Dm il led microscopy

To evaluate cytotoxicity, RDV hydrogels were extracted using 5 mL of DMEM/F12 medium to obtain a standard leach liquor [25 (link)]. The HCECs were seeded onto a 96-well plate with 1 × 10⁴ cells per well and then co-cultured with 100 μL leaching liquor. The morphological appearance of HCECs was observed using a DM IL LED microscopy (Leica, Germany) to obtain a sense of immediacy. Cell proliferation capacity was measured following the instructions of the Cell Counting Kit-8 (https://www.dojindo.eu.com/TechnicalManual/Manual_CK04.pdf, accessed on 12 December 2021, CCK-8, Dojindo, Japan).
The rate of apoptosis in HCECs was measured in all groups using the TUNEL assay. This assay identified DNA breaks by labeling the 3′-OH terminals of DNA with modified nucleotides to TdT or terminal nucleotidyltransferase. Briefly, HCECs were cultured on slides and then treated with the leach liquor. After an incubation period of 48 h, the existence of apoptotic cells in the samples was investigated using a DeadEnd™ Fluorometric TUNEL System (Promega, Madison, WI, USA) in accordance with the protocol of the manufacturer.

HBMEC endothelial cells kindly donated by Dr. Lecanda (Laboratory of Cell Adhesion and Metastasis, CIMA) were grown on 0.2% gelatine-coated plates in EBM-2 medium supplemented with 5%FBS. MiR-942/Caki-2 cells and HBMEC were co-cultured in 0.4 µm pore size 6-well Boyden chambers. HBMEC (1.5×10⁵ cell/well) were plated at the bottom of the plates in EBM-2 medium. After 2 h, 8×10⁴ miR-942-overexpressing Caki-2 cells or control cells (mock-transfected) were seeded in McCoy's 5a Medium in the inserts. Co-culture was incubated for 72 hours and HBMEC cells were then harvested to perform MTT (with sunitinib doses up to 35 µM), western blot for p-VEGFR2, total VEGFR2, p-p44/p42 MAPK (Erk1/2) and total Erk1/2 (S3 Text), and for migration assays.
Migration was performed in 24 well Boyden chambers. HBMEC (5×10⁴ cells) were seeded in the chamber in a final volume of 150 µl in serum free medium. The lower compartment was filled with 450 µl of 5% serum containing EBM-2 medium to chemoattract the cells. 48 h later, cells from the insert were removed with a swab and cells that migrated to the lower compartment were fixed with 4% formalin, stained with crystal violet and counted with a Leica DMIL LED microscopy.