Comparing cell activity, LDH release and viability of Malme-3M cells cultured on flat, round and V-shaped well plates at 10⁴ cells/well density over 5 days and at fixed growth volume and fixed growth surface area.

<p>Group A: Mitochondrial activity of Malme-3M cells cultured at 10⁴ cells/well density, A1) cell activity cultured on round and V-shaped well plates and relative to flat at FV; A2) cell activity cultured on round and V-shaped well plates and relative to flat at FSA. Group B: LDH release of Malme-3M cells cultured at 10⁴ cells/well density, B1) LDH release on round and V-shaped plates and relative to flat at FV; B2) LDH release on round and V-shaped plates and relative to flat at FSA. Group C: cell viability/ml of Malme-3M cells cultured at 10⁴ cells/well density l, C1) round and V-shaped cell viability relative to flat well plates at FV; C2) round and V-shaped cell viability relative to flat well plates at FSA. Data are represented as mean + SD of three independent repeats (n = 3). Any significant difference in results is shown as <i>p</i> ≤ 0.05*, <i>p</i> ≤ 0.01**, <i>p</i> ≤ 0.001*** and is relative to flat plates.</p

Attachment and spreading of ARPE-19 cells on different surface topographies and at different growth conditions.

<p>ARPE-19 cultured at 1 x 10⁴ cells per well density on flat, round and v-shaped well plates over 8 days and at fixed growth volume and fixed growth surface. Group A and B represent cells at FV and FSA respectively and images 1, 2 and 3 represent flat, round and v-shaped well plates respectively. Samples were photographed using GX CAM digital camera at X 4 magnification (100 μm = scale bar). Each images is accompanied by analysis of the cells in the dot plot display mode of forward scatter (FCS) versus side scatter (SSC) on a logarithmic scale, and the core population of the cells is surrounded by a gate.</p

Calculation of surface area (SA) based on 100 μl cell suspension volume (V) on flat well plates.

<p>Calculation of surface area (SA) based on 100 μl cell suspension volume (V) on flat well plates.</p

Attachment and spreading of A549 cells on different surface topographies and at different growth conditions.

<p>A549 cultured at 1 x 10⁴ cells per well density on flat, round and v-shaped well plates over 8 days and at fixed growth volume and fixed growth surface. Group A and B represent cells at FV and FSA respectively and images 1, 2 and 3 represent flat, round and v-shaped well plates respectively. Samples were photographed using GX CAM digital camera at X 4 magnification (100 μm = scale bar). Each images is accompanied by analysis of the cells in the dot plot display mode of forward scatter (FCS) versus side scatter (SSC) on a logarithmic scale, and the core population of the cells is surrounded by a gate.</p

Cell specific marker expression for RPE-65, CRALBP, CD74 and HLA-A2 using flow cytometry via indirect staining.

<p>Median Fluorescent Intensity (MFI) data for cell specific marker expression of ARPE-19, A549, and Malme-3M cell lines cultured at 1 x 10⁴ cells per well density on flat, round and v-shaped well plates over 5 days and at fixed growth volume and fixed growth surface area. Samples incubated with 2 μl per well primary antibodies and labelled with goat anti-mouse IgG FITC-tagged secondary antibody (1:500). Data is representative of 3 independent repeats and is shown as mean + SD (n = 3). Group 1: Fixed volume growth condition and Group 2: Fixed surface area growth condition showing cell specific marker expression of A) RPE-65 (ARPE-19 cell line), B) CRALBP (ARPE-19 cell line), C) CD74 (A549 cell line) and D) HLA-A2 (Malme-3M cell line) marker proteins.</p

ECM induces MR and DC-SIGN up-regulation.

<p>After mRNA extraction and conversion to cDNA, MR and DC-SIGN expression was analyzed using Q-PCR. Relative expression levels of both genes were compared to that of a house-keeping gene (GAPDH). Results depict one representative out of three independent experiments.</p

ECM-treatment causes a decreased expression of some DC maturation markers.

<p>DC membrane phenotype was analyzed after 48 h of culture in the presence of FN or LMN (both at 10 µg/ml) compared with immature DC. Filled histograms represent isotype controls, control (1% BSA treated) DC are depicted with the solid black line, FN treated DC are depicted with the dashed black line and LMN treated DC are depicted with the solid gray line. Results depict one representative out of four independent experiments. <b>*</b> P<0.05.</p

FN and LMN induce an increase in the endocytic ability of DC.

<p>Dextran-FITC uptake at 37°C was evaluated by flow cytometry. DC were incubated for 48 h in ECM-coated plates before performing the assays. n = 4 independent experiments. Error bars represent standard deviations. <b>*</b> P<0.001.</p

MR and DC-SIGN expression by DC is increased after ECM treatment.

<p><i>A</i>, Following 48 h of culture in FN or LMN-coated plates (both 10 µg/ml), DC were stained for MR and DC-SIGN expression and analyzed by flow cytometry. Filled histograms (gray) represent isotype controls. <i>B</i>, The endocytic receptors MR and DC-SIGN were blocked using mannan before addition of dextran-FITC and analysis by flow cytometry. Filled histograms (gray) represent cells without dextran-FITC and results depict one representative out of three. <i>C</i>, The uptake of specific MR and DC-SIGN ligands by ECM-treated DC was also evaluated by flow cytometry. The sulphated sugar SO₄-3-galactose-FITC is specifically bound by MR and Lewis-X-FITC by DC-SIGN. Filled histograms (gray) represent cells incubated without ligand. Results show one representative out of three independent experiments.</p

ECM-treated DC are able to fully mature after stimulation with maturation stimuli.

<p>Membrane phenotype of control, FN and LMN treated DC was analyzed after 48 h of incubation with 200 ng/ml LPS (n = 3) and 1 µg/ml CD40L (n = 4). Error bars represent standard deviations. MFI values for isotype controls for each antibody was deducted from the values shown. <b>*</b> P<0.05.</p