Summit software version 5

Three GC cell lines (KATO-III, SNU216, and SNU601) were purchased from the Korean Cell Line Bank and maintained in RPMI1640 medium (Hyclone, Logan UT, USA) supplemented with 10% (v/v) calf serum (Hyclone) at 37 °C in a 5% (v/v) CO₂ humidified atmosphere. The cells were harvested at 300×g for 5 min, incubated in cell-staining buffer containing phycoerythrin (PE)-labeled anti-CD133/1(AC133) antibody (1:10; Miltenyi Biotec, Bisley, UK) for 10 min in a dark refrigerator, and washed with 0.5% (w/v) bovine serum albumin in phosphate-buffered saline, pH 7.2, with 2 mM EDTA. An isotype-matched PE-labeled control antibody (Miltenyi Biotec) was used to label the samples and set gating levels. MoFlo XDP flow cytometry (Beckman Coulter, Brea, CA, USA) was also used to sort cell lines into CD133+ and CD133- populations. The data were analyzed using Summit software, version 5.2 (Beckman Coulter).

For analysis of immunomarkers, the cells were suspended in PBS with 0.5% serum and stained with fluorescent antibodies (eBioscience, Inc., San Diego, USA) against CD11b (Cat: 25-0118-42), Gr1 (Cat: 48-5931-82), Ly6C (Cat: 12-5932-82), Ly6G (Cat: 11-9668-82), CD4 (Cat: 11-0041-82), and CD8 (Cat: 48-0081-82). For analysis of fibrosis markers, cardiac cells were isolated as previously described. The cells were stained with primary antibodies (Affinity, Inc., USA) against collagen I (Cat: AF0134) and α-SMA (Cat: BF9212) and secondary fluorescent antibodies. Flow cytometric analysis or sorting was performed by a flow cytometer (MoFlo Astrios EQ, Beckman Coulter, Indianapolis, IN, USA). All the flow cytometric results were analyzed with Summit Software Version 5.2 (Beckman).