Lsm 510 confocal microscopy system

LNCaP cells were plated at a density of 100,000 cells/plate on Ibidi μ-dishes (Ibidi, LLC, Verona, WI) and subcultured at 37°C in RPMI 1640 medium supplemented with 10% FBS and 2 mM L-glutamine. After 48 h in culture, cells were transfected with GFP-tagged recombinant constructs using Lipofectamine Plus reagent (Invitrogen) according to the manufacturer’s recommendations. After 24 hours, the cells were treated with 1000 nM of PMA in confocal medium (Dulbecco’s Modified Eagle Medium without phenol red supplemented with 1% FBS), and time-lapse images were collected every 30 s using the Zeiss AIM software. Imaging was performed with a Zeiss LSM 510 confocal microscopy system (Carl Zeiss, Inc.) with an Axiovert 100 M inverted microscope operating with a 25 mW argon laser tuned to 488 nm. A 63×1.4 NA Zeiss Plan-Apochromat oil-immersion objective was used together with varying zooms (1.4 to 2×). Imaging was performed in the Center for Cancer Research Confocal Microscopy Core facility.

LNCaP cells were plated at a density of 100,000 cells/plate on Ibidi μ-dishes (Ibidi, LLC, Verona, WI) and subcultured at 37 °C in RPMI 1640 medium supplemented with 10% FBS and 2 mm l-glutamine. After 48 h in culture, cells were transfected with GFP-tagged recombinant constructs, using X-tremeGENE HP DNA transfection reagent (Sigma) according to the manufacturer’s recommendations. After 24 h, the cells were treated as indicated with 100 nM, 1 μM and 10 μM of PMA in confocal medium (Dulbecco’s Modified Eagle Medium without phenol red supplemented with 1% FBS), and time-lapse images were collected every 30 s using the Zeiss AIM software. Imaging was with a Zeiss LSM 510 confocal microscopy system (Carl Zeiss, Inc.) with an Axiovert 100 M inverted microscope operating with a 25 mW argon laser tuned to 488 nm. A 63 × 1.4 NA Zeiss Plan-Apochromat oil-immersion objective was used together with varying zooms (1.4 to 2×). Imaging was performed in the Imaging Core Facility, Center for Cancer Research, Bethesda, MD.

All reactions utilizing air- or moisture-sensitive reagents were performed in dried glassware under an atmosphere of nitrogen using commercially supplied solvents and reagents unless otherwise noted. CH₂Cl₂ was distilled from CaH₂ and stored over molecular sieves. For thin-layer chromatography (TLC), precoated silica gel plates (Merck 60F254) were employed. TLC plates were visualized by fluorescence quenching under UV light and by staining with phosphomolybdic acid, p-anisaldehyde, or ninhydrin. Flash chromatography was performed with Wakogel C-200 (Wako Pure Chemical Industries, Ltd.) and silica gel 60 N (Kanto Chemical Co., Inc.). ¹H NMR (400 or 500 MHz) and ¹³C NMR (125 MHz) spectra were recorded on a Bruker Avance III 400 spectrometer or a Bruker AVANCE 500 spectrometer. Chemical shifts are reported in δ (ppm) relative to Me₄Si (in CDCl₃) as the internal standard. Infrared (IR) spectra were recorded on a JASCO FT/IR 4100 and are reported as wavenumber (cm⁻¹). Low- and high-resolution mass spectra were recorded on a Bruker Daltonics micrOTOF-2focus (ESI-MS) spectrometer in the positive or negative detection mode. For confocal laser scanning fluorescence images, cells were observed with a FluoView FV10i laser scanning confocal microscope (OLYMPUS, Japan) or a Zeiss LSM 510 confocal microscopy system (Carl Zeiss, Inc.).

LNCaP cells were plated at a density of 100 000 cells per plate on Ibidi microdishes (Ibidi, LLC, Verona, WI) and cultured at 37 °C in RPMI-1640 medium supplemented with 10% FBS and 2 mM L-glutamine. After 48 h in culture, cells were transfected with a GFP-tagged recombinant PKCε construct using X-tremeGENE HP DNA transfection reagent (Sigma) according to the manufacturer’s recommendations. After 24 h, the cells were treated as indicated with 1 μM of PMA and 3 μM of DAG–lactone derivatives in confocal medium (Dulbecco’s modified Eagle medium without phenol red supplemented with 1% FBS), and time-lapse images were collected every 30 s using the Zeiss AIM software. Imaging was with a Zeiss LSM 510 confocal microscopy system (Carl Zeiss, Inc.) with an Axiovert 100 M inverted microscope operating with a 25 mW argon laser tuned to 488 nm. A 63 × 1.4 NA Zeiss Plan-Apochromat oil-immersion objective was used together with varying zooms (1.4 to 2×). The imaging was performed using the resources of the Confocal Microscopy Core Facility, Center for Cancer Research, National Cancer Institute.