Magnetic cell sorting device

A375-SM tumor cells were subcutaneously injected into the right flanks of female nude mice. TECs and NECs were isolated from the tumor xenografts and the dermis of tumor-free counterparts, respectively. ECs were isolated using CD31 microbeads and a magnetic cell sorting device (Miltenyi Biotec, Bergisch Gladbach, Germany), followed by flow cytometry (FACS, Aria II; BD Biosciences, San Jose, CA, USA) with CD31 and CD45 antibodies. CD31⁺ cells were cultured in EGM-2 MV (Lonza) containing 15% FBS at 37 °C in a humidified atmosphere with 5% CO₂. The remaining human tumor cells carrying the DT receptor were eliminated using diphtheria toxin (DT, 500 ng/mL; Calbiochem, San Diego, CA, USA). Isolated ECs were further purified using FITC-BS1-B4 lectin (Vector Laboratories, Burlingame, CA, USA). TECs and NECs were determined to be positive for EC markers (Pecam1, Eng, Cdh5, Icam1, Flt1, and Kdr) and negative for hematopoietic markers (Itgam and Ptprc). cDNA from MS1 cells was used as a control for EC markers, and the cDNA from CD31-negative cells as a control for non-EC markers. All antibodies used are listed in Additional file 1: Table S1.

Cell suspensions were treated in a sorting buffer, which contained MACS Buffer A (autoMACS^® Rinsing Solution) and Buffer B (MACS^® BSA Stock Solution) in a 20:1 ratio. Centrifuge cell suspensions at 1000 g for 10 min. Add 20 µL of CD44 Microbeads (MiltenyiBiotec, Bergisch Galdbach, Germany) and 80 µL of sorting buffer per 1 × 10⁷ total cells. Mix well and incubated at 4 °C for 15 min in the dark, followed by MACS buffer washes and centrifugation for 10 min at 1000 rpm. Cells were suspended in 500 µL of sorting buffer per 1 × 10⁷ total cells. CD44(+) cells and CD44(-) cells were dissociated using a magnetic cell sorting device (MiltenyiBiotec, Bergisch Galdbach, Germany). Briefly, the CD44(+) cells are magnetically labeled with CD44 microbead before they were run through an LS magnetic column, CD44(+) cells were eluted when the magnet was taken away from the column. In addition, to prevent the differentiation of CD44(+) cells into CD44(-) cells after multiple passages, second-generation CD44(+) cells were used in all the experiments, i.e., after sorting, the CD44(+) cells were passaged only once to maintain their cancer stem cell properties.