Tween 20

Fifty-eight peptides, which cover the extracellular domain of CD44v3–10 [26 (link)], were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). We immobilized them on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific Inc) at 1 µg/mL for 30 min at 37 °C. The palate washing was performed using the HydroSpeed Microplate Washer (Tecan, Zürich, Switzerland) with phosphate-buffered saline (PBS) containing 0.05% (v/v) Tween 20 (PBST; Nacalai Tesque, Inc.). After the blocking with 1% (w/v) bovine serum albumin (BSA) in PBST for 30 min at 37 °C, C₄₄Mab-1 (10 µg/mL) was added to each well. Then, the wells were further incubated with anti-mouse immunoglobulins peroxidase-conjugate (1:2000 diluted; Agilent Technologies Inc., Santa Clara, CA, USA) for 30 min at 37 °C. One-Step Ultra TMB (Thermo Fisher Scientific Inc.) was used for enzymatic reactions. An iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA, USA) was used to measure the optical density at 655 nm.

Collected single protist cells were washed several times in solution U and then transferred to water droplets containing 0.01% Tween 20 (Nacalai Tesque, Kyoto, Japan). Bacterial cells that leaked from the ruptured host cell were collected with a glass capillary attached to the micromanipulation system and subjected to WGA using Thermo Scientific EquiPhi29 DNA Polymerase. The reaction was performed at 10-µL scale at 45 °C for 3 h (see Supplementary Methods for details). PCR was conducted using primer sets specific to the respective 16S rRNA phylotypes (Table S2), to check for the presence of their genomes. The WGA samples containing the target were re-amplified using EquiPhi29 DNA Polymerase at 50-µL scale. The reaction was performed at 45 °C for 2 h.

Ninety-six-well immunoplates (Thermo Fisher Scientific, Inc., Waltham, MA, USA) were coated with 1 mg/mL OVA diluted in PBS and left overnight at 4 °C. The solution in the plates was removed, and 1% (w/v) BSA (Nacalai Tesque, Inc.), dissolved in PBS, was added to the plates and incubated at room temperature for 2 h. Plates were washed 3 times with wash buffer (0.05% [v/v] Tween 20 [Nacalai Tesque, Inc.] in PBS). Serum samples, diluted with 1% (w/v) BSA and 0.05% (v/v) Tween 20 in PBS, were plated in serial dilutions and then, incubated for 2 h at room temperature. Goat anti-mouse IgG, IgG1, IgG2b, and IgG3, conjugated with horseradish peroxidase (SouthernBiotech, Inc., Birmingham, AL, USA), were diluted at a dilution of 1:4000 with 1% (w/v) BSA (Nacalai Tesque, Inc.) and 0.05% (v/v) Tween 20 in PBS, and then, incubated for 1 h at room temperature. Plates were washed 3 times with wash buffer. Tetramethylbenzidine peroxidase substrate (SeraCare Life Sciences, Inc., Milford, MA, USA) was added to the plates and then, incubated for 2 min at room temperature. Next, 0.5 N HCl (Nacalai Tesque, Inc.) was added to the plates. The absorbance of serum samples was measured at 450 nm with an iMark™ Microplate Absorbance Reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

CD44ec was immobilized on Nunc Maxisorp 96−well immunoplates (Thermo Fisher Scientific Inc.) at a concentration of 1 µg/mL for 30 min at 37 °C. After washing with PBS containing 0.05% (v/v) Tween 20 (PBST; Nacalai Tesque, Inc.), wells were blocked with 1% (w/v) bovine serum albumin (BSA)−containing PBST for 30 min at 37 °C. Culture supernatants were added to each well, followed by peroxidase−conjugated anti−mouse immunoglobulins (1:2000 diluted; Agilent Technologies Inc., Santa Clara, CA, USA). Enzymatic reactions were conducted using 1 Step Ultra TMB (Thermo Fisher Scientific Inc.) followed by the measurement of the optical density at 655 nm, using an iMark microplate reader (Bio−Rad Laboratories, Inc., Berkeley, CA, USA).

Formalin-fixed, paraffin-embedded sections (4 μm thick) of the skin were deparaffinized in d-limonene and graded alcohol. For antigen retrieval, the sections were incubated in 1 mM EDTA buffer (pH 8.0) for 20 minutes in a 95°C water bath, followed by incubation with 3% hydrogen peroxide in methanol for 5 minutes at room temperature to block endogenous peroxidase activity. The slides were rinsed with tris(hydroxymethyl)-aminomethane–buffered saline containing 0.1% Tween 20 (Nacalai Tesque) and blocked with goat serum for 1 hour. Subsequently, the sections were incubated with rabbit anti-mouse IgG antibody (Invitrogen 31194) diluted to 1:2,000 overnight at 4°C, followed by incubation with the envision system–labeled polymer, horseradish peroxidase anti-rabbit antibody (DAKOCytomation K4003) for 20 minutes. Finally, the sections were incubated with 3,3-diaminobenzidine and counterstained with hematoxylin.

Proteins were extracted with 1% Triton X100-containing lysis buffer: 50 mM HEPES (pH: 7.5); 150 mM NaCl; 10 mM glucose; 2 units/mL hexokinase (Sigma); and protease inhibitor cocktails. Detergent-soluble and insoluble fractions were separated by centrifugation at 18,000× g at 4 °C for 10 min. Protein concentration of the soluble fraction was determined by a standard Bradford assay system. For pulldown experiments, the cell lysate was treated using anti-Flag agarose beads (Sigma), as previously described [22 (link)]. Protein samples were subjected to reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA). The membrane was blocked with tris-buffered saline containing 5% skim milk and 0.05% Tween 20 (Nacalai, Kyoto, Japan) at 4 °C overnight. Next, the proteins were detected using indicated primary antibodies and secondary antibodies. Finally, they were visualized with enhanced chemiluminescence and the ChemiDoc system (Bio-Rad, Hercules, CA, USA). Densitometric analysis was performed using the ImageJ software version 1.53 (National Institute of Health, Bethesda, MD, USA). The expression of proteins of interest was normalized to that of αTubulin. The error bars in figures represent the standard deviation for at least three independent experiments.

Skin samples were homogenized in PBS containing 0.1% Tween 20 (Nacalai Tesque) and 1% Protease Inhibitor Cocktail Set III (Merck) and centrifuged. Blood samples were collected under anaesthesia, and serum was isolated after coagulation and centrifugation. The concentrations of IFN‐α, heat shock protein 70 (HSP70) and IL‐18 were measured by enzyme‐linked immunosorbent assay (ELISA) using VeriKine‐HS Mouse IFN‐α All Subtype ELISA Kit (PBL Assay Science), Mouse HSP70 ELISA kit (CUSABIO) and Mouse IL‐18 SimpleStep ELISA kit (Abcam). The concentrations of IL‐1β, IFN‐γ, tumor necrosis factor (TNF)‐α and IL‐17A were measured using Bio‐Plex Multiplex Immunoassay System (Bio‐Rad) with Bio‐Plex Pro Mouse Cytokine IL‐1β Set and Bio‐Plex Pro Mouse Cytokine Th1/Th2 Assay (Bio‐Rad).