Hek blue lps detection

The gut barrier was determined by fluorescein isothiocyanate-dextran (FITC-dextran) and the spontaneous elevation of lipopolysaccharides (LPS) and (1→3)-β-D-glucan (BG) in the serum (gut translocation) without systemic infection [23 (link)]. The detection of FITC-dextran, a gut nonabsorbable molecule at a molecular weight of 4.4 kDa, in the serum after an oral administration indicates gut barrier permeability (leaky gut) [41 (link)]. Then, FITC-dextran (Sigma-Aldrich) was orally administered at a concentration of 25 mg/mL in 0.25-mL phosphate buffer solution (PBS) at 3 h before sacrifice, and the serum FITC-dextran was measured by fluorospectrometry (microplate reader; Thermo Scientific, Wilmington, DE, USA). In parallel, serum LPS (endotoxin) BG were measured by HEK-Blue LPS detection (InvivoGen, San Diego, CA, USA) and Fungitell (Associates of Cape Cod, Falmouth, MA, USA). The values of LPS < 0.01 EU/mL and BG <7.8 pg/mL were recorded as 0 due to the limitation of the standard curves.

Gut permeability was determined by fluorescein isothiocyanate dextran (FITC-dextran) assay, endotoxemia, and immunofluorescent detection of a tight junction protein (zonaoccludens-1; ZO-1) following previous publications^{33 (link)–35 (link)}. As such, FITC-dextran, a nonabsorbable molecule with 4.4 kDa molecular mass (Sigma-Aldrich, St. Louis, MO, USA) at 12.5 mg per mice was orally administered at 3 h before the detection of FITC-dextran in serum by Fluorospectrometer (NanoDrop 3300; Thermo Fisher Scientific, Wilmington, DE, USA). Serum endotoxin (LPS) was measured by HEK-Blue LPS Detection (InvivoGen, San Diego, CA, USA) and the data were recorded as 0 when LPS values were less than 0.01 EU/ml because of the limited lower range of the standard curve. Also, the cecum was used as a representative of the intestine to determine gut tight junction. Accordingly, cecum in Cryogel (Leica Biosystems, Richmond, IL, USA) were cut into 5 μm-thick frozen sections, fixed in acetone, blocked by blocking buffer, stained with a fluorescent antibody against ZO-1 and a green secondary antibody (Alexa Fluor 488) (Life Technologies, Carlsbad, CA, USA) before visualization and scoring by ZEISS LSM 800 (Carl Zeiss, Germany).

Gut permeability was determined by the detection of fluorescein isothiocyanate-dextran (FITC-dextran), a non-absorbable molecule through the intestine, in serum after oral administration¹⁸ . Briefly, 0.5 ml of FITC-dextran (molecular weight 4.4 kDa; FD4; Sigma, St. Louis, MO, USA) at a concentration of 25 mg/ml in sterile phosphate buffer solution (PBS) was orally administered and collected blood through tail vein at 3 h later. Serum FITC-dextran was measured by fluorospectrometry (NanoDrop 3300; Thermo Scientific, Wilmington, DE). In addition, leaky-gut was also determined by the spontaneous increased endotoxin (LPS) and (1→3)-β-D-glucan (BG) in serum. Serum LPS and BG was analyzed with HEK-Blue LPS Detection (InvivoGen, San Diego, CA, USA) and Fungitell assay (Associates of Cape Cod, East Falmouth, MA, USA), respectively. Values of LPS < 0.01 EU/mL and BG <7.8 pg/mL were recorded as 0.