Fluoroblok insert

Migration assays were performed on cells in log phase growth stage as previously described [17 (link)]. Briefly, the cells were harvested, centrifuged, suspended in medium with the addition of 10 μg/ml DilC12(3) fluorescent dye (Becton Dickinson) and stained 1 hour in standard condition. Next, cells were harvested, centrifuged and suspended in FBS free culture medium to the final density 8 × 10⁵ cell/ml. 250 μl of the prepared cells suspension (2 × 10⁵ cell) was added on the 3 μm FluoroBlok inserts (Becton Dickinson) and placed in 24-well plate. Chambers under the inserts were filled with 750 μl of culture medium (DMEM with 10% addition of mothers-mice sera). As negative control serum-free culture medium was used. After 24 h incubation in standard conditions (37°C, 5% CO₂), the inserts were relocated into new 24-well plate containing 1 ml of PBS and the fluorescence was measured directly in the plate with inserts (excitation 549 nm emission 565 nm) using the reader with bottom optic option. The results were presented as relative fluorescence units – RFU (mean ± SEM).

The cells (in log phase growth stage) were harvested, cen-trifuged, suspended in medium (DMEM, 4.5 g/ml glucose with L-glutamine, 10% FBS, 50 U/ml penicillin, 50 pg/ml streptomycin) with the addition of 10 pg/ml DilC₁₂ (3) fluorescent dye (Becton Dickinson) and stained for 1 hour in standard conditions. Next, cells were harvested, centrifuged and suspended in FBS-free culture medium to the final density of 8 × 10⁵ cell/ml. 250 µl of the prepared cells suspension (2 × 10⁵ cell) was added on the 3 µm FluoroBlok inserts (Becton Dickinson) and placed in a 24-well plate. Chambers under the inserts were filled with 750 µl of culture medium (DMEM, 4.5 g/ml glucose with L-glutamine, 5% FBS, 50 U/ ml penicillin, 50 µg/ml streptomycin). 10 or 50 µM of resveratrol were added to the lower chamber. As a control, culture medium without resveratrol was used. As a negative control, FBS-free culture medium was used. The fluorescence (excitation 549 nm and emission 565 nm) was measured after 24 h exposure directly in the inserts using the FLUOstar Omega reader with “bottom optic ” option. The results were shown as the relative fluorescence units - RFU (mean ± SEM).

Chemotaxis of purified neutrophils was assessed by means of FluoroBlok inserts (Falcon; BD). Cells (5 × 10⁶/ml) were labeled with calcein-AM (1 µM final concentration; Molecular Probes) for 30 min at 37°C, washed twice, and resuspended in Hepes buffer at a concentration of 2 × 10⁶/ml. Chemoattractant solution (PAF, IL-8, and C5a, all at 10 nM; Sigma-Aldrich) or medium alone (0.8 ml/well) was placed in a 24-well plate, and 0.3-ml cell suspension was delivered to the inserts (3-µm pore size) and placed in the 24-well plate. Cell migration was assessed by measuring fluorescence in the lower compartment at 2.5-min intervals for 45 min with the HTS7000+ plate reader (Perkin Elmer) at an excitation wavelength of 485 nm and emission wavelength of 535 nm. Maximal slope of migration was estimated over a 10-min interval.