa were adjusted to 5 × 10⁶ cells/mL, and then incubated at 37 °C for 15 min. Fluorescence intensity of Rh123 was measured by flow cytometry (Dual-Laser FACS Aria II, BD Biosciences, San Jose, CA, USA) with 488 nm excitation and 525 nm emission wavelengths and analyzed. For each of the three independent replicate experiments, three samples were used.

HaCaT keratinocytes were treated with triterpenoids (20 μM), and the mitochondrial function was evaluated after indicative times. According to manufacturer’s instructions the following fluorescent probes were used to examine mitochondrial function43 (link): Rhodamine 123 (Rh123, Sigma-aldrich) and MitoTracker Red CMH₂XRos (MTR, Molecular Probes) to monitor mitochondrial inner transmembrane potential (ΔΨm); MitoTracker Green FM (MTG, Molecular Probes) to measure cellular mitochondria content. MTG is a cell-permeant mitochondrial-specific dye that becomes fluorescent only on sequestration by mitochondria44 (link). However, unlike Rh123 and CMH₂XRos, MTG covalently binds to mitochondrial proteins and thus can be used as a measure of mitochondrial mass independent of ΔΨm44 (link). To quantify the fluorescence emission of MTG or Rh123 we collected at least 30,000 events to further cytofluorometric analysis (BD FACS Verse^™). To analyze the data we used the FlowJo software. Alternatively, we used confocal microscopy to monitor ΔΨm regarding MTR fluorescence.

To determine whether the antifungal AF₄ affected the mitochondrial membrane potential (ΔΨm) of the cells, we analyzed the cells with Rh123 (Sigma-Aldrich, USA). Generally, changes to membrane potential are observed by a shift in the fluorescence of Rh123 in flow cytometry. Cell suspensions with ~5 × 10⁶ CFU/mL were treated with AF₄ (8 and 16 mg/L) for 18 h in RPMI 1640 at 37°C under shaking conditions. Heat-treated (121°C, 15 min) cell suspension, sodium azide-treated (40 mM) samples, untreated cells, and unstained cells were analyzed as controls. After treatment and incubation, cells were harvested at 10,000 rpm and Rh123 was added at a concentration of 1 mg/L for 15 min at 35°C; cells were then washed twice and incubated at 35°C for an additional 30 min and analyzed immediately (67 (link)) using a BD FACScan flow cytometer and FlowJo version 10.8.1 software. The decrease in fluorescence peak intensity due to the sequestration of Rh123 in the mitochondrial membrane indicates a change in the ΔΨm (22 (link)). The peaks obtained for each sample were compared to determine the effect of AF₄ on the ΔΨm. Additionally, live and dead cells were distinguished by observing the side scatter when they were stained with Rh123.