Annexin 5 pi detection kit

For apoptosis and cell cycle analysis, 2 × 10⁵ MLE-12 cells and MenSCs were seeded into the lower and upper transwell chambers of 6-well plates, respectively. An Annexin V/PI detection kit (BD Biosciences, CA, USA) was used to detect MLE-12 apoptosis, and a Cell Cycle and Apoptosis analysis kit (Beyotime Biotechnology, Haimen, China) was used to detect the MLE-12 cell cycle. Both experiments were performed according to the manufacturer’s instructions using a FC500 flow cytometer, and the data were analyzed using FlowJo software (Tree Star, OR, USA).
To determine the number of lung epithelial cells in lung tissues, flow cytometry was performed. The lung tissues were cut into small pieces and digested with type 1 collagenase for 1 h at 37 °C. Then a single-cell suspension was obtained by grinding the digested tissue through a 100-μm filter (BD Biosciences, CA, USA) before being centrifuged at 300×g for 10 min at 4 °C, and red blood cells were lysed twice. Subsequently, the cells were resuspended with stain buffer (BD Biosciences) and incubated with antibodies listed in Additional file 5, Table S1 for 30 min at 4 °C. Subsequently, the cells were washed twice with stain buffer before being analyzed with a multicolor flow cytometer (BD Biosciences). The data were analyzed using FlowJo software.

LSS-11 was synthesized by Meng’s laboratory (Peking University Health Center, Beijing, China). Paclitaxel was obtained from Dalian Meilun Co. (Dalian, China) and dissolved in DMSO to make a stock solution of 50 mM. Primary antibodies against DR5, PARP1, cleaved PARP1, Bax, Bcl2, GAPDH, and secondary antibodies including FITC-linked anti-rabbit and HRP-linked anti-rabbit or anti-mouse were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-P-gp (Santa Cruz, #sc-55510), anti-MRP1 (Santa Cruz, #sc-7774), anti-p-STAT3 (Ser 727, Santa Cruz, #sc-8001-R), and anti-STAT3 (Santa Cruz, #sc-482), were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Chemiluminescent agents were purchased from Bio-Rad (Hercules, CA, USA). Annexin V/PI detection kit were obtained from BD Biosciences (San Diego, CA, USA). Ethidium bromide, propidium iodide (PI), rhodamine123 were brought from Sigma-Aldrich (St. Louis, MO, USA).

Cells were plated in 6-well plates at the concentration of 2 × 10⁵/well and treated with 5-FU (10 μM) or sorafenib (5 μM) combined with or without exosomes (50 ng/μL). At 48 h after treatment, cell apoptosis and cell cycle were detected using an Annexin V/PI detection kit (BD Biosciences) and cell-cycle staining kit (MultiSciences), in accordance with the manufacturer’s instructions and then analyzed on a BD Accuri® C6 flow cytometer.

To analyze the apoptosis population of SH-SY5Y cells, flow cytometry using annexin V-FITC and propidium iodide (PI) staining was performed using an Annexin V/PI detection kit (#559763, BD Biosciences, San Jose, CA, USA) with a FACSCalibur flow cytometer. Neuronal cells were trypsinized, collected, and washed with PBS. Cells were counted and 1 × 10⁶ cells were suspended in 1 ml cold binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2). Cells were aliquoted into 1.5 ml tube at 1 × 10⁵ cells per tube, and were incubated with 5 μl of annexin V-FITC and 2 μg/ml of PI at room temperature for 15 min. After incubation, 400 μl of binding buffer was added and flow cytometric analysis was performed. FITC and PI fluorescence were passed through 520- and 630-nm bandpass filters, respectively, and the data were analyzed using Flowing Software. The apoptotic rate was calculated as a percentage of Q2 + Q4 quadrants. Six replicates were performed for each group.

After collection and washing twice with stain buffer (BD Biosciences, CA, USA), MenSCs were incubated with antibodies of surface markers including CD29, CD34, CD45, CD73, CD90, CD105, CD117, and HLA-DR (Becton Dickinson, NJ, USA) for 20 min. The stained cells were washed twice with stain buffer and resuspended in 500 μl of stain buffer to be analyzed by FC500 flow cytometer (Beckman Coulter, CA, USA).
HCC cells were seeded in six-well plates and cocultured with MenSCs, MRC-5, and medium only for 72 h, respectively. Cell apoptosis was detected using Annexin V/PI detection kit (BD Biosciences, CA, USA) by FC500 flow cytometer. Data were analyzed using Flowjo software (Tree Star, OR, USA).