Expo32 adc

MMP (Δψ_m) was measured with flow cytometry using TMRM staining. TMRM accumulates in normal mitochondria due to its positive charge whereby the reduction of MMP leads to the release of TMRM. Following various treatments, cells were incubated with 100 nM TMRM at 37°C for 30 min. Harvested cells were immediately analyzed for potential using Beckman Coulter Epics XL flow cytometer (650 nm long pass filter) equipped with Expo32 ADC.

Cells were incubated with FITC-CD11c, PE-CD86, and PE-MHC-II for 30 min at 4 °C. All washing steps were performed in PBS or PBS supplemented with 2% FBS to avoid unspecific binding. Flow cytometry analyses were performed on a Coulter Epics XL flow cytometer (Beckman Coulter, Marseille, France) equipped with the analysis software Expo32 ADC (Beckman Coulter) [7 (link)].

The cell apoptosis rate was detected using the TUNEL and Annexin V-FITC/PI staining kits after different treatments. For TUNEL analysis, BMSCs were grown on gelatin-precoated microscopy slides and fixed with 4% paraformaldehyde at room temperature for 15 min. The samples were then covered with 100 μL of mixed TUNEL reagent for 1 h at 37 °C in a thermostatic water bath. DAPI solution (5 μg/mL) was then added to reveal the number of nuclei. The TUNEL signals were observed with an FV1000 Olympus confocal microscope (Olympus, Tokyo, Japan). At least 100 cells/fields (5 fields in general) were randomly counted to determine the ratio of the number of TUNEL-positive nuclei to the number of DAPI-positive nuclei and calculate apoptosis rates.
For Annexin V-FITC/PI staining, BMSCs were seeded in six-well plates at 6 × 10⁵ cells/mL for overnight stabilization. After administration, the cells were gently collected with trypsin solution. The suspension was centrifuged at 300 g and 4 °C for 5 min. In total, 1 × 10⁵ cells were resuspended in 195 μL of FITC-conjugated annexin V binding buffer containing 5 μL of Annexin V-FITC and 10 μL of PI at room temperature for 20 min in the dark and maintained on ice when processing. The samples were analyzed with EPICS XL-MCL (Beckman Coulter, Brea, CA, USA), and analysis was performed with EXPO32 ADC within 30 min.

For apoptosis, the cells (1 $\times {10}^6$ cells) were suspended with 500 $\mu L$ binding buffer and with 5 $\mu L$ of Annexin V-FITC and 10 $\mu L$ of PI (FITC Annexin V Apoptosis Detection Kit I, Sigma Aldrich, St. Louis, USA) added, and were later mixed. Next, the suspended cells were incubated at 4 ${}^{\circ }$ C for 30 min in dark. For the cell cycle, the cells were suspended with 500 $\mu L$ of binding buffer and assayed by PI/RNase Staining Buffer (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instruction. The apoptotic cells were analyzed using EXPO32 ADC, an analysis software (Beckman Coulter, Brea, CA, USA). The cell cycle was analyzed using ModFit 3.0 analysis software (Verity Software House, Inc. Topsham, ME, USA).