Laminarin

The reaction mixtures (500 μl each) were prepared with 0.01 mg/ml purified protein, 1% (w/v) soluble or insoluble polysaccharides, such as chitin (Nacalai Tesque Inc.), chitosan (from crab shell; Nacalai Tesque Inc.), curdlan, cellulose (α-cellulose; Nacalai Tesque Inc.), carboxymethyl cellulose sodium salt (Nacalai Tesque Inc.), xylan (from beechwood; Nacalai Tesque Inc.), laminarin (Nacalai Tesque Inc.), sodium alginate (500 cps), pectin (from citrus; Nacalai Tesque Inc.), heparin sodium, xanthan gum (Nacalai Tesque Inc.), or gellan gum (Nacalai Tesque Inc.), and 50 mM Tris buffer pH 7.5. These reaction mixtures were incubated at 37 °C for 12 h. Then, the reaction mixtures were centrifuged at 15,000 g for 10 min and the clear fractions were transferred to clean tubes for assays. The degradation of the polysaccharides was determined using the DNS assay^{43 (link)}. The DNS reagent consisted of 1% (w/v) DNS, 30% (w/v) potassium sodium tartrate, and 0.4M NaOH. The aliquots (100 μl) were mixed with equal volumes (100 μl) of DNS reagent, boiled for 5 min, cooled to 20 °C, and then observed for a red colour or measured at an absorbance of 525 nm^{43 (link)}.

Phagocytosis inhibition assay was performed by dispensing mouse BMDCs into 24-well plates at a concentration of 2.5 × 10⁵ cells/well and incubating with 1 mg/mL mannan from Saccharomyces cerevisiae (Sigma-Aldrich), 1 mg/mL d(+)-mannose (FUJIFILM Wako Pure Chemical), 1 mg/mL laminarin (Nacalai Tesque, Kyoto, Japan), 1 mg/mL dextran from Leuconostoc spp. (M_r 450000–650000; Sigma-Aldrich), 1 mg/mL d(+)-galactose (FUJIFILM Wako Pure Chemical), 1 mg/mL N-acetyl-d(+)-glucosamine (FUJIFILM Wako Pure Chemical), or PBS at 37°C under an atmosphere of 5% CO₂ for 30 min, followed by addition of CFW-labeled rLdpA–chitin complex (rLdpA, 1 μg/mL, chitin, 50 μg/mL). After 4-h incubation, the MFI of rLdpA–chitin complex-phagocytosed cells was measured by flow cytometry (BD FACSVerse; BD Biosciences). Data were analyzed using FlowJo version 10 (BD Biosciences).