Fluoroblok insert

Chemotactic activity and cell migration were assessed using Falcon^® 24-well Companion plates, and Corning^® FluoroBlok Inserts with 8 µm pore size (both: Corning GmbH, Kaiserslautern, Germany). To determine cell motility toward a soluble chemotactic agent, an FCS gradient was employed. For chemotaxis, inserts were left unaltered. To score cell migration, inserts were pre-coated with 200 µg/mL of collagen G overnight at 4 °C.
Aliquots of 6 × 10⁵ parental and sunitinib-resistant RCC cells, pre-stained with CellTracker Green CMFDA Dye (Invitrogen™, Thermo Fisher Scientific, Darmstadt, Germany) and 72 h pre-treated with SHI or diluent, were seeded per insert. The companion plates were loaded with cell growth media containing 30% FCS and inserts with the cell suspension in media with 10% FCS. After 24 h incubation, the inserts were washed with PBS, containing Mg²⁺ and Ca²⁺, and fixed with 2% glutaraldehyde. To quantify the number of cells migrating through the insert membrane toward the 30% FCS stimulus, mean fluorescent intensity on the lower surface of the inserts was determined using a Sapphire Imager (Azure Biosystems, Munich, Germany). Values were presented as a percentage compared to untreated controls, set to 100%.

Primary microglia were seeded onto FluoroBlok™ Inserts (8 μm pore size, Corning Incorporated) at a density of 3 × 104 cells/Transwell insert. Both the insert and the well underneath contained cell culture medium supplemented with 250 μM aleglitazar or vehicle (DMSO 0.5%). One hundred micromolar ATP (Sigma-Aldrich) was added to the well below the insert. After 6-h incubation at 37 °C and 5% CO2, the membranes of the inserts were stained with 10 μm CFSE dye (Sigma-Aldrich), fixed with 4% paraformaldehyde (PFA), and counterstained with 2 μm 4′,6′-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Migrated cells below the FluoroBlok membranes were visualized using an inverted fluorescence microscope at × 200 magnification (Leica DMI3000).

Lymphoid malignant cells were treated with the conditioned medium derived from macrophages for 3 hours, and then cells were seeded into 96-well plates that were precoated with fibronectin (BD Biosciences). Thirty minutes later cells were washed twice with PBS to remove the non-adherent cells, and adherent cells were measured by CellTiter-Glo luminescent cell viability assay kit. For relative adhesion determination, the group treated with LPS alone was set to be 1 and the other group was normalized to this group.
For cell invasion and migration assay, tumor cells suspended in serum free medium were pre-labeled with calcein-AM (Sigma) for 30 minutes, then cells were plated into FluoroBlok™ inserts (Corning), which were precoated with fibronectin or not. The inserts were placed in a 24-well plate containing conditioned medium from macrophages. Twelve hours later, tumor cells in the lower chambers were measured by plate reader at bottom reading mode and visualized by an inverted fluorescence microscope (TCS-SP2, Leica Microsystems).

NB4 neutrophil chemotaxis and adhesion were assessed, as previously reported (53 (link)). 5 × 10⁶/mL NB4 neutrophils were labeled with 1 μM calcein AM (Molecular Probes) for 30 min at 37°C. Cells were thoroughly washed and resuspended in 2 × 10⁶/mL in HEPES+ medium. For chemotaxis, 8 μm-pore-size FluoroBlok inserts (Corning Inc.) were used and cells were stimulated with 10 nM complement component 5a (C5a; Sino Biological) or 10 nM IL8 (Sigma). For adhesion, calcein-labeled cells were stimulated with 100 ng/mL PMA (Sigma-Aldrich) or 10 mM dithiothreitol (DTT; Sigma-Aldrich) and incubated for 30 min at 37°C, 5% CO₂, in an uncoated, fibronectin-coated (Sanquin Plasma Products), or ICAM-1-coated (Sino Biologicals) coated MaxiSorp plate (Nunc). Where indicated, cells were preincubated with 10 μg/mL monoclonal antibody against CD18 (IB4, ATCC) and CD11b (44a, ATCC), for 10 min at room temperature. NB4 neutrophil adhesion was assessed in the presence of 5 μM latrunculin B (LatB; Merck Millipore). The cells were preincubated with the inhibitor for 20 min at 37°C and 5% CO₂, prior to further stimulation. Dimethyl sulfoxide (DMSO, 0.1%) was used as vehicle control. Adhesion was determined in a GENios plate reader after lysis in 0.5% (w/v) Triton X-100 for 5 min at room temperature at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

VO-PyMT, GFP⁺ cells were suspended at 1 × 10⁴ cells per 200 μl of DMEM, high glucose (11965; Gibco) with 1% penicillin–streptomycin and 0.5% puromycin. 700 μl of DMEM with 2% FBS, 1% penicillin–streptomycin, and 0.5% puromycin along with 0.375 mg/ml of IgG control or SDS3 was added into each well of a 24-well flat bottom plate. FluoroBlok inserts (351152; Corning) were lined with 25 μl of 1:2 diluted growth factor reduced Matrigel (354230; Corning) and incubated for 45 mins at 37°C. VO-PyMT, GFP⁺ cells were seeded into FluoroBlok insert and imaged 24 h postseeding. Images were taken with an inverted fluorescent microscope for GFP⁺ signal using Leica software. Each well was split into four quadrants and a random image was taken within each quadrant at 10× magnification. The number of migrated, GFP⁺ cells were counted and compared against the total number of cells seeded.