Ckx41 f32fl fluorescence microscope

NRK-52E cells transfected with pmRFP-LC3 were exposed to HBSS and bafilomycin A1 with rhein alone or co-treatment of rhein and metformin, or HBSS and bafilomycin A1 with PD098059 or SB203580 for 2 hours. The confocal images were captured at 200× magnification using the Olympus CKX41-F32FL fluorescence microscope (Olympus, Tokyo, Japan).

According to the report of Robinson et al. [14] (link), we assessed intracellular ROS levels by DCFH-DA to investigate whether the MEHP exposure induces oxidative stress in HUVEC cells. The fluorescence intensity is corresponding to the ROS generation in the treated cells. HUVEC cells (1×10⁴ per well) were plated into 48-well plates for 24 hours, and incubated with MEHP of various concentrations (0, 6.25, 12.5, 25, 50 and 100 µM) for another 24 hours. Then 10 mM DCFH-DA was supplemented and the cells were incubated at 37°C for 20 min. When washed three times with PBS, the plates were immediately inserted into an Olympus CKX41-F32FL fluorescence microscope to detect and photograph the fluorescence.

We used the JC-1 Detection Kit in order to investigate the mitochondrial membrane potential (MMP) changes induced by MEHP in HUVEC cells according to the manufacturer's protocol. JC-1 is a kind of fluorescent carbocyanine dye. Depend on the status of MMP, JC-1 may assemble on the mitochondrial membrane in monomer forms or dimer forms. When the mitochondrial membrane is highly polarized, JC-1 becomes dimmers and transmits red fluorescence. If the mitochondrial membrane is depolarizeded, JC-1 converts into monomers and transmits green fluorescence. Therefore, the green fluorescence represents the MMP loss. HUVEC cells (1×104 per well) were cultured in 48-well plates for 24 hours, and incubated with MEHP of various concentrations (0, 6.25, 12.5, 25, 50 and 100 µM) for another 24 hours. Controls were treated with DMSO only. After incubated with JC-1 for 30 min at 37°C in the dark, the treated cells were immediately photographed by the Olympus CKX41-F32FL fluorescence microscope.