Binding buffer

HeLa and CaSki cell apoptosis was measured using fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI) (BD Pharmingen; Becton-Dickinson and Company, Franklin Lakes, NJ, USA). The cells were washed twice in cold PBS and then resuspended in Annexin V-binding buffer (BD Pharmingen; Becton-Dickinson and Company) at a concentration of 3×10⁶ cells/ml. Additionally, 100 µl of the suspension was incubated with 5 µl Annexin V-FITC and 5 µl PI in dark at 37°C for 15 min. After gentle vortexing, the cells were incubated for 15 min at room temperature in the dark. Following the addition of 400 µl binding buffer (Sigma-Aldrich; Merck KGaA) to each tube, the cells were analyzed using FACSAria™ III Cell Sorter flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo V10 software (FlowJo LLC, Ashland, OR, USA).

For apoptosis analysis, Jurkat cells were treated with different concentrations of ex-LAC, and 0.1% Triton X-100 was added as a positive control. Following 48 h of incubation, all cells were washed using phosphate-buffered saline (PBS; Biochrom; Merck Millipore), resuspended in a binding buffer (Sigma-Aldrich) and stained with 5 µl Annexin V-fluorescein isothiocyanate (FITC) and 10 µl propidium iodide (PI), according to the manufacturer's protocol of the Annexin V-FITC Apoptosis Detection Kit (Sigma-Aldrich). Cells were incubated for 10 min in the dark, and immediately analyzed with FACSCalibur™ (BD Biosciences, San Jose, CA, USA). Live cells served as a negative control.

Three milligrams of epoxidized magnetic particles were added to 1 mL of prepared coupling buffer(Aladdin Crop, U.S.A) [9 (link)]. The sample was mixed well and placed on a magnetic separation rack. When it was observed that the magnetic particles were obviously adsorbed on the magnet, the supernatant was gently discarded. This step was repeated 3 times to clean the magnetic particles. After cleaning, 600 μL of prepared coupling buffer and 300 μg of SNA were added and shaken at 20 °C and 180 rpm. After the reaction time, the SNA-magnetic particle complex was washed. Then, 1 mL of preprepared blocking solution(Sigma-Aldrich Corp, U.S.A) was added to the cleaned SNA-magnetic particle complex, shaken for 1 h. Next, the blocked magnetic particle complex was washed with 1 mL of binding buffer(Sigma-Aldrich Corp, U.S.A) 3 times, 600 μL of the sample system were added, and the mixture was shaked 3 h at 25 °C, 120 rpm. After the reaction, the glycoprotein-magnetic particle complex was washed at least 6 times. After washing, 300 μL of elution buffer(Sigma-Aldrich Corp, U.S.A) were added to the system and placed on a shaker for 1 h, and the eluted solution was collected. We added 300 μL of elution buffer to the original system, repeated the steps, and collected the solution to obtain a total of 600 μL of glycoprotein solution.

Transfected and untransfected A549/DDP cells (3.5×10⁵ cells/ml) were seeded into 12-well plates and incubated with 0, 1.0 or 2.0 µg/ml DDP for 24 h. The A549/DDP cells were then washed with 1X PBS and resuspended in 100 µl binding buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Next, Annexin V-FITC and PI (Sigma-Aldrich; Merck KGaA) were added for 30 min at 37°C in the dark. Following dilution with 400 µl binding buffer, staining was analyzed within 1 h by flow cytometry. The fluorescence intensity (green, FL1-H; red, FL2-H) was measured using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). CellQuest Pro software 5.1 (BD Biosciences) was used for acquisition and analysis of data.