phase-contrast microscope with fluorescence accessories  (Nikon)

Journal: Biochemical pharmacology

Article Title: Connexins and apoptotic transformation

doi:

Figure Lengend Snippet: Time course of the development of apoptosis measured in HeLa parental cells and HeLa cells stably expressing Cx43-EFGP. (A) Shown is an image of HeLaCx43-EGFP cells obtained by overlap of fluorescence (in green) and phase contrast (in gray) images. Cx43-EGFP forms large junctional plaques (white arrows) located at the appositional areas between neighboring cells. Yellow arrows indicate vesicles composed of internalized junctional plaques. (B) Shown are percentages of HeLa parental (black bars) and HeLaCx43-EGFP (gray bars) cells distributed among five categories (see Section 2). Apoptosis was induced by bath application of anisomycin, 1 µg/mL. The percentage of NVA cells was significantly higher in HeLaCx43-EGFP than HeLa parental cells.

Article Snippet: Apoptosis assay Fluorescence microscopy (Olympus IX-70 microscope equipped with accessories for phase contrast and broad field fluorescence) was used to evaluate apoptosis.

Techniques: Stable Transfection, Expressing, Fluorescence

Journal: Biochemical pharmacology

Article Title: Connexins and apoptotic transformation

doi:

Figure Lengend Snippet: HeLaCx43-EGFP cells (gray bars) show a higher percentage of NVA cells than HeLa parental cells (black bars) in response to a variety of agents that induce apoptosis. Apoptotic features were examined 48 hr following treatment. Shown are the distributions (in %) of cells evaluated with AO and EB fluorescence staining.

Article Snippet: Apoptosis assay Fluorescence microscopy (Olympus IX-70 microscope equipped with accessories for phase contrast and broad field fluorescence) was used to evaluate apoptosis.

Techniques: Fluorescence, Staining

Journal: Biochemical pharmacology

Article Title: Connexins and apoptotic transformation

doi:

Figure Lengend Snippet: (A, B) Anisomycin inhibits EB uptake through Cx43-EGFP hemichannels. (A) EB uptake over time measured as normalized fluorescence intensity in HeLaCx43-EGFP (open circles) and HeLa parental (filed circles) cells; cells were incubated (5% CO2 and 37°) and perfused with DMEM containing 5 µM EB. The experiments were performed in co-cultures of HeLa parental and HeLaCx43-EGFP cells. Petri dishes with thin glass bottoms containing the cocultures were positioned into a micro-incubator adapted onto the stage of inverted microscope. Time-lapse imaging started just before addition of EB (control) to the perfusion medium. Excitation and emission wavelengths of EGFP and EB do not overlap. (B) The same as in (A), but in addition perfusion medium contained anisomycin, 1 µg/mL. (C, D) Changes in Cx43-EGFP expression and distribution after treatment with apoptotic agents. Time-lapse imaging was used to evaluate changes in total fluorescence and the number of junctional plaques in HeLaCx43-EGFP cells incubated (5% CO2 and 37°) on the stage of an inverted microscope for 48 hr. (C) Changes in total EGFP fluorescence. Each trace is averaged from measurements performed in four experiments for each apoptotic agent; in each experiment fluorescence intensity was measured from six randomly selected fields of view. (D) Changes in the number of Cx43-EGFP junctional plaques. Junctional plaques were assessed in the same experiments and the same fields of view as in (C). The data are averaged and normalized to the data values measured at the beginning of the experiments. (E) Phase-contrast (left) and fluorescence (right) images of HeLaCx43-EGFP cells 16 hr after treatment with anisomycin. HeLaCx43-EGFP cells undergoing apoptosis maintain contact through long cytoplasmic extensions (tube-like structure between cells 1 and 2 shown by two arrows in the phase contrast image). In the example shown, a junctional plaque remains within the cytoplasmic extensions (arrowhead in the fluorescence image).

Article Snippet: Apoptosis assay Fluorescence microscopy (Olympus IX-70 microscope equipped with accessories for phase contrast and broad field fluorescence) was used to evaluate apoptosis.

Techniques: Fluorescence, Incubation, Inverted Microscopy, Imaging, Expressing