Facsaria 3 flow cytometer

Cancer stem-like cells from BxPC3 cancer cell line were labelled using the ALDEFLUOR assay kit according to the manufacturer’s recommendations (StemCell Technologies). The enriched subpopulation of CSCs was characterized using a FACSAria III flow cytometer (Becton Dickinson).
To analyse CSC markers treated and not treated BxPC3 CSCs were disaggregated by tryple and washed twice in PBS supplemented with 1% bovine serum albumin (Sigma-Aldrich). The cell surface Fc receptor was blocked using IgG (Santa Cruz Biotechnology) on ice for 15 min. Cells were stained for 30 min at 4 °C with anti-CD44-PE and anti-CD326 FITC and CxCR4-APC monoclonal antibodies (BD Biosciences). After washing, cells were analyzed using a FACSAria III flow cytometer (Becton Dickinson).

Apoptosis was detected using an Annexin V-FITC Apoptosis Detection Kit (Koma Biotech, Seoul, South Korea). Briefly, cells transfected for 48 h were harvested through trypsinization, rinsed in PBS, and resuspended in 1× binding buffer before being incubated with 1.25 μL of Annexin V-FITC solution at 22–25 °C for 20 min in the dark, stained with 10 μL propidium iodide (PI; Sigma-Aldrich), and analyzed using a FACSAria III flow cytometer (BD Biosciences).
Apoptotic cells were evaluated using Hoechst 33342 and PI staining. The transfected cells were fixed in 4% paraformaldehyde (Biosesang) for 10 min and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 20 min. The cells were then treated with Hoechst 33342 (10 μg/mL; Invitrogen) for 10 min, stained with PI (20 μg/mL; Sigma-Aldrich) for 20 min, and observed using an Olympus IX71 microscope (Olympus).
To analyze the cell cycle, transfected cells were collected, fixed using 70% ethanol for 3 h at 4 °C, washed in PBS, and stained using 200 μL PBS containing 10 μg/mL RNase A, 1% Triton X-100, and 30 μg/mL PI (Sigma-Aldrich) for 30 min in the dark. The proportion of cells at each stage of the cell cycle was determined using a FACSAria III flow cytometer (BD Biosciences). Experiments were performed in triplicate.

For ABCs detecting, PBMCs/B cells to be detected were incubation with FcR blocking reagent (Miltenyi Biotec) for 10 minutes at 4 °C, then stained with BV421 anti-CD11c (BioLegend) and percp-Cy5.5 anti-CD19 (BioLegend) at 4 °C for 30 minutes. For intracellular staining, cells were surface stained, fixed using eBio fix/perm kit (eBioscience), washed and stained with PE-Cyanine7 anti-T-bet (BioLegend) in 1 × eBio fix/perm buffer for 1 hour at 4 °C. Mouse IgG1 kappa Isotype Control, PE-Cyanine7 (eBioscience) was used as isotype control for T-bet staining.
For RNASE2 detecting, PBMCs to be detected were incubation with FcR blocking reagent (Miltenyi Biotec) for 10 minutes at 4 °C, then stained with APC anti-CD14 and viability dye efour 506 (Invitrogen) at 4 °C for 30 minutes. For intracellular staining, cells were surface stained, fixed with fixation/permeabilization solution (Cytofix/Cytoperm kit, BD), washed and stained with RNASE2 antibody (Invitrogen) in 1× Perm/Wash buffer for 1 hour at 4 °C, then stained with AlexaFlour 488 Goat anti-rabbit IgG (FCMACS). Cells stained with CD14 and AlexaFlour 488 Goat anti-rabbit IgG were used as isotype control for RNASE2 staining.
Then Cells were analyzed on a BD FACSAria III flow cytometer (BD Biosciences) using FACSDiva software. Cells were analyzed on a BD FACSAria III flow cytometer (BD Biosciences) using Flowjo VX software.