Dpbs ca mg

MSCs were characterized via flow cytometry as previously described at the beginning of the second passage^{32 (link)}. Briefly, cells were harvested and washed two times with wash buffer (DPBS (Ca^-/Mg^-) (Gibco, Grand Island, NY), 1% bovine serum albumin (Gibco, Grand Island, NY) and aliquoted into tubes containing 1 × 10⁵ cells in 100 µL wash buffer. Primary antibodies were added to the aliquots according to Supplementary Table 1 and incubated for 30 min in the dark on ice. Tubes were washed twice with wash buffer and centrifuged at 1000 rpm for 2 min at 4 °C. Flow was performed on an Attune NxT Flow Cytometer (Thermo Fisher, Waltham, MA) and analyzed using Attune Nxt software. Percent positive events were determined against samples stained with matching isotype controls.

The flow-cytometric method was employed to identify the apoptosis of spermatozoa. For this purpose, fluorescent probes Annexin V-FITC (Annexin V Apoptosis Detection Kit, Canvax, Cordoba, Spain) and propidium iodide (PI; Molecular Probes, Eugene, OH, USA) were used. The assay was performed following the method described by Kuzelova et al. [40 (link)] with slight modifications. Briefly, the semen samples were washed and centrifugated in Dulbecco’s phosphate-buffered saline + Ca; Mg (DPBS + Ca; Mg, Gibco, Waltham, MA, USA) at 600× g for 7 min. The supernatant was discarded, and aliquots of 10⁶ cells were used for staining. Annexin V staining was done according to the manufacturer’s instructions. Immediately prior to analysis, 4 μL of the PI were added to the cell suspension. The cells were evaluated using FACS Calibur^TM (BD Biosciences, Franklin Lakes, NJ, USA) and Cell Quest Pro^TM (BD Biosciences, USA) software. A minimum of 10,000 cells was analyzed for each sample. Cells found in the lower left quadrant (AnV⁻/PI⁻) were identified as live. Cells located within the lower right quadrant (AnV⁺/PI⁻) were determined as apoptotic. Both upper quadrants (AnV⁻/PI⁺ and AnV⁺/PI⁺) included dead sperm cells [41 (link)]. The evaluation of spermatozoa viability is illustrated in Figure 1.