Matrigel coated insert

Cell migration assays were conducted by using transwell chambers (Corning Life Sciences, NY, USA) as previously described.^{57 (link)} Briefly, transfected cells were starved for 24 h without serum, then 2 × 10⁴ cells were plated in the upper chamber. The lower chamber was added with DMEM plus 20% FBS. Forty-eight hours later, non-migrated cells from the upper side of the chamber were removed, and cells on the lower side of the chambers were fixed by methanol and stained with 0.1% crystal violet.
For cell invasion assays, 2 × 10⁴ MDA-MB-231 cells were seeded in the upper compartment of Matrigel-coated inserts (Corning Life Sciences). After 72 h, invaded cells were collected. The rest of the protocol was similar as cell migration assays. The mean number of the five fields represented migrated/invaded cells’ amount. All the fields were photographed with the use of a BX51 microscope (× 400 magnification). Each experiment was performed in triplicate independently.

A number of 1 × 10⁵ tumor cells or macrophages in 200 μL serum-free RPMI-1640 were seeded onto the transwell inserts (for migration assay, 8 μm, Corning, USA) or Matrigel-coated inserts (for invasion assay, 8 μm, Corning, USA) of the 24-well plates. The lower chamber was filled with 600 μL conditioned media as the chemoattractant. After 12 h of incubation, the cells on the upper chamber were carefully removed. The cells that passed the membrane were fixed with 4% formaldehyde and stained with 1% crystal violet. The evaluation of migration and invasion were performed by counting the number of stained cells in five random fields (400x magnification).

After serum starvation, 5 × 10⁴ of 786‐O and 1 × 10⁵ of Caki‐2 cell lines were seeded into a modified Boyden chamber (MilliporeSigma, Burlington, MA, USA) or Matrigel‐coated inserts (Corning Inc., NY, USA) with 8 μM pore size to evaluate cell migration and invasion, respectively. The lower compartment was filled with cell growth media supplied with 10% FBS. The cells were allowed to migrate under treatment with DMSO or sunitinib for 12 h. The cells on the outer side of the inserts were fixed in 75% ethanol and stained with crystal violet (Carl Roth GmbH, Germany). The cells were counted under a microscope at 10× magnification.

Transfected cells were diluted to 50.000 cells/mL in RPMI media containing serum. Then, 500 μL (corresponding to 25.000 cells) were transferred to Matrigel-coated inserts (Corning^®, cat. no. 354230; Bedford, MA). This was followed by a 24-hour incubation period at 37°C, 7.5% CO₂. After carefully removing RPMI media and replaces by 500 μL of RPMI without serum, the invasion was triggered by adding 750 μL of RPMI medium with FCS as a chemoattractant factor into the lower compartment of the chamber. After 24 hours, all media was removed, and the cells on top of the Matrigel were removed with “cotton-wool” sticks and washed in PBS for 1 minute. After removing the PBS, the cells were stained in 1% toluidine blue in BORAX (Sigma, cat. No. T3260) for 6 minutes and washed with H₂O. Two non-overlapping pictures were taken under a Zeiss Axiophot (Zeiss, Jena, Germany) bright-field microscope (magnification 10X) and invaded cells were counted.

T24 and UMUC3 cells in serum free media were seeded in 8-μm pore size cell culture inserts (#353097, BD Falcon, Franklin Lakes, NJ, USA) and in Matrigel coated inserts (#354480, Corning, NY, USA) for migration and invasion, respectively. Media containing 10% FBS was added to the lower chamber. The cells were removed from the insert after 24 h incubation, and cells on the chamber were stained with crystal violet solution. The stained cells were dissolved in DMSO, and measured with a microplate spectrophotometer (wavelength 590 nm, BioTek Instruments, Winooski, VT, USA).

We employed 8-μm pore transwell inserts for migration (Ref.354578, Corning), and Matrigel coated inserts for invasion experiments (Ref. 354480, Corning). The insert was fixed and stained 24 h after seeding using the Diff-Quick method (QCA, Spain). Two different fields were photographed at 20 × magnification for the subsequent cell count using the ImageJ program (National Institutes of Health, USA).
For the study of migration and invasion capacity in 3D, 4 day spheroids were placed on a previously solidified Matrigel layer for migration, or embedded in Matrigel for invasion assays.. The images were taken and quantified after 3 to 7 days using ImageJ (NIH).Two experiments were performed and the measures obtained in two fields are represented as a ratio with the non-treated control cells.

Migration and invasion assays were performed using transwell inserts (Corning) or Matrigel-coated inserts (Corning), respectively. Cells were subjected to MLK4 knock-down or overexpression, then serum-starved, trypsinized and seeded into upper chamber. Chemoattractant in the lower chamber was medium with 20% serum. After no more than 24 h, cells were fixed, stained with crystal violet and five pictures per condition were taken. Quantitative analysis of was performed using ImageJ.