Yo pro 1 iodide

Cells were seeded at 1 × 10⁴ cells for NIH3T3 and HUVEC and 2 × 10⁴ for A549, MCF7, HT29 cells per well, in 100 µL complete medium, into 96-well plates and treated with garlic juice or allicin to obtain the final concentrations as indicated. The cells were incubated with allicin for 30 or 60 min, washed in PBS and subsequently incubated in the presence of 1 µM YO-PRO-1 iodide in 100 µL PBS (Molecular Probes, Eugene, OR, USA) for 20 min at room temperature. Fluorescence of YO-PRO-1 iodide is used as a measure for the relative quantity of apoptotic cells. Fluorescence was measured by spectrofluorimetry (VICTOR 2; PerkinElmer, Paris, France) at 490 nm excitation and 510 nm emission as described in [34 (link)].

Cells were plated in black 96-well plates with clear bottom. When a confluence around 80–90 % was reached, the media was changed to a physiological buffer (140 mM NaCl, 10 mM HEPES, 1 mM MgCl₂, 0.4 mM KH₂PO₄, 1.6 mM K₂HPO₄, 1.5 mM CaCl₂) with 11.1 mM of glucose for AsPc-1 and BxPC-3 and 25 mM for Panc-1. Cells were incubated with AZ10606120 (10 μM) or A438079 (10 μM) for 1 h. Then cells were incubated with Yo-Pro-1 Iodide (2.5 μM, Molecular Probes) for 15 min. ATP was dissolved in a physiological buffer and the indicated concentrations were added. Ionomycin (5 μM) was added at the end of each experiment. Fluorescence was read every 10 min by FLUOstar OPTIMA.

Poly‐d‐Lysine (PDL) (P0899), DNase 1 type IV, Triton X‐100, FITC‐NHS, Rhodamine 6G (Rh6G), fluorescein sodium salt (FITC), and 1,1′‐Dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate were purchased from Sigma‐Aldrich. Alexa‐Fluor 488 goat anti‐mouse, 4′,6‐diamidino‐2‐phenylindole, paraformaldehyde (PFA, 7 230 681), goat serum, Dulbecco's modified eagle medium (DMEM), penicillin/streptomycin (Pen/Strep), B27 supplement (17 504 001), GlutaMAX, neurobasal medium, Fluoromount‐GTM mounting medium (00‐4958‐02), fetal bovine serum (FBS), CMFDA, and YO‐PRO‐1 iodide were purchased from Life Technologies. Mouse anti‐Tuj1 antibody (801 202) was purchased from Biolegend. Rabbit anti‐GFAP (Z0334) was obtained from DAKO. Papain suspension was purchased from Worthington. Cell strainer (70 µm) was obtained from BD, Biosciences, USA.

All cell culture materials were obtained from Gibco Inc. Recombinant Human Activin-A (GFH6) was purchased from Cell Guidance Systems. RepSox (#72392), a cell permeable, selective inhibitor of the TGF-β type 1 receptor (TGFβRI) ALK5 was obtained from Stemcell Technologies. Recombinant forms of FSH (Gonal-F) and hCG (Ovitrelle) was purchased from Merck Global (Darmstadt, Germany). SAPK/JNK Kinase Assay Kit (#8794, nonradioactive), Hoechst 33342 (#4082), anti c-Jun (#9165), anti-phospho-c-Jun^Ser73 (#3270S), Anti-Phospho-SAPK/JNK^{Thr183/Tyr185} (#9251), Smad2 (#3122) and Phospho-Smad2^{Ser465/Ser467} (#18338) antibodies were obtained from Cell Signaling. Oil Red O was purchased from Sigma Inc. (USA). All western blotting buffers and reagents were purchased from Bio-Rad. Anti-Vinculin Antibody (V9264) was purchased from Sigma-Aldrich. Mouse antihuman monoclonal antibodies were purchased from Santa Cruz Biotechnology for the detection of human 3β-HSD Type II (sc-100466), 17β-HSD type-I (sc-376719), StAR (sc-166821), and P450 SCC (CYP11A1, sc-292456). Aromatase (CYP19A1, ab34193) monoclonal mouse antibody was from Abcam Inc. YO-PRO®-1 Iodide (491/509) was obtained from Life Technologies. Texas Red™-X Phalloidin was obtained from Thermo Fisher Scientific.

For YOPRO-1 dye uptake experiments cells were plated at a density of 2 × 10⁴ cells/well in complete RPMI 1640 media (100 µL per well) in poly-D-lysine coated 96-well plates. Media was removed using a manual multichannel pipette and replaced with a low divalent cation buffer (145 mM NaCl, 2 mM KCl, 13 mM D-glucose, 10 mM HEPES and 0.1 mM CaCl₂, pH 7.3) containing 2 µM YO-PRO-1 iodide (Life Technologies catalogue number Y3663). For most experiments, ginsenosides (10 µM) were co-injected simultaneously with the agonist using a Flexstation 3 microplate reader (Molecular Devices, UK). Ginsenosides and agonist were prepared at 10X final concentration in the compound plate. Dye uptake over time was recorded using an excitation wavelength of 488 nm and an emission wavelength of 520 nm on the Flexstation 3 (6 reads/well, PMT setting medium). Basal fluorescence measurements were acquired for 40 sec followed by automatic injection of agonist and the kinetic measurement of fluorescence intensity was performed for 300 sec using Softmax Pro v5.4 software (Molecular Devices). Measurements were performed in triplicate and repeated in three independent experiments. Dye uptake responses were calculated as area under the curve from 50–300 sec using zero baseline normalised data.