Dead and whole cells were detected with propidium iodide (PI) and Hoechst 33342 (Dojindo, Kumamoto, Japan) 3 or 7 days after the injection [16] (link). Endplates were removed with a scalpel blade, and NP tissues were harvested using a biopsy punch. Harvested NP tissues were stained with 4 µM PI and 4 µM Hoechst 33342 for 1 h. PI emits red fluorescence in dead cells, whereas Hoechst 33342 emits blue fluorescence in all cells, regardless of live or dead status. The stained NP tissues were washed with phosphate-buffered saline (PBS) and examined using a confocal laser scanning microscopy system (Nikon A1 and Ti-E, Tokyo, Japan) equipped with a Plan Fluor 20× objective lens (N.A., 0.45; Nikon). The number of dead and all cells in each NP tissue was measured from >3 randomly chosen stacks, each representing a 100-µm projection at a 5-µm interval. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used for cell number quantification [12] (link), [13] (link). All the experiments were performed on 6 discs from each treatment group and time point.
Plan fluor 20 objective lens
Manufactured by Nikon
Sourced in Japan
The Plan Fluor 20× objective lens is a high-performance optical component designed for use in various laboratory equipment and instruments. It provides a 20× magnification with a wide field of view and flat image plane to facilitate accurate observations and measurements. The lens is optimized for fluorescence microscopy and other applications requiring superior optical quality and performance.
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4 protocols using plan fluor 20 objective lens
1
Nucleus Pulposus Cell Viability Assay
2
Cell Area and Circularity Analysis
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Cells were examined on an Eclipse Ti microscope fitted with a Plan Fluor 20× objective lens (NA 0.45; Nikon Instruments Inc.). After binary image processing, the cell area and circularity were measured using ImageJ. Five randomly selected fields were considered.
3
3D Culture of MCF10A Cells on PA Gels
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Matrigel overlay culture on PA gel was performed as previously described, with slight modifications (Debnath et al., 2003) (link). MCF10A cells were detached with trypsin at 37 °C for 5 min to ensure complete collection of the cells, and then resuspended in culture medium (DMEM/F12 supplemented with 20 ng/mL of EGF, 100 ng/mL of cholera toxin, 0.01 mg/mL of insulin, 500 ng/ mL of hydrocortisone, 5% horse serum, 100 U/mL penicillin, and 100 μg/mL streptomycin). The cells were washed with assay medium (DMEM/F12 supplemented with 100 ng/mL of cholera toxin, 0.01 mg/mL of insulin, 500 ng/mL of hydrocortisone, 2% horse serum, 100 U/mL penicillin and 100 μg/mL streptomycin), and then counted and diluted to 1 × 10 4 cells/mL in assay medium at RT. We prepared 4% Matrigel solution in assay medium at 4 °C. Then, the cells and Matrigel solution were combined in 1:1 ratio. PA gels were transferred to 12-well plates, washed with assay medium three times, and 1 mL of the cell-Matrigel mixture (5 × 10 3 cells/well) was applied to the PA gel. The cells were refed assay medium containing 2% Matrigel every 4 days. After 10 days, the cells were examined on an Eclipse Ti microscope fitted with a Plan Fluor 20× objective lens (NA 0.45; Nikon Instruments Inc.).
4
Immunofluorescence Analysis of FAK and YAP
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MCF10A cells (5 × 10 4 ) were cultured on collagen-coated PA gels for 24 h. The cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 1% (for p-FAK) and 0.1% (for YAP) Triton X-100 in PBS for 10 min, and then blocked with 3% BSA in PBS for 30 min at RT. The cells were incubated with 1:50 solution of primary antibodies, for p-FAK and YAP, overnight at 4 °C, and then incubated with secondary antibodies and Hoechst 33,342 (Thermo Fisher Scientific) at RT for 30 min. Finally, the cells were mounted with VECTASHIELD ® and examined on an Eclipse Ti microscope fitted with a Plan Fluor 20× objective lens (NA 0.45; Nikon Instruments Inc.).
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