Calcein am

The PET membranes (8 µm pore size) of FluoroBlok™ cell culture inserts (351152, Corning, NY, USA) were coated with 90 µL of diluted Matrigel (0.3 mg/ml) (356234, Corning), and incubated at 37 °C for 2–3 hours until solidified. Next, 1 × 10⁴ of 4T1, BT20 or MDA-MB231 cells suspended in serum-free DMEM media were seeded on the solidified Matrigel layer. Then, 700 µL of chemo-attractant medium (DMEM, 1% P/S and 10% FBS) was added to the lower chambers (353504, multiwell 24 well companion plate, Corning), and the plate was incubated in a 37 °C incubator. After 24 hours of incubation, the insert bottoms were dipped in 1X PBS and stained in diluted Calcein AM (354217, Corning) in PBS for 10 min. Fluorescence images of invaded cells were captured with an EVOS inverted microscope, and analysis was done with ImageJ software.

The procedure was followed and modified as previously reported [35 (link)]. Briefly, human umbilical vein endothelial cells (HUVECs, ATCC, Cat# CRL-1730) were incubated with endothelial cell medium (ECM) (Sciencell Research Laboratory, CA, 1001) at passage 6–8 when the cells reached to 80–100% confluency. Then, the cells were trypsinized and resuspended in the following conditions: tenfold diluted CM from an infertile patient and a volunteer, M199/0.5% BSA as a negative control, and M199/0.5% BSA with recombinant VEGF (5 ng/ml) as a positive control. The cells were then seeded in 96-well plates (Corning, USA) pre-coated with 30 μl growth factor reduce (GFR) Matrigel (Corning, USA, 354230) at a density of 2 × 10⁴ cells/well. After 18–20 h, gels were stained with Calcein AM (Corning, USA) to quantify covered area using fluorescence microscopy (BZ-X700, KEYENCE, Japan). Each experiment was performed in triplicate.