Pore polycarbonate membrane insert

5.0 μm Pore Polycarbonate Membrane Inserts(3421,Corning) were placed into the wells of a 24-well plate and 600 μl serum-free F-12K medium containing 0, 100, 1000ng/ml recombinant MFGE8 (10853-H08B, Sino Biological Inc.) was added to the outer compartment of each well. A 400μL suspension of 1X10⁵ PY8119 cells/ml in serum-free F-12K medium was gently added onto the cell culture inserts. Cells were then incubated at 37 °C for 18h. For bone cell and tumor cell chemotaxis experiments, 2.5X10⁵ bone marrow stromal cells from mouse vertebrae and long bones were seeded at the bottom of the wells in serum-free αMEM medium, and a 400μL suspension of 1X10⁵ PY8119 cells/ml in serum-free αMEM medium was gently added onto the cell culture inserts. Cells were then incubated at 37 °C for 18h. Cells that did not undergo migration on the top of membrane were carefully removed with cotton swabs. Migrated cells on the inserts were fixed in 4% paraformaldehyde for 10min and washed with PBS 3 times followed by staining with 0.5% crystal violet solution in 25% methanol at room temperature for 10 minutes. Cells were then washed 4 times in water and dried at room temperature.

Cores 4 mm in diameter were punched from sacrificed murine tumors using a biopsy punch (#7424; RoyalTek, TWN) and cut into 1 mm organotypic tumor slices. Tumor slices were cultured individually on 12 mm Transwell with 0.4 μm pore polycarbonate membrane inserts (#3401; Corning) using 12-well plates. Slices were cultured in 1× Advanced Dulbecco’s Modified Eagle Medium (#12491023; Thermo Fisher) supplemented with 5% FBS, 1× GlutaMAX (#35050061; Thermo Fisher), 0.5× Penicillin-Streptomycin, 1× Insulin-Transferrin-Selenium supplement (#41400045; Thermo Fisher), and 15 mM HEPES (#15630130; Thermo Fisher) as previously described [38 (link)] and maintained at 37° in 10% CO₂. After 24 h in culture, slices were exposed to vehicle or 40 μM HMA for 24 h. Tumor slice viability was measured using RealTime Glo (#G9711; Promega) according to the manufacturer’s instructions. Images were taken before (day 0) drug treatment and 24 h after drug treatment with a ChemiDoc MP Imaging System (BioRad) and analyzed with Image Lab software version 1.2 to quantify the RealTime Glo signal.

To detect the recruitment of CD8+ T cell by tissue lysates and BPH-1 cells, the tissue lysates or 1 × 10⁵ of BPH-1 cells of different treatment were plated into the lower chamber of the transwells with 5 μM pore polycarbonate membrane inserts (3421 Corning, MA, USA). 1 × 10⁵ of CD8+ T cells isolated by flow cytometry from 6 healthy persons and Molt-3 cells were plated onto the upper chamber. After 6 hrs, the cells migrated into the lower chamber media were collected and counted by the Bio-Rad TC10 automatic cell counter. Each sample was assayed in triplicate and each case was repeated twice.

Cells transfected with IGFBP2-siRNA or control-siRNA for 24 h were seeded into 6.5 mm Transwell® plates (8.0 μm Pore Polycarbonate Membrane Inserts, Corning) or 24-well BioCoat™ Matrigel®Invasion upper Chamber (8.0 μm PET Membrane, BD Biosciences). The lower chamber was filled with culture media containing 10% FBS. 24 h post-seeding, non-migrating or non-invading cells from the upper surface of membrane were removed, stained with crystal violet, and quantitated using an inverted light microscope (Nikon). The lower surface of the membrane was dissolved in methanol; fluorescence was read at 540 nm using a SynergyTM HTX Multi mode reader (BioTek).

S. tropica cells were grown to late exponential phase in 10 ml of MB before washing them three times with sterile mineral media, as appropriate for each phototroph, and finally resuspending the washed cell pellet in 10 ml of mineral media. Axenic phototroph cells grown to late exponential phase and the washed S. tropica were co‐inoculated in fresh media to a concentration of 10% (v/v) and 20% (v/v), respectively. S. tropica cells were also washed and resuspended in a conditioned Synechococcus supernatant (SUPSYN), MB or ASW when required for the metabolomic and proteomic analyses. To obtain the conditioned supernatant, Synechococcus cultures were incubated for 35 days as described above before centrifugation (4000 g for 10 min at room temperature) and further filtration through 0.22 μm pore size filters to remove cells and particulate organic matter. Washed S. tropica cells were used to inoculate SUPSYN, MB or ASW and cultures were incubated at 22°C with shaking (140 rpm) and a light intensity of 10 μmol photons m^‐2 s^‐1. For the physically separated Synechococcus‐S. tropica co‐cultures using the porous filters, cells were grown in 24 mm transwell with 0.4 μm pore polycarbonate membrane inserts (Corning, New York, USA). Synechococcus cells were inoculated in the well to a concentration of 20% (v/v) and S. tropica in the insert to a concentration of 55% (v/v).

To investigate the impact of M1NVs on the migration and invasion capacities of EM-ESCs, we performed cell migration and invasion assays. Transwells (Corning, USA) with 8.0 µm Pore Polycarbonate Membrane Inserts were precoated with (invasion assay) or without (migration assay) 100 μL Matrigel Matrix (Corning) on ice and incubated at 37°C for 30 min before seeding the cells. To investigate the direct effects of M1NVs, ESCs were pre-incubated with 20 μg/mL NVs derived from different types of macrophages for 24 h. The ESCs were then seeded in the upper chambers, which contained 5% FBS, whereas the lower chambers contained 600 μL medium supplemented with 20% FBS. After 24 h of incubation, cells above the membrane were removed using a cotton swab. The remaining cells were fixed with 4% paraformaldehyde for 10 min, stained with 0.1% crystal violet, and photographed under a microscope (Olympus BX53; Olympus, Tokyo, Japan). To investigate the indirect effects of M1NVs, the macrophages were induced with LPS, IL-4, or M1NVs; ESCs were seeded into the upper chambers of the inserts, whereas different types of macrophages were seeded into the lower chambers. Medium containing 10% FBS was added to both the upper and lower chambers. Cells that migrated or invaded were counted using Image J v1.8.0 (National Institutes of Health, Bethesda, MD, USA).