Eclipse ts100 microscope

Cell migration was evaluated by wound healing assay and Transwell migration assay. HemSCs cells were seeded in 6-well-plates, and the cell monolayer was scratched with a sterile tip in the middle when cell confluence reached 80%. Then, the cell debris was washed away with culture medium. The pictures of cell morphology at different time points were taken with the Nikon Eclipse TS100 microscope. The narrowest distance of the gap between the front lines was measured by ImageJ software. Transwell migration assay was performed using the 8 μm pore size membrane chamber (Corning) in 24-well plates. Briefly, 2 × 10⁵ cells were plated in the upper chamber with EBM-2 medium supplemented with 0.5% FBS. For 6 hours, cells were allowed to migrate to the bottom chamber, which contained RTK growth factors (100 ng/mL EGF, FGF2, or VEGF) in EBM-2 medium supplemented with 0.5% FBS or full growth medium. After fixation with 4% paraformaldehyde and staining with 0.1% crystal violet and PBS, images of cells that migrated across the pore membrane were captured with Nikon Eclipse TS100 microscope.

PI staining (Sigma-Aldrich Corporation, St. Louis, MO, USA) was used to examine the cell viability in the 1 cm circle, as well as the 2 and 3 cm bands of the irradiation area, providing more spacial information of the cell response to our designed irradiation intensities [34 (link)]. At 2, 24, and 48 h after irradiation, cancer cells were incubated with 7.5 μM PI buffer for 15 min at 37°C and then washed twice with PBS. PI fluorescence was monitored via Nikon Eclipse TS 100 microscope (Nikon Corporation, Tokyo, Japan). The setup for fluorescence imaging of PI fluorescence was as follows: Xenon lamp power (LPS-100, Photon Technology International, Inc. (PTI), Birmingham, NJ, USA); DeltaRAM X High-Speed Multi-Wavelength Illuminator LPS-100 (Photon Technology International, Inc. (PTI), Birmingham, NJ, USA); Nikon Eclipse TS 100 microscope and CCD camera (Nikon Corporation, Tokyo, Japan); ET excitation, 535 ± 15 nm; ET emission, 617 ± 37.5 nm, objective × 20. The emitted signal was captured and presented as an image of 1392 × 1040 pixels on a computer monitor using Macro-ImageJ software (National Institutes of Health, Bethesda, MD, USA). Three random areas were selected from each band for the cell viability assay. The mean fluorescence was analyzed using Adobe Photoshop CS6 (64 Bit) software (Adobe Systems Inc., San Jose, CA, USA) to determine the cell viability.

Capsule size in vitro was measured as described [25 (link)] and repeated twice. Briefly, 1 × 10⁵ cells/mL of H99W and P15 were added to Dulbecco’s modified Eagle medium (DMEM, Invitrogen, catalog # 10,566–016) and incubated at 37°C + 10% CO₂ for 18 h. Cells were collected, suspended in 10 μL PBS and added to a microscope slide with India Ink (Fisher Scientific, catalog # 14–910-56). Cells were imaged on an Eclipse TS100 Nikon microscope (Tokyo, Japan) with a 100× objective. For each strain, pictures of 30 cells were taken. The diameter of the cell body and capsule were measured using Nikon Elements software. Capsule diameter was calculated by subtracting the cell body diameter from the diameter of the entire cell + capsule and dividing by 2.