Mtt assay reagent

Matrigel (Corning, Glendale, AZ) was reconstituted with DEME-LG at a concentration of 40 μg/mL. A 96-well plate was coated with reconstituted Matrigel at 2 μg/50 μL/well and incubated for 18 h at 25°C. After washing the plate with PBS, canine melanoma cells were seeded at 3,000 cells/200 μL in each well of a 96-well plate and incubated for 60 min at 37°C, 5% CO₂. Unattached cells were washed and discarded by PBS. The attached cells were stained by MTT assay [43 (link)]. MTT assay reagent (Dojindo, Tokyo, Japan) was dissolved in PBS at a concentration of 5 mg/mL, and 20 μL of the reagent was added to each well for 1 h with 5% CO₂ at 37°C. Following incubation, PBS (100 μL) was added to each well. After 1 min, the supernatant was discarded, and the MTT formazan crystals were dissolved in 200 μL of 0.04 M hydrochloric acid (HCl) in 2-propanol. The optical density (O.D.) was detected by a microplate reader (Fluoroskan Ascent FL, Thermo Fisher Scientific K.K., Kanagawa, Japan) at 570 nm wavelength.

UE7T-13 human immortalized MSCs were purchased from the JCRB Cell Bank (Osaka, Japan). AC-GlcNAc-coated wells were blocked with 0.002% BSA for 1 h at 37°C. After blocking, 2 × 10⁴ cells/well in serum-free DMEM were seeded in each well of a 96-well plate and incubated at 37°C with 5% CO₂ for 1 h. After incubation, nonadherent cells were removed by washing with PBS, and 100 μL of 10% methylthiazole tetrazolium (MTT) assay reagent (Dojindo Laboratories, Kumamoto, Japan)/DMEM containing 10% fetal bovine serum (FBS) was added to each well, followed by incubation for 2 h at 37°C with 5% CO₂. After 2 h of incubation, MTT formazan was solubilized with 100 μL of dimethyl sulfoxide. The absorbance at 570 nm of these solutions was measured using a microplate reader. The number of adherent cells was determined by the standard curve based on formazan production.