Facscallibur

Cells were seeded into 24-well plates at 1.2 × 10⁵ cells/well and left to adhere overnight. On the next day, cells were exposed to 0, 0.1 µM, and 1 µM DOX in normoxic (CCM_FBS) or hypoxic conditions (CCM_FBS + CoCl₂, 100 µM) for 24 h, following which apoptosis was assessed by flow cytometry using eBioscience™ Annexin V Apoptosis Detection Kit FITC (88-8005-72) according to manufacturer’s guidelines. Briefly, cells were washed with 1× Binding Buffer and incubated with a 1/20 dilution of fluorochrome-conjugated Annexin V for 10 min, after which fluorescence was measured at 485 nm excitation and 535 nm emission wavelengths using BD FACSCallibur. Results are shown as percentage of mean Annexin V signal intensity of normoxic untreated condition.

After 7-day induction, induced MDSCs were tested or sorted on Beckman Coulter MoFlo Astrios^EQ (Beckman Coulter, CA, USA) or FACS AriaIII (BD Biosciences, San Diego, CA, USA) by using mAb CD11b (clone: M1/70), Ly-6G (clone: 1A8), Ly-6C (clone: AL-21), and CD127 (clone: A7R34) (all antibodies were purchased from BD Biosciences or eBioscience company). Flow cytometry verified that all the isolated MDSCs yielded a pure population of more than 90%. The sorted MDSCs were stored in PBS + 10% FBS for further experiments.
Splenocytes and peripheral blood mononuclear cells were obtained from spleens and peripheral blood of mice by Ficoll density gradient centrifugation. Cells were tested by using mAb CD45 (clone: 30-F11), CD3 (clone: 17A2), CD4 (clone: GK1.5), CD8 (clone: 53-6.7), and Foxp3 (clone: FJK-165) (all antibodies were purchased from BD Biosciences, eBioscience or Biolegend company) on BD FACSCallibur (BD Biosciences, San Diego, CA, USA) and analyzed by Flow JoX software.

Oxidative stress was measured using DCFDA—Cellular Reactive Oxygen Species (ROS) Detection Assay Kit (ab₁₁₃₈₅₁) in a microplate format and by flow cytometry. DCFDA is fluorogenic dye that measures ROS activity within the cell. After diffusion in to the cell, DCFDA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into DCF, a highly fluorescent compound with maximum excitation and emission spectra of 495 nm and 529 nm respectively. Cells were seeded into 96-well plates with clear bottom and black sides at a density of 2.4 × 10⁴ cells/well for the microplate assay, and into 24-well plates at 1.2 × 10⁵ cells/well for flow cytometry and left to adhere overnight. On the next day, cells were stained with 25 µM DCFDA for 45 min at 37 °C, according to manufacturer’s guidelines. Following this step, cells were exposed to 0, 0.1 µM and 1 µM DOX in normoxic (CCM_FBS) or hypoxic conditions (CCM_FBS + CoCl₂, 100 µM). After 6 h of treatment, fluorescence was measured at 485 nm excitation and 535 nm emission wavelengths using a Fluostar Omega plate reader and BD FACSCallibur. Results of the microplate assay are shown as fold change fluorescence. Results of the flow cytometry measurements are shown as percentage of mean DCF signal intensity of control conditions.

When the isolated cells reached the third passage in the culture conditions, their cell surface antigen expressions were evaluated by flow cytometric analysis for characterization. For this purpose, MSC-specific markers CD44-PE and CD90-APC were analyzed as positive markers, and endothelial CD34-FITC and hematopoietic CD45-APC-Cy were used as negative markers for MSCs (BD the Biosciences, USA) [18 (link)]. Isotype antibodies determined for each antibody were used as negative control (Biolegend, San Diego; APC mouse IgG1, κ isotype control, APC/Cyanine7 mouse IgG1, κ isotype control, PE mouse IgG1, κ isotype control, FITC mouse IgG1, κ isotype control). Briefly, the cells were first collected by trypsinization and pelleted by centrifugation at 1500 rpm for 5 min. The number of viable cells was determined by staining the cells with trypan blue and counting. 10⁶ of cells were pipetted through a 70-μm filter, and the cells were separated from each other for flow cytometry analysis. After washing twice in staining buffer (2% FBS in PBS), primary and isotype antibodies were added to the mixtures and incubated in the dark for 30 min. After incubation, the cells were washed two more times with staining buffer and then analyzed by BD FACS Callibur [6 , 18 (link)]. This analysis was repeated three times, and the results are given as mean + SD.