Matrigel matrix

A wound-healing assay was performed to observe the migratory capacity of PC-3 and SIRT5-KO cells. Cells (0.4 × 10⁶ per well) were seeded in a 6-well plate (Corning, Corning, NY) and cultured to ∼ 90% confluence. A sterilized 1000 μl tip was used to scratch the cell monolayer artificially, and debris was then removed by washing with phosphate-buffered saline (PBS). Next, wounded cells were observed under a microscope at 12 h intervals and imaged with a Leica Las EZ microscope (Olympus, Tokyo, Japan).
To evaluate the invasiveness of PC-3 and PC-3 SIRT5-KO cells, a transwell invasion assay was performed with 200 μg/ml matrigel matrix (Corning) and 8 μm pore-permeable supports (Corning). Cells were resuspended in serum-free medium and seeded (5 × 10⁴) in the upper chamber, and the medium containing 10% FBS was added into the lower chamber. After 24 h, a cotton swab was used to remove the non-invading cells in the upper chamber. Cells that migrated through the matrigel matrix were fixed with 4% paraformaldehyde for 10 min and stained with 0.5% crystal violet (Catalog No. 61135, Sigma-Aldrich, St. Louis, MO) for microscopy. Finally, cells attached to the membrane were added. Optical density was measured at 570 nm.

Transwell tissue culture inserts (diameter, 6.5 mm; pore size, 5.0 µm) and 24-well plates were purchased from Corning Costar Corp (Corning, NY). RPMI 1640 medium and endothelial growth medium were obtained from Gibco (Grand Island, NY). Heat-inactivated fetal bovine serum was obtained from HyClone (Logan, Utah). Mouse anti-human claudin-1 was obtained from Cell Signaling Technology (America, Boston). The Limulus amebocyte lysate test kit, polymyxin B-agarose, anti-human ICAM-1 monoclonal antibody (MAb), anti-human E-selectin (MAb), Calcein AM, phalloidin, tetramethylrhodamine bisothiocyanate, matrigel matrix, and MCP-1 were purchased from Sigma-Aldrich (America, St Louis, MO). Recombinant T. pallidum protein Tp0965(rTp0965)was purified on nickel-nitrilotriacetic acid (Ni-NTA) chromatographic column from E. coli lysates frozen in our laboratory. The human MCP-1 enzyme-linked immunosorbent assay (ELISA) kit was purchased from R&D Systems, Inc. (Minneapolis, MN). Anti-mouse immunoglobulin G and horseradish peroxidase (HRP)-tagged antibody were purchased from Amersham (Piscataway, NJ). Anti-rabbit HRP-tagged antibody was purchased from Zymed (San Francisco, CA).

Migration and invasion abilities were assessed by wound healing and by Matrigel matrix assays, respectively, as previously described [38 (link)].
In brief, for the wound healing experiment, cells were plated in 6-well culture plates and grown to confluence. A scratch was generated on a confluent cell monolayer and the closing of the wound was monitored and measured using Cell^a software (Olympus Biosystem Gmb, London, UK).
For the invasion assays, transfected cells were seeded into the upper Transwell chambers precoated with Matrigel matrix (BD Biosciences, San Jose, CA). After incubation for 24 h, cells that had invaded through the chamber membrane were fixed with 11% glutaraldehyde solution (Sigma-Aldrich) for 90 min, stained with crystal violet solution and, after elution, quantified by spectrophotometer measuring optical density (O.D.) 550 nm.
Before the wound healing and Matrigel matrix assays, cells were treated with mitomycin (Sigma-Aldrich) to inhibit cell proliferation during these experiments.

The invasion assays were conducted in 24‐well plates with permeable supports on 8‐μm Pore Polycarbonate Membrane (Corning Transwell 3422; Corning, Inc., Corning, NY, USA) for 24 and 48 hours.⁽²⁸⁾ Eight micrograms/microliter (8 μg/μL) of Matrigel matrix (Sigma‐Aldrich, St. Louis, MO, USA; E1270) was coated on the permeable supports (upper chambers) and incubated at 37°C for 1 hour. Cell suspensions prepared in serum‐free media were seeded onto the upper chamber (1 × 10⁴ cells/chamber) and culture media containing 10% FBS was loaded in the lower chamber. After the incubation, invaded cells in the lower chamber were washed with PBS and stained with 0.05% crystal violet for 1 hour. The invaded cells were captured under the microscope and counted in five random fields in triplicate.

Migration assay was performed with uncoated Transwell chambers (Corning Life Sciences, Corning, NY).200000 cells in 100 μl serum-free DMEM were added to the upper chamber. The lower chamber was filled with 800 μl DMEM supplementary with 10% FBS. The transwell chambers were cultured for 48 hours. The cells were fixed with 4% PFA and stained with 0.5% crystal violet. Invasion assays were carried out as described above with chambers which were pre-coated with Matrigel matrix (Sigma-Aldrich, USA). Images were obtained by a microscope and analyzed by Image J [24 (link)].