Falcon matrigel invasion chambers

Cell invasion was assayed using the BD Falcon matrigel invasion chambers (BD Biosciences, CA, USA). The assay was performed according to manufacturer’s instructions. In brief, DMEM was added to the transwell inserts in 24 well plates for 2 hours at 37° to rehydrate the matrigel. Media was removed and 2.5 X 10⁴ cells in 500 μl of serum free DMEM were seeded in each transwell insert. Quadruplicate samples were used for each of the three experiments. The lower chamber of the plate was filled with 500 μl of NIH-3T3 conditioned media. After 24 hour incubation at 37°, the cells remaining on the interior of the transwell insert were gently removed, while the invasive cells on the bottom surface of the insert were fixed with 100% methanol and stained with 1% Toluidine blue. The membranes were removed from the insert and cells from five fields per membrane were counted using a Nikon TMS microscope. Images were acquired using an Olympus IX73. The number of invasive cells was determined for each cell line and then normalized to the NB_1 cell line.

Cell invasion was assayed using the BD Falcon matrigel invasion chambers (BD Biosciences, San Jose, CA, USA) and the accompanied manufacturer’s instructions were followed. HuNB(-MGAT2) cells transiently transfected for 72 h were used for the cell invasion assay. Briefly, the matrigel of 24-well plates were rehydrated by addition of DMEM for 2 h at 37°. Cells (5.0 × 10⁴) in serum-free DMEM (500 µL) were plated in each transwell insert. NIH-3T3 conditioned media (500 µl) was added to the lower chamber of the plate and cultured at 37 °C for 24 h. Cells in the interior of the transwell insert were gently removed, and cells on the bottom surface of the insert were fixed with 100% methanol and stained with 1% Toluidine blue. Three experiments were conducted in quadruplicate. Cells from five fields per membrane insert were counted using a Nikon TMS microscope (Nikon, Chiyoda, Japan). Each of the cell lines were normalized to the HuNB cell line. Images were captured using an Olympus IX71 microscope (Olympus, Center Valley, PA, USA).

BD Falcon cell chambers (BD biosciences, CA, USA) were employed to evaluate cell migration. 2.5 X 10⁴ cells in 500 μL serum free DMEM were added to each transwell insert without matrigel. 500 μL NIH-3T3 conditioned media was added to the lower chambers and cells were cultured for 20 h at 37°. BD Falcon Matrigel invasion chambers (BD biosciences, CA, USA) were used to measure cell invasion following the manufacturer’s instruction and with modifications as described [18 (link)]. For both assays, samples were done in quadruplet for 2 experiments. Membranes without and with Matrigel were removed from inserts, and then washed, fixed with 100% methanol, and stained with 1% Toluidine blue. Each membrane containing cells were counted using a Nikon TMS microscope. An Olympus IX73 was used to image the cells, to determine the number of either migratory or invasive cells per well.