F7250 sigma

The EVs of Paenalcaligenes hominis were isolated, as described by Kim et al. [47 (link)]. Briefly, Paenalcaligenes hominis was cultured in GAM broth at 37 °C for 24 h and centrifuged (5000×g, 4 °C, 20 min). The resulting supernatant was centrifuged (100,000×g, 4 °C, 2 h) using sucrose solution (0.8 and 2.5 mol/L). The collected interlayer was centrifuged at 150,000×g for 2 h. The resulting precipitate was used as EVs. The characteristics of EVs were analyzed via tandem mass spectrometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protein assays, and Limulus amebocyte lysate (LAL) assays (Supplement Figures S16 and S17, Supplement Table S1). Paenalcaligenes hominis LPS was purified as described previously (Supplement Methods) [26 (link)]. FITC (F7250 Sigma, Aldrich)-conjugated EVs (FITC to protein ratio, 0.01) and LPS (FITC to LPS ratio, 0.1) were prepared as described by Park et al. [48 (link)].

Pediococcus acidilactici, Enterococcus faecium and Bifidobacterium longum anaerobically cultured in the GAM broth at 37 °C were collected, washed with distilled water, suspended in distilled water, and sonicated according to the methods of Kim and Kobashi⁵⁵ . The sonicated suspension was centrifuged (10,000g, 4 ℃, 30 min). The supernatant faction was freeze-dried and used as cytosolic fraction (CF). EPSs were purified from their precipitates according to the method of Smitinont et al.^{56 (link)}Klebsiella oxytoca was anaerobically cultured in the GAM broth at 37 °C, and centrifuged for 20 min at 5000 g, and washed twice with saline. The collected cells (1 × 10¹⁰ CFU/mL) were suspended in distilled water. Its LPS was purified using a LPS extraction kit (iNtRON Biotechnolgy, Seongnam, Republic of Korea)⁴² . FITC (F7250 Sigma, Aldrich)-conjugated LPS (FITC to LPS ratio, 0.2) was prepared as described by Park et al.^{57 (link)}.