Fortasa flow cytometer

Flow cytometry was performed using a previously described method (Fang et al., 2016 (link)). In brief, HN4 and HN30 cells were incubated with the indicated treatment for 48 h. Cells were suspended in 400 μl 1 × binding buffer, then added 5 μl Annexin V-FITC (BD Biosciences, Franklin Lake, NJ, USA) to the cell suspension, and incubated on ice for 30 mins. Then 5 μl propidium iodide (PI) was added and incubated on ice for 5 mis. All the procedure should protect from prolonged exposure to light. After PBS wash, cells were resuspended in 100 μl of binding buffer and analysed on a BD FORTASA flow cytometer. The final results were analysed with FlowJo software. For the cell cycle analysis, cells that had been exposed to the indicated treatment were fixed with 70% ethanol overnight. Cells were then washed and incubated with a PI/RNase staining kit (BD). For the Dc and natural killer cell analysis, peripheral blood monocytes (PBMC) were extracted from nude mice and analysed with the following antibodies and Fixable Viability Dye (eBioscience, San Diego, CA, USA), a FITC-I-A/I-E antibody, APC-CD11c antibody, and PE-CD49b antibody (all purchased from BD Biosciences). The subsequent steps performed as described above.

Flow cytometry was performed as described previously 16 (link). Briefly, HN4 and HN30 were treated with 0, 2, 20 ng/ml IFNα in 12 well-plate for 72 h. The cells were collected, washed and incubated with CD44 and ALDH1 antibodies at 1:100 dilution. After 1 hour incubation, cells were washed and incubated with FITC-labeled secondary antibody for 30 min. Then cells were washed twice and resuspended in 100 μl of FACS buffer. The stained cells were analyzed on BD FORTASA flow cytometer. The final results were operated using FlowJo software (Tree Star).