Costar transwell plate

DCs from different groups were transferred to a Corning Costar Transwell plate (Pore Size: 5.0 μm; 6.5 mm Diameter; 0.33cm² Growth Area) that included an upper and lower chamber. The upper chamber had cells in 100 ul media without cytokines and serum. The lower chamber contained 500 ul of media with serum, CCL19 (250 ng/ml), CCL21 (250 ng/ml) and CXCL3 (250 ng/ml). The upper chamber was cast off after 5 h and the migrated cells into the lower chamber were counted on hemocytometer. In the in vitro migration experiments, CXCR2 activity was inhibited by treating cells for one hour before the migration assay with mouse anti-CXCR2 (at 1 to 50 dilution, Clone242216, R&D Systems) [8 (link)]. For in vivo application, SB225002 (selective non-peptide antagonist of CXCR2, Sigma-Aldrich) was dissolved in vehicle (NaCl 0.9% solution plus Tween-80 0.33%) according to manufacturer’s instructions. SB225002 was injected into the mice through intraperitoneal (IP) at 50 μg (1.4 × 10–7 mol) in 200 μl per animal one hour prior each DC vaccine injection [9 (link), 10 (link)]. In the human DC in vitro migration experiments, cells were treated with SB225002 at a concentration of 10 μM for one hour prior to the migration studies [11 (link)].

Transwell migration assays were performed following protocols from Justus et al. (2014) , with slight modifications for Jurkat T cells. Jurkat T cells (250 μL, 1 × 10⁴ cells well⁻¹) were carefully spread on top of a FN-treated 3 µm pore upper chamber of a Costar transwell plate (Corning, United States). The upper chamber was maintained with serum-reduced RPMI medium. Inhibitors were applied on both the upper and the lower chambers at the indicated concentration. The bottom chamber was loaded with media containing cytokines (IL-4, 20 ng/ml⁻¹; TNF-α, 10 ng ml⁻¹) and 10% FBS in RPMI media as a chemoattractant (Leung et al., 1994 (link); Xia et al., 1996 (link)). Serum-free RPMI was used in the bottom chamber as a negative control. Cells were allowed to transmigrate for 21 h at 37°C in a humidified incubator with 5% CO₂. Cells were then collected from the bottom chamber and counted using a flow cytometer (BD Accuri C6, BD Biosciences). Normalized transmigration was calculated using the formula: Normalized transmigration, $Tr=\left(\frac{\text{N}21}{N_B}\right)\times 100$ . Where N₂₁ was the number of live cells per 50 µL of medium in the bottom chamber after 21 h, and N_B was the number of live T cells per 50 µL of medium in the bottom chamber of the control well.

The coculture system was set up as reported previously [28 (link)]. Briefly, neuronal cells or SH-SY5Y cells were cultured on the bottom side of a Costar Transwell plate (0.4-µm pore size; Corning, NY, USA) in a humidified 5 % CO₂ incubator at 37 °C for 24 h. Microglia in the culture inserts were treated with MNPs@SiO₂(RITC) or LPS for 12 h. Then, the inserts were placed in the plate and incubated for another 12 h. To measure the viability of the neuronal cells, the upper inserts were removed, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium solution (CellTiter 96® AQueous One; Promega, WI, USA) was added to each well containing the microglia. The assay plate was incubated at 37 °C for 1 h. The amount of soluble formazan produced via cellular reduction was measured using a plate reader (Molecular Devices, CA, USA) at 490 nm. Values were normalized to the corresponding total protein. Images of cell morphology and density were acquired before the cell viability assay under an Axiovert 200 M fluorescence microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) at the Three-Dimensional Immune System Imaging Core Facility of Ajou University. The excitation wavelength for the MNPs@SiO₂(RITC) was 530 nm.