Tms microscope

Migration of breast cancer cells was examined with the help of wound-healing assays, which is standard procedure in monitoring cell migration [5 (link)]. Approximately 0.8 ×10⁶ cells were plated in each well of a 6-well plate and incubated overnight in a medium (RPMI-1640 or DMEM) containing fetal bovine serum (FBS, 10%). Cells were allowed to incubate overnight in 5% FBS-supplemented medium. It was observed that 5% FBS (instead of 0 or 10%) was optimal to monitor the effect of AA-induced migration of MDA-MB-231 cells. The monolayers were wounded by scratching with a sterile 10-μL pipette tip [5 (link)] and treated individually or in combination with AA (100 μM), NDGA (5μM), zileuton (100 μM), LTB₄ (100 nM) PGD2 (10 ng/ml), and methyl-β-cyclodextrin (MBCD, 1 mM) for 24 h. The cell images that had migrated between wounded regions were captured using a Nikon-TMS microscope equipped with a Nikon F-601 camera and then counted. The cells treated with NDGA or AA+NDGA were also supplemented with either PGD₂ or LTB₄.

Prepared hydrogels were transferred to nonadhesive 24-well cell culture plates (Corning Inc., Corning, NY) and rinsed overnight in 1 mL DMEM at 4 °C. Cell proliferation experiments used MC3T3-E1 preosteoblast cells (passage 24, ATCC CRL 2593, Manassas, VA) seeded at 5000 cells per cm². Cells were seeded onto the surface of the hydrogels, cultured in 0.5 mL of Minimum Essential Medium Alpha (αMEM, Thermo Fisher Scientific) with 10% fetal bovine serum (FBS, Fisher Scientific) changed every other day, and observed at 12 h, 4 days, and 7 days. Cells were photographed on a TMS Microscope (Nikon, Tokyo, Japan) using a Nikon CoolPix camera. At these time points, cells on the hydrogels were incubated for 1 h with phanazine methosulfate (PMS) and 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, (MTS) (Promega, Madison, WI), which can be reduced in the presence of metabolically active cells.^{53 (link)} The assay was performed per manufacturer’s guidelines, and the absorption of resulting formazan product was measured at 490 nm using a VersaMax plate reader (Molecular Devices, Sunnyvale, CA), with N = 4 samples per condition at each time point. Absorbance readings were normalized to the calcium-cross-linked hydrogels at the 12 h time point, allowing a relative measure of cell number changes over time.