Facsfortessa multicolor flow cytometer

All antibodies used for flow cytometry were obtained from BioLegends. PBMCs were incubated with the following Ab-conjugates on ice in the dark: anti-CD66b-PE-Cy7 (G10F5, 305115, 1:100), anti-CD3-BV510 (UCHT1, 300447, 1:100), anti-CD56-BV711 (HCD56, 318335, 1:100), anti-CD8a-APC-Cy7 (RPA-T8, 301015, 1:100), anti-CD57-PB (HCD57, 322315, 1:100). Incubation was completed for 30 min, then samples were washed twice with FACS buffer and resuspended in 300 μL of the same buffer. Furthermore, 7-AAD (BioLegend, 420404, 1:100) was added to PBMCs immediately before analysis. The flow cytometry was carried out using a BD FACSFortessa Multicolor Flow Cytometer (BD Biosciences). The CD57⁺CD8⁺T cells/T cells ratio was calculated using the following formula: $\frac{\mathrm{CD}57+\mathrm{CD}8+\mathrm{CD}3+\mathrm{cells}}{\mathrm{CD}3+\mathrm{cells}}100\%$ . The CD57⁺CD8⁺ T cells/CD8⁺ T cells ratio was calculated using the formula below: $\frac{\mathrm{CD}57+\mathrm{CD}8+\mathrm{CD}3+\mathrm{cells}}{\mathrm{CD}8+\mathrm{CD}3+\mathrm{cells}}100\%$ . As well, the total CD8⁺ T cells/T cells ratio and the total CD57⁺ T cells/T cells ratio were calculated as the proportion of CD8⁺CD3⁺ cells among total CD3⁺ cells and the proportion of CD57⁺CD3⁺ cells among total CD3⁺ cells, respectively.

To assess the effect of exosomes on the apoptosis of 3D4/21 cells, cells were pretreated with Exo and Ba-Exo for 24 h, and the apoptosis rate was detected by flow cytometry in accordance with the manufacturer’s instructions. Cells were collected and suspended in permeabilization buffer (2% FBS and 0.2% Tween-20 in DPBS) for 10 min at room temperature. After washing with cold DPBS, the cells were suspended in 100 μL of 1× binding buffer and mixed by gentle back-pipetting until a single-cell suspension was obtained. The cells were incubated with 5 μL of Annexin V-FITC and 5 μL of propidium iodide staining solution for 5 min at room temperature, protected from light. After washing with cold DPBS, the apoptosis rate was determined using a BD FACSFortessa multicolor flow cytometer (BD Biosciences, USA) at 488 and 561 nm. At least 10,000 events were collected from the cell gates. The data were then analyzed using FlowJo software (BD Biosciences).