Eclipse ts2r fl microscope

We inoculated rBMSCs (1.5 × 10⁵) into 6-well plates for a 24-h period or grew then till reaching 60% confluency. Cell co-culture with 200ng/ml mineralized ZIF-8 was completed within the osteogenic induction solution-free culture medium for a 14-day period, followed by 30-min fixation within 4% paraformaldehyde (PFA). After removing fixation solution, cells were stained with Alizarin Red reagent for indicating ECM mineralization. Excessive Alizarin reagent was later discarded with DI water. Images were taken with the Nikon Eclipse Ts2R-FL microscope.

To examine the effect of ZOL on cell morphology, ZOL-treated cells were stained with Giemsa. Briefly, cells seeded in six-well plates were allowed to adhere overnight onto cover slips followed by ZOL treatment. The cells were fixed with methanol for 10 min and stained with Giemsa (10% in PBS) for 15 min, followed by washing with tap water. Images were acquired using a Nikon Eclipse Ts2R-FL microscope.

To examine the effect of mesima + IR on cell morphology, treated cells were fixed with methanol for 10 min and then stained with Giemsa (10% in PBS) for 15 min, followed by washing with tap water. Images were acquired using a Nikon Eclipse Ts2R-FL microscope (Tokyo, Japan).

After 4% paraformaldehyde fixation, the liver tissue underwent gradient dehydration and was embedded with paraffin. The sample blocks were cut into 6 μm slices and stained with hematoxylin and eosin (H&E). The stained sections were observed and captured under the Eclipse Ts2R-FL microscope (Nikon, Tokyo, Japan).

3T3 proliferation assays of MEFs^{15 (link)} have been previously described. In total, 3000 mouse ccRCC 2020 cells per well were seeded in 96 well plates in six replicates and incubated for 6 days. Cells were cultivated in K-1 medium + 10% FCS, medium was changed after 3 days of incubation. Cell proliferation was measured using a sulforhodamine B (SRB) colorimetric assay^{15 (link)}. After the indicated time points the cells were fixed with 10% (w/v) trichloroacetic acid and stained with 0.057% (w/v) SRB solution. Overall, 10 mM Tris base solution (pH 10.5) was used to solubilise SRB, followed by OD measurement at 510 nm in a microplate reader (Tecan Spark 10 M plate reader). For sphere-forming assays, cell suspensions were filtered through a 40-µm cell strainer and 1000 cells were seeded in six-well low attachment plates (Corning). The cells were cultivated in K-1 medium + 10% FCS. Every 3 days fresh medium was added to the wells. After 14 days, microscopy pictures of the formed spheres were captured with DKM 23U274 camera connected to Eclipse Ts2R-FL microscope (Nikon) at ×20 magnification. Images were acquired with IC Capture 2.4 software and analysed using ImageJ software.

Intracellular ROS were measured using a fluorescent probe, 2′,7′-dichloro-dihydrofluorescein diacetate (DCFDA) [43 (link)]. Peritoneal macrophages (1 × 10⁶) were incubated in serum-free media for 1 h at 37 °C, then stimulated with LPS (1 μg/mL) for 30 min. Then, DCFDA (5 μM) was added, and the cells were incubated for 15 min before fluorescent cells were measured using a Nikon Eclipse Ts2R-FL microscope with a 20X objective lens.

To examine the effect of irradiation on cell morphology, the irradiated cells were stained with Giemsa. The cells were seeded in six-well plates and allowed to adhere overnight onto cover slips followed by irradiation treatment. The cells were then fixed with methanol for 10 min and stained with Giemsa (10% in PBS) for 15 min, followed by washing with tap water. Images were acquired using a Nikon Eclipse Ts2R-FL microscope (Tokyo, Japan).