Jurkat t cells

Human 293T cells, Jurkat-T cells and HeLa cells were obtained from the China Center for Type Culture Collection and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 100 U mL^–1 penicillin, and 100 μg mL^–1 streptomycin. Cells were maintained in a humidified atmosphere with 5% CO₂ at 37 °C. As for the D₃-Met labeling, 30 mg of D₃-Met was added into the l-methionine-free DMEM-KO medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 100 U mL^–1 penicillin, and 100 μg mL^–1 streptomycin.
A total of 10 pairs of hepatocellular carcinoma (HCC) tissues and matched tumor-adjacent normal tissues were collected from Hubei Cancer Hospital. An approval was granted by the Hubei Cancer Hospital Ethics Committee and the study met the requirements of the declaration of Helsinki. All the experiments were performed in accordance with Hubei Cancer Hospital Ethics Committee's guidelines and regulations.

Jurkat T-cells were purchased from the China Center for Type Culture Collection and cultured with 5% fetal bovine serum (Gibco Thermo Fisher, USA) in RPMI 1640 (Gibco) under 5% CO₂ in an incubator (Thermo Scientific). Only cells in the logarithmic growth phase were used in these experiments. The average cell diameter was 11.5 μm, consistent with previous reports.^{30,31 (link)} Dynabeads M270 Amine (Thermo Fisher) were incubated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide for 2 h to activate the amine group. The peptide GRGDNP was synthesized by a solid phase peptide synthesis method (Yuanpeptide Biotech Co. Ltd.). The beads were then incubated with 1 μM peptide, resulting in conjugation of the amine and the carboxyl group of the peptide. The beads were then washed and suspended in RPMI 1640 medium. Jurkat cells were washed with RPMI 1640 containing 10% IgG-free serum and then mixed with magnetic beads coated with polypeptides. The mixture was centrifuged at 500 rpm for 5 min, followed by incubation at 37 °C for 45 min to form rosettes (Fig. 2).^{32 (link)}