Polyoxyethylene sorbitan monolaurate

A lysing solution, containing 9.0 g/L NH₄Cl, 1.0 g/L KHCO₃, and 0.037 g/L EDTA-4Na, was used to lyse the red blood cells. A 300-mM mannitol solution (Sigma Aldrich, St. Louis, MO, USA) was used for dielectrophoresis because it had suitable conductivity and appropriate osmotic pressure to allow living cells to maintain their shape. The surfaces of the microwell arrays were pre-coated with 0.1% bovine serum albumin (BSA; Sigma Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS). Distilled water containing 0.01% poly-L-lysine was used for the cell attachment procedure. The chamber-washing solution consisted of PBS with 1% BSA and 0.05% polyoxyethylene sorbitan monolaurate (Wako Pure Chemical Industries, Kyoto, Japan).
A fluorescein isothiocyanate (FITC)-conjugated anti-CK monoclonal antibody (mAb), CK3-6H5 (mouse IgG₁), a phycoerythrin (PE)-conjugated anti-CD45 mAb, 5B1 (mouse IgG_2a), and an FcR-blocking reagent were purchased from Milenyi Biotec (Bergisch-Gladbach, Germany). An Alexa Fluor 488-conjugated anti-Pan-CK mAb, AE1/AE3 (mouse IgG₁), was purchased from Thermo Fisher Scientific (Santa Clara, CA, USA). A 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) solution was purchased from DOJINDO Laboratories (Kumamoto, Japan).

Probenecid and polyoxyethylene sorbitan monolaurate (equivalent to Tween-20) were purchased from Wako Pure Chemical Industries (Osaka, Japan). 5-BDBD, AZ11645373, MRS2179, MRS2578, NF157 and MRS2211 were from Tocris Bioscience (Bristol, UK). BAPTA-AM solution was purchased from Dojindo (Kumamoto, Japan). SQ22536 was purchased from Merck (Darmstadt, Germany). Phospho-Akt rabbit monoclonal antibody (#4060), Akt rabbit monoclonal antibody (#4691), goat horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (#7074) and goat HRP-conjugated anti-mouse IgG antibody (#7076) were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Anti-P2X7 receptor antibody (APR-004), anti-P2Y11 receptor antibody (APR-015) and anti-P2Y13 receptor antibody (APR-017) were purchased from Alomone Labs (Jerusalem, Israel). Mouse anti-β-actin antibody (sc-47778) and goat anti-rabbit IgG-FITC (sc-2012) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-connexin 43 antibody (ab11370) was purchased from Abcam (Cambridge, UK). Panx1 polyclonal antibody (12595-1-AP) was purchased from Proteintech, Inc (Chicago, IL). Unless otherwise stated, all other reagents were obtained from Sigma-Aldrich (St Louis, MO). All other chemicals used were of the highest purity available.

Western blot analysis was carried out by a procedure described previously [9 (link)]. In brief, the PANC-1 cells were treated with three different concentrations of compound 3 (20, 40, and 60 μM) for 6 h in NDM. Proteins were then separated by gel electrophoresis on a polyacrylamide gel containing 0.1% sodium dodecyl sulfate and transferred to polyvinylidene fluoride membranes. The membranes were blocked with Block Ace (DS Pharma Medical, Suita, Japan), washed with PBS containing 0.1% polyoxyethylenesorbitan monolaurate (Wako Pure Chemical, Osaka, Japan), and incubated overnight with caspase-3, cleaved caspase-3 and GAPDH antibodies (Cell Signaling Technology, Danvers, MA, USA). After washing, the membranes were incubated for 45 min at room temperature with horseradish peroxidase-conjugated anti-rabbit or anti-goat immunoglobulins as the secondary antibody. The bands were detected with an enhanced chemiluminescence solution (Bio-Rad, Hercules, CA, USA).