Cck 8 kit

MC3T3-E1 cells (5×10³) were cultured in 96-well plates. PEN/MT01 nanocomposites solution with different solute mass ratios (1.0, 2.0, 4.0, 6.0, 8.0, 10.0, w/w) were incubated at 37 °C for 4 h. The control group was treated with PBS. Solution was discarded, DMEM (containing 10% FBS) was added, and the cells were incubated for 24 h. The above procedure was repeated the next day. Cytotoxicity of PEN/MT01 nanocomposites was analyzed using a CCK-8 kit (Invitrogen, United States), and absorbance was recorded at 450 nm using a microplate reader (Bio Tek, United States).

The viability of retinal cells was assessed using a cell counting kit-8 (CCK-8) kit (Invitrogen, Carlsbad, CA, USA). A quantity of 1 × 10⁵ primary cultured retinal neurocytes were seeded in 96-well plates. After 24-h adhesion, the retinal cells were treated with white light or blue light illumination for 2 h. The cells were then transferred to a dark environment to proliferate. Twenty-four hours later, the cells were incubated with a CCK-8 agent for 2 h. The absorbance was measured at 450 nm using a fluorescence plate reader (Power Wave XS; BIO-TEK, Winooski, VT, USA). Cell viability was determined based on the optical density ratio of a treated culture relative to an untreated control.

Butaprost and AH6809 were purchased from Caymen (USA), Recombinant human TGF-β1 was purchased from PeproTech (UK), The CCK-8 kit and Trizol RNA extracting kit were purchased from Invitrogen (USA). Mouse cAMP and PGE2 ELISA kit were purchased from Weston Biology Company (Shanghai, China). The reverse transcription (RT) kit was purchased from Fermentas (USA). The real-time quantitative PCR (RT-qPCR) kit was from Roche (USA). The Annexin V-FITC cell apoptosis assay kit was purchased from Beyotime (Shanghai, China). Nephrin, podocin, and CD2AP primers were purchased from Invitrogen (USA). Rabbit anti-mouse polyclonal antibodies for Nephrin, podocin and caspase3 were purchased from Abcam (UK). Rabbit anti-rat monoclonal antibodies of GAPDH, CD2AP, PI3K-p85 and Akt were purchased from Cell Signaling (USA), as well as phosphorylated PI3K-p85 and Akt monoclonal antibodies. The horseradish peroxidase (HRP) labeled goat anti-mouse and goat anti-rabbit IgG secondary antibody were purchased from SantaCruz (USA). Alexa Fluor 488 labeled goat anti-rabbit IgG (H+L) was purchased from Fcmacs (Nanjing, Jiangsu province, China).

Briefly, 5 × 10³ cells/well were initially seeded into a 96-well plate. After transfection and incubation, a cell counting kit (CCK)-8 kit (Invitrogen) was then used to detect cell viability. Finally, the absorbance values were measured at 450 nm in the indicated timepoints by a microplate reader (Bio-Rad, Hercules, CA, USA).