7 aminoactinomycin d 7 add

For analysis of cell growth curve, cells were seeded into 12-well plates at the initial density of 2 × 10⁴ cells/well. The cells were then harvested every 24 h until 96 h and cell numbers were counted by trypan blue exclusion assay. For cell cycle analysis, cells were seeded into 6-cm dishes at the initial density of 2 × 10⁵ cells/dish. Before cell harvesting, 10 μM BrdU was added to dishes and incubated at 37 °C for two h. The harvested cells were then fixed, permeabilized, and incubated with anti-BrdU- fluorescein isothiocyanate (FITC) antibody according the manufacturers’ protocol (BD Biosciences, San Jose, CA, USA). Before fluorescence-activated cell sorting (FACS) analysis, the cells were stained with 7-aminoactinomycin D (7-ADD) (BD Biosciences) on ice for 10 min. The fluorescence signals were detected by Epics XL flow cytometry (Beckman Coulter, Inc., Atlanta, GA, USA) and data were analyzed by WinMDI software (J. Trotter, Scripps Research, La Jolla, CA, USA).

The used antibodies include: CD34 (FITC, clone 8G12, BD Bioscience) (5 μL/1× 10⁶ cells), CD3 (PE/CY5, UCHT1, BioLegend) (2 μL/1× 10⁶ cells), CD56 (FITC, clone 3G8, BD Bioscience) (2 μL/1× 10⁶ cells), CD57 (FITC) (4 μL/1× 10⁶ cells) and CD105 (FITC) (4 μL/1× 10⁶ cells). Flow cytometry was performed on day 15. Briefly, the collected cells were centrifuged at 300 g and washed twice with cold PBS plus 5% FBS (staining buffer). Then the cells spun down and were re-suspended in 50 μL of staining buffer. After adding the appropriate volume of antibodies, the cells were incubated in 4°C in the darkroom. Following 20-30 minutes incubation, the stained cells were washed with 1ml of staining buffer to remove unbounded antibodies. Finally, the cells were re-suspended in 500 μL of staining buffer and were evaluated by flow cytometry. 7-Amino-actinomycin D (7ADD, (5 μL/1 × 10⁶ ells, BD Bioscience) were used for excluding dead cells. Approximately, 10 000 to 30000 events were tracked for each samples by using BD caliber (BD eBioscience) and data were analyses by FlowJo (7.6.1) software.

Flow cytometry was performed to test the tumor cells migrated to draining lymph nodes, the diLNs and dpLNs were removed from footpad tumor-bearing mice after the euthanize was performed. Then, all lymph nodes were ground separately and digested with collagenase in a 37°C incubator for 20 minutes. The concentration of collagenase was 1 mg/ml. Collagenase was neutralized with DMEM media, and the liquid with lymph node tissue was filtered. 5 × 10⁵ cells from each cell suspension were stained with 7-amino-actinomycin D (7-ADD, 0.25 ^µg/105 cells, BD Bioscience) to eliminate dead cells before flow cytometry. Equal amounts of cells (50,000) were harvested from the suspension of each sample by FACS (Becton, Dickinson and Company, USA). The GFP positive cells were tumor cells migrated from tumor tissues to lymph nodes. The percentage of GFP positive cells may represent the metastasis of tumor.