24 well matrigel invasion chamber

The invasive potential of wild-type and XB130-silenced germinal center cells was assessed using an invasion assay through a 24-well Matrigel Invasion Chamber (BD Biosciences, San José, CA, USA). A total of 5 × 10⁴ cells per ml in 0.5 ml of serum-free medium were added to the upper chamber, and 0.75 ml of medium supplemented with 5% fetal bovine serum was added to each lower chamber. After 18 h of incubation, the invasive cells in the lower chamber were fixed with 3.7% paraformaldehyde, and the cells remaining in the upper chamber were removed by scratching. The cells were then washed twice with PBS, stained with hematoxylin at room temperature for 1 h, and photographed under a microscope.

Quantitative cell migration and invasion assays were performed using a 24-well Matrigel Invasion Chamber (BD Biosciences, San Jose, CA, USA) containing a cell culture insert consisting of a PET membrane with a pore size of 8 μm and coated with a thin layer of Matrigel basal membrane matrix. We measured the number of cells after trypsinisation and diluted them to have an equal amount of cells in each assay. After incubation at 37 °C for 24 h, the filters were collected and the cells that had adhered to the lower surface were fixed, stained with a Diff-Quik Stain Kit (Sysmex, Kobe, Japan), and counted. We quantitated the invasion rate in at least four independent experiments. The mean and s.d. (error bar) of at least four independent experiments are shown. The invasion assays were performed in Hec108 cells into which the siRNA against dlg1 was transfected. They were also performed in the stable DLG1 knockdown KLE, Hec1B, and Hec59 cells.

PANC-1 cell invasion assays were performed using 24-well Matrigel invasion chambers (BD Biosciences, CA, USA). The detailed protocol was performed as previously described^{23 (link)}.