Plasma fibronectin

Human proteins used for ELISA include: plasma fibrinogen (Code: F3879), plasma fibronectin (Code: 11051407001), Glu-plasminogen (Code: P7999), vitronectin (Code: SRP3186), laminin (Code: L6274), and lactoferrin (Code: L1294) which were all supplied by Sigma.
Binding affinity measured by ELISA was performed as described previously^{17 (link)}. Recombinant protein RP15 was produced as described^{55 (link)} and both C-terminal peptides were synthesised by Chempeptide Limited (China). P1-30 (¹⁵⁹⁷TSAAKPGAPRPPVPPKPGAPKPPVQPPKKPA¹⁶²⁷) without any tags, but P1-15 (¹⁶¹³PGAPKPPVQPPKKPA¹⁶²⁷) was sequenced with an N-terminal biotin tag.
15 µg/ml of C-terminal P1 fragments were bound to wells and incubated with different host proteins. Wells were then incubated with different antiserum raised against the different host proteins at the following dilutions (all from Sigma): anti-fibrinogen 1:3000, anti-fibronectin 1:1000, anti-plasminogen: 1:2500, anti-vitronectin 1:5000, anti-laminin 1:750, and anti-lactoferrin 1:5,000. These incubations were followed by incubations with anti-rabbit IgG (Dako) or anti-goat IgG (both 1:2,000). Detection was measured by adding Tetramethylbenzidine (Sigma) followed by 1 M HCl, and absorbance was measured at 450 nm (620 nm as reference).

Clonal cultures of mouse embryonic DRG cells were performed by a modification of methods described previously (Ito et al., 1993 (link)). DRGs were dissected and cut into small fragments. These fragments were explanted into 35-mm culture dishes coated with collagen gel (Cellmatrix, Nitta Gelatin). Two days later, DRG cells derived from these fragments were resuspended by trypsinization. This essentially single cell suspension (>95% single cells) was diluted to 100 cells/ml. One milliliter aliquots of this diluted cell suspension were plated to 35-mm culture dishes that were coated with a collagen gel (PureCol, Advanced BioMatrix, San Diego, CA, USA) and conditioned with culture medium (see the ‘Culture medium and signaling molecules’ section) containing 10 µg/ml plasma fibronectin (Sigma, St. Louis, MO, USA). The clone founder cells were identified at 8 h after seeding cells. The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO₂, and the culture medium was changed every day for the first 2 days and then every other day.

Liver endothelial cells were starved overnight in 0.5% FBS/DMEM F12 (Invitrogen). Fluroblock transwell inserts (BD Biosciences, Franklin Lakes, NJ) were coated with either 0.02 mg/ml plasma fibronectin or cellular fibronectin (Sigma) on both sides for 30 min at room temperature. Cells were plated in the top chamber of transwell filters in media with 0.5% FBS and the filters were placed atop a 24-well plate with media containing 10% FBS. Inserts were incubated at 37°C for 18 h and then stained for 1 h at 37°C with calcein AM (BD Biosciences). Fluorescence intensity was measured from the bottom with a BioTek Synergy 2 microplate reader (Winooski, VT). There were three replicates for each condition.

Freshly harvested lymph nodes were harvested from C57BL/6 mice, and incubated in digestion mixture containing RPMI-1640, 0.25 mg/mL Liberase TL (Roche, Cat: 5401020001), 200 Kunits/mL DNAse I (Sigma-Aldrich, Cat: 10104159001) at 37° C for 1h with occasional shaking. Single cell suspensions were strained using a 70 µm cell strainer (Corning), centrifuged and resuspended in αMEM. Cells were plated on 6-well plates coated with 10 µg/mL PureCol (Collagen, Sigma-Aldrich) and human 10 µg/mL plasma Fibronectin (Sigma-Aldrich). After 3 hours non-adhering cells were removed. 5-7 days later, when >80% confluence was achieved, cells were detached using Accutase (Biological Industries). Under these conditions blood endothelial cells die and the remaining cells are composed of fibroblastic reticular cells (FRCs) (~40%) and LECs. LECs were purified by staining with APC-conjugated anti-mouse CD31 (Biolegend, clone 390) and positively selected using anti-APC microbeads according to manufacturer’s instructions (Miltenyi Biotec).

The binding of bacteria to plasma and/or ECM proteins was analyzed as described elsewhere (Agarwal et al., 2013 (link)) with modifications. Briefly, black polystyrene 96-well microtiter plates were treated (18 h at 4°C) with each human protein [plasma fibronectin, plasma fibrinogen, plasma plasminogen, or type I collagen from human fibroblast (Sigma-Aldrich, United States); 5 μg/ml in PBS pH 7.2]. Afterward, the plates were washed (three times with PBST) and incubated (2 h at room temperature) with blocking solution (50 mM Tris–HCl – pH 8, 150 mM NaCl, 0.1% Tween 20, 3% fish gelatin). Next, 100 μl of suspensions of FITC-labeled bacteria in carbonate buffer (0.15 M NaCl, 0.1 M Na₂CO₃; pH 9.6) (containing 1 × 10⁸ ufc) was added per well, and plates incubated during 1 h at 37°C. Plates were then washed three times with washing buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.1% Tween 20) for removal of unbound bacteria, and fluorescence signal quantified with the help of a microplate fluorescence reader (Synergy H1, BioTek, CA, United States). The control samples for each strain included wells treated with each human protein, but not incubated with FITC-labeled bacteria, and wells treated with BSA followed by the incubation with the bacterial suspensions.