Ethidium homodimer

Gels were immersed in PBS containing 20 μg of fluorescein diacetate (Sigma-Aldrich) and 4 μM ethidium homodimer (Sigma-Aldrich) for 10 min at 37°C, after which they were placed on a glass slide. Gels were imaged using a Zeiss LSM610 confocal microscope.

Juvenile greenlip abalone (Haliotis laevigata) were farmed in Tasmania and distressed for a week without feeding in 4000-l tanks filled with filtered sea water at 14–18 °C. Dispase, sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl₂), CelLytic M reagent, ethidium homodimer, phenazine methosulfate (PMS), 3,3′,5,5′ tetramethylbenzidine (TMB), bovine serum albumin (BSA) and ammonium sulphate were purchased from Sigma-Aldrich, Australia. Phosphate buffer solution (PBS pH 7.4), antibiotic–antimycotic solution (anti-anti) 100 × , fetal bovine serum, KnockOut™ serum replacement, minimum essential medium (MEM) vitamin solution 100 × , minimum essential medium (MEM) amino acid solution 100 × , chemically defined lipid concentrate, (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) assay kit, Insulin-Transferrin-Selenium-Ethanolamine (ITS -X) (100 ×), fluorescein isothiocyanate (FITC)-conjugated anti-rabbit antibody from goat and Qubit 2.0 Fluorometric Protein Quantification kit were supplied by Invitrogen (US). F-12 nutrient mixture ham medium, Leibovitz’s L-15, modified Eagle’s medium (MEM) and Dulbecco’s modified Eagle’s medium (DMEM) were supplied by Gibco, Lipimax® by Selbourne Biological Service (Aus), magnesium chloride and magnesium phosphate by MERCK. Rabbit anti-hemocyanin polyclonal antibody was produced in-house.

OKT 9 and H25B10 hybridoma cells were washed 3 times with plain DMEM before encapsulation into droplets. Either OKT 9 or H25B10 cells were then injected into the droplet production chip as shown in Figure 1Bi, however, instead of K562 and fluorescently labeled antibodies, plain DMEM was injected. The aqueous phases were injected at a flow rate of 500 μL/hr, whereas Novec 7500 oil (Iolitec Liquids Technologies) with 1% PS-2 Surfactant (Sphere Fluidics) was injected at a flow rate of 4,000 μL/hr to produce droplets. After the cell encapsulation, the droplets were stored in the incubator at 37°C under a 5% CO₂ atmosphere. At various time intervals (2, 4, 6, 12, and 24 hr), 200 μL of emulsion was broken with an equal volume of 1H,1H,2H,2H-Perfluoro-1-octanol (PFO; Sigma), and cells were recovered from the aqueous phase. The recovered cells were then stained for 30–40 min with a staining solution containing Calcein-AM (2 μM, Thermo Fisher Scientific) and Ethidium Homodimer (4 μM, Sigma) in PBS. After incubation, images of the viable (green) and non-viable (red) cells were captured using a Nikon Ti-Eclipse microscope. The cells were counted within 4 different fields of view (>100 cells) for each time interval, from 3 independent experiments and plotted as mean viable cells ±SD.

As described above, hippocampal neurons were derived from E17–18 pups and incubated for seven days. The neurons were pretreated with QBA (0–30 µM) for 48 h and subsequently exposed to hypoxia (<0.3% O₂) for 48 h. The cultures were stained with calcein AM (Invitrogen), a marker of live cells, with ethidium homodimer (Sigma-Aldrich), a marker of cell death, and with tetramethylrhodamine ethyl ester (TMRE; Sigma-Aldrich), a marker for mitochondrial health [27 (link)]. The fluorescence of each of these markers was then quantitated with a fluorimeter.

Plumbagin and DMSO, with purity >97%, were purchased from Sigma-Aldrich (St. Louis, MO, USA). A 100 mM stock solution of Plumbagin was prepared in DMSO, stored as small aliquots at −20°C, and then diluted as needed into cell culture medium. Penicillin-streptomycin solution, RPMI-1640 medium and fetal bovine serum (FBS) were obtained from CellGro (Manassas, VA, USA). Antibodies against AKT, BIM, PARP1, NF-κB, cyclin B1, BCL2, cleaved PARP1, caspase 3, cleaved caspase 3, caspase 9 and cleaved caspase 9 were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibody to cyclin D1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); the FAK and Src antibodies were from BD Bioscience (San Jose, CA, USA), and the antibodies against p53 and p21^WAF1/CIP1 were obtained from Epitomics (Burlingame, CA, USA) and EMD Millipore (Billerica, MA, USA), respectively. Ethidium homodimer was obtained from Sigma-Aldrich.