Isolated chondrocytes were seeded at 2.5 × 105 cells/cm2 in a treated 12 well tissue-culture plate and cultured in a 37 °C cell culture incubator under an atmosphere of 5% CO2 at 100% humidity. Culture media were changed every 2 days. Chondrocytes were examined daily using an Olympus CKX53 light microscope equipped with 4×, 10×, and 20× objectives. Light microscopic images of chondrocytes in monolayers were recorded with a digital camera (Olympus LC30) installed on the microscope. We selected without preference 3 areas of chondrocyte monolayers in each well of a plate for imaging.
Ckx53 light microscope
Manufactured by Olympus
Sourced in Japan
The CKX53 is a light microscope designed for routine observation and documentation of samples in a laboratory setting. It provides clear, high-contrast images with its advanced optical system. The CKX53 is a versatile and reliable tool for basic microscopy tasks.
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Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Fluoroshield mounting medium with dapi, by Abcam (1 mentions)
Deadend fluorometric tunel system, by Promega (1 mentions)
Glass slide, by Matsunami (1 mentions)
Harris hematoxylin and eosin, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Sartorius (1 mentions)
H dmem, by Thermo Fisher Scientific (1 mentions)
Transwell insert, by Corning (1 mentions)
9 protocols using ckx53 light microscope
1
Chondrocyte Monolayer Culturing
2
TUNEL Assay for Apoptosis Detection
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Cells (5×105) were seeded in 6 cm dishes and incubated for 18 h at 37°C in a humidified incubator containing 5% CO2 in air. Following incubation, cells were treated with DMSO (0.1%) as a control vehicle and 0.5 and 1 µM MTZ for 2 h. Cells were fixed with 4% paraformaldehyde solution for 15 min at room temperature and permeabilized with Triton X-100 (0.2%). Apoptosis was determined by enzymatic labeling of DNA strand breaks with a TUNEL assay kit (the DeadEnd Fluorometric TUNEL System; Promega Corporation, Madison, WI, USA), according to the manufacturer's procedures. Fluoroshield Mounting Medium with DAPI (Abcam, Cambridge, UK) was used, and 3 fields of view were imaged using the Olympus CKX53 light microscope (Olympus Corporation, Tokyo, Japan; magnification, ×100).
3
Scratch Assay of Macrophage Migration
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The RAW264.7 macrophages were plated at a density of 0.5 × 106 cells/well in 6-well plates in the complete growth medium. After 24 h, boron carbide preparations were added for 24 h at a concentration of 100 µg/ml. Next, when 100% cell confluency was achieved, a gentle scratch was made with a 200 µl tip in the center of the cell monolayer in the wells. The floating cells were removed by washing with phosphate-buffered saline and then the complete medium was added. The macrophages were incubated at 37 °C and imaged over time for 24 h using an Olympus CKX53 light microscope. The experiment was performed in three repetitions, but the images show a selected part of the well for each sample at 0, 4 and 24 h.
4
Quantification of Bent Mouse Sperm
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Mature spermatozoa collected from the cauda epididymis (from mice at age of 8–17 weeks) were mounted on glass slides (Matsunami) and observed with an Olympus CKX53 light microscope for counting of bent spermatozoa.
5
Histological Analysis of Tibial Bone
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For histological analysis, 2 cm fragments of the proximal part of the tibia were used. The bone material was decalcified in EDTA solution, embedded in a Cryomatrix gel and then frozen in liquid nitrogen. The frozen bone material was cut in cryotome into slices of 10–12 μm. The sections were stained with Harris hematoxylin and eosin (Sigma-Aldrich, St. Louis, MO, USA). Microscopic images were collected using a Olympus CKX53 light microscope (Olympus, Munster, Germany), magnification ×400, and a camera Olympus EP50 (Olympus, Munster, Germany).
6
Isolation and Culture of Rat Bone Marrow Stromal Cells
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A total of 6 of the aforementioned rats were anesthetized with 1% pentobarbital sodium injected intraperitoneally (40 mg/kg) and the rats were then euthanized by dislocation of the spine. Bone marrow was subsequently harvested via gentle puncture to the tibia and femur. The total bone marrow samples were combined (~8.0 ml) and resuspended in H-DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) and supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.), 100 UI/ml penicillin (Invitrogen; Thermo Fisher Scientific, Inc.) and 100 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were subsequently plated on cell culture plates for use in the studies. The cells were kept in humidified incubators at 37°C and 5% CO2. Cultures were rinsed with PBS (Biological Industries) after 2 days to remove nonadherent cells and fresh growth media was added. After 10–12 days, the cells reached ~80% confluence and were used in subsequent experimental studies. Growth and proliferation of BMSCs were observed using a CKX53 light microscope (Olympus Corporation).
7
Hydrogel Scaffold Cell Culture
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The precursor hydrogel solutions (70 μL) were added to the 96-well plate and allowed to gel for 1 h. Hydrogels were then washed with PBS (pH 7.2) and swollen in fresh media. NIH 3T3 cells (100 μL) in fresh medium (7500 cells/well) were added to the prepared hydrogels and cultured for 48 h at 37 °C with 5% CO2. The cellular adhesion was qualitatively evaluated using a CKX53 light microscope from Olympus® (Shinjuku, Tokyo, Japan).
8
Macrophage Migration Assay with B4C
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The RAW264.7 macrophages were incubated with boron carbide preparations at a concentration of 100 µg/ml for 24 h. After this time, macrophages alone (0.4 × 105 cells) and loaded with B4C nanoparticles were suspended in 150 µl of medium without FBS and placed in Transwell inserts with a pore size of 8 μm (Corning Costar). 500 µl RPMI-1640 with 5% FBS as a control medium and 72-hour supernatant from MC38 mouse colorectal cancer cells (1.5 × 105 cells/well in 1 ml of RPMI-1640 with 5% FBS in a 24-well plate) were added to the lower chambers. After 16 h of incubation at 37 °C and 5% CO2, the upper part of the inserts was wiped using a cotton swab to remove cells that did not migrate. Then, the cells on the inserts were stained with the RAL 555 kit (RAL Diagnostics) according to the manufacturer’s instructions. Migrated cells were observed by an Olympus CKX53 light microscope and counted using ImageJ software from 7 images of the central part of each insert.
9
Lipid Droplet Quantification in 3T3-L1 Cells
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The cell culture medium was removed from the differentiated 3T3-L1 cells and washed twice with PBS. Follow the instructions for Oil Red O (ORO) staining solution (Cell-Specific) Kit (Solarbio, China). Add ORO fixative fixation solution to the cells for 20–30 min and then discard the fixation solution and wash it twice with distilled water; 60% isopropanol was added for 5 min and then discarded. Add freshly prepared ORO stain, stain for 10–20 min, discard the staining solution, and wash with distilled water 2–5 times until there is no excess staining solution. Add Mayer hematoxylin staining solution, stain for 1–2 min, then discard the staining solution, and wash with distilled water 2–5 times. Add ORO buffer for 1 min and discard. Add distilled water to cover the cells. Images were acquired with CKX53 light microscope (Olympus, Japan) and analyzed using Image J (Image J V1.8.0.112, United States). Three images were randomly selected to evaluate the percentage of lipid area.
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