Er tracker red bodipy tr glibenclamide

HUVEC were stained for different intracellular compartments using the following markers:
ER‐Tracker Red (BODIPY TR Glibenclamide) (Thermo Fischer, E34250) was added to cells at 1 µm for 30 min at 37 °C in Na‐Tyrode's buffer. The solution was then washed off before imaging.
LysoTracker Green DND‐26 (Thermo Fischer, L7526) was added at 150 nm for 30 min at 37 °C in Na‐Tyrode's buffer to cells. The solution was replaced with a fresh buffer before imaging.
MitoTracker Green FM (Thermo Fischer, M7514) was added to cells at 0.1 µm for 30 min at 37 °C in Na‐Tyrode's buffer. The solution was then washed off before imaging.

In total 5 pmol (67.6 ng) of human YAP-siRNA (sc-38637, Santa Cruz Biotechnology, Dallas, TX, USA) or 5 pmol of Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA) was transfected into human neurons differentiated from normal iPS cells (ASE-9203, Applied StemCell, Milpitas, CA, USA) using Viromer® BLUE (TT100300, OriGene, Rockville, MD, USA). Before transfection, siRNA was labeled with Label IT® siRNA Tracker™ Fluorescein Kit without Transfection Reagent (MIR7216, Mirus, WI, USA) according to the manufacturer’s procedures. Eighteen hours later, cells were stained with ER-Tracker™ Red (BODIPY™ TR Glibenclamide) (E34250, Thermo Fisher Scientific, MA, USA) and NucRed™ Live 647 ReadyProbes™ Reagent (R37106, Thermo Fisher Scientific, MA, USA) for 60 min at 37 °C. Time-lapse images of iPS-derived neurons were acquired at ×60 magnification on an Olympus FV10i-W (Olympus, Tokyo, Japan) at 15 min intervals for 24 h. The chamber was kept at 37 °C with 5% CO₂. The ratio of cell death patterns was counted cells transfected labeled-siRNA. To validate knockdown efficiency of siRNA, cells were fixed and collected. Each sample was used for Immunocytochemistry and western blot.

Myc-DDK1-tagged SLC15A3, MAVS, STING, pCMV6-AC-GFP-SLC15A3, and pCMV6-AN-HA were purchased from OriGene Technologies (Rockville, MD). HA-tagged SLC15A3 was constructed by standard molecular biology techniques. RFP-PXMP2 was a generous gift from Dr. Hong-Bing Shu (Wuhan University, China). Rabbit anti-human SLC15A3 and anti-human Rab11 were purchased from Abcam. Anti-human Rab5 and Rab7 were purchased from Santa Cruz. Alexa Fluor 594 horse anti-mouse or rabbit secondary antibodies and Alexa Fluor 488 goat anti-mouse or rabbit IgG (H + L) antibody were purchased from Thermo Fisher Scientific. Anti-human β-actin, anti-Flag, and anti-HA antibodies were purchased from Sigma-Aldrich. Lyso Tracker™ Red DND-99 and ER-Tracker™ Red (BODIPY™ TR Glibenclamide) were purchased from Invitrogen (Carlsbad, CA).

ER membranes were imaged using the cell-permeable live-cell dye ER-Tracker Red (BODIPY TR Glibenclamide) (Invitrogen, ThermoFisher, Inc., USA). HCT-116 cells were seeded at 5 × 10³/well into 96-well plates and treated with different concentrations of NB extract. After treatment, ER-Tracker (1 μM) and DNA staining Hoechst 33342 (Sigma-Aldrich) (10 μg/ml) were incubated for 30 min in Hankʼs Balanced Salt Solution with calcium and magnesium (HBSS, Gibco, ThermoFisher, Inc, USA) at 37°C, 5% CO₂. After incubation, the staining solution was replaced with fresh medium and the fluorescent values of ER-Tracker (587/615 nm) and Hoechst 33342 (361⁄497 nm) were obtained using the Cell Imaging Multi-Mode Reader Cytation (BioTek Instruments, Inc., United States).

Seedling, inflorescence, and trichome images were acquired on an Olympus SZ61 microscope. Epifluorescence and DIC microscopy were carried out on a NIKON Eclipse E800 microscope equipped with a SPOT camera (Molecular Diagnostic). Filters from Chroma were used: green fluorescence, Ex460-500/DM505/BA510-560; red fluorescence, Ex546(10)/ DM565LP/EM590LP; yellow fluorescence, Ex490-510/DM515/BA520-550. Confocal imaging was carried out on a Zeiss Meta510 or a Nikon A1. Comparative studies were based on identical imaging conditions and followed procedures described in Duan et al. (2010 (link)). Use of ER-Tracker Red (BODIPY TR Glibenclamide; Life-Technologies) to stain ER followed Cui et al. (2012 (link)), Yang et al. (2013 (link)), and the manufacturer's protocol.