Eclipse te2000 inverted microscope

The assay was applied as described (Woriedh et al., 2013 (link)). Briefly, freshly collected pollen was immediately distributed onto Petri dishes containing droplets of 10 µL PGM at different pH values. Pollen tubes started to burst or to grow after about 10min. Pollen tubes were subjected to further studies only if germination rates exceeded 80%. After optimization, PGM at pH 5 was applied to all pollen tube assays. A 10 µL solution of the above peptides or acetonitrile as a negative control was added to each droplet of germinated pollen using a Cell-TramH Air pump (Eppendorf) and a glass micropipette. During this assay, droplets were observed using an Eclipse TE2000 inverted microscope (Nikon) equipped with a 1.4 Megapixel digital AxioCam MRm camera (black and white) and AxioVision digital image processing software (Release 4.8).

Changes in the morphology of cervical cells incubated with various concentrations of SW-AgNPs were analysed my microscopy 24 hours post treatment. Bright field images (20x) of the cells were captured using Eclipse TE2000 Inverted Microscope (Nikon).

A total of 400,000 BV-2 microglial cells were grown in six-well plates as 80% confluent monolayers and were wounded with a sterile 100 μl pipette tip. Thereafter, the cells were stimulated with 50 ng/ml LPS, 50 μM XBD173, 50 ng/ml LPS + 50 μM XBD173, or ethanol as solvent control. Migration into the open scar was documented with microphotographs taken at different time points after wounding using a Nikon ECLIPSE TE2000 inverted microscope (Nikon, Tokyo, Japan). The number of migrating cells was quantified by counting all cells within a 0.4 mm² region in the center of each scratch. The number of migrated cells was then normalized to the average cell density to account for changes in proliferation. A minimum of five individual cultures was used to calculate the mean migratory capacity of each cell culture condition.

Cells were imaged on the 6-well plate at 10× magnification with a set scale of 100 μm using Nikon eclipse TE2000 inverted microscope. Another set of cells was used for Oil Red O staining. Briefly, cells were washed twice with 1X DPBS. Cells were then fixed in 10% phosphate-buffered formalin for 30 min at room temperature. After fixation, the cells were washed twice again with 1X DPBS and then treated with Oil Red O, four parts ddH₂O with six parts Oil Red O solution (Sigma-Aldrich), for 15 min. Each well was gently washed 5 times with ddH₂O and then imaged (22 (link)).

To monitor single‐cell ROS dynamics, time‐lapse imaging experiments were performed using a Nikon Eclipse TE‐2000 inverted microscope equipped with a Nikon Perfect Focus System and a Hamamatsu Orca ER camera under conditions of controlled temperature (37°C), atmosphere (5% CO₂), and humidity (90–100%). Cells were excited at both 432 and 475 nm (5% intensity and 150 ms exposure) with 432/36–25 nm and 475/25–25 band‐pass filters, a 550‐nm dichroic mirror, and a 536/40–25 nm emission filter. Images were acquired every 15 min with a 20× Nikon plan apo objective (NA 0.75). Ratiometric readouts of F475 nm/F432 nm were acquired to report ROS levels. To quantify cell death kinetics, time‐lapse imaging experiments were performed using the IncuCyte S3 Live‐Cell Analysis System (Essen BioScience) with a 10× objective at 37°C and 5% CO₂. Bright‐field and green fluorescent channels were used to capture cellular morphology and YOYO‐1 iodide fluorescence signal, respectively. To monitor the intracellular Ca²⁺, Fluo‐4 AM, cells were excited at 475 nm (5% intensity and 200 ms exposure) with 475/35–25 band‐pass filter, a 500‐nm dichroic mirror, and a 536/40–25 nm emission filter. Image analyses and quantifications of cell death based on YOYO‐1 iodide fluorescence signal were performed using the analysis tool in the IncuCyte S3 Live‐Cell Analysis System.