Mouse Blood Sample Analysis and Iron Measurement

To determine white blood cell (WBC) count from peripheral blood, 15 µL of blood was mixed with 250 µL of 3% acetic acid, a hemolytic solution, and the total number of leukocytes was counted with a hemocytometer. Meanwhile, 10 µL of blood was smeared on a glass slide for Wright stain and counted with x100 magnification in 20 fields to determine the percentage of polymorphonuclear cells (PMN) and monocyte. The total number of PMN was calculated by total leukocyte count from hemocytometer multiplied by the percentage of cells from the Wright stain glass slide.33

A micro-hematocrit method with a Hitachi 917 automated biochemistry analyzer (Roche Diagnostics, Indianapolis, IN, USA) was used for hematocrit (Hct) measurement. Meanwhile, BioAssay Systems (Haywood, CA, USA) was used for serum creatinine (Scr) (DICT-500), blood urea nitrogen (BUN) (DIUR-500), aspartate transaminase (AST) (EASTR-100), and alanine transaminase (ALT) (EALT-100). Serum cytokines were measured by enzyme-linked immunosorbent assay (ELISA) (PeproTech, NJ, USA). To determine blood bacterial burdens, mouse blood samples in several dilutions were directly spread onto blood agar plates (Oxoid, Hampshire, UK) and incubated at 37°C for 24 hours before enumeration of bacterial colonies. In addition, endotoxin (LPS) in serum was evaluated as a sepsis parameter using HEK-Blue LPS Detection Kit 2 (InvivoGen, San Diego, CA, USA).34 Values less than 0.01 EU/mL were recorded as 0 due to the lower limit of the test. For organ iron accumulation, the organs including kidney, liver, spleen, and intestine (cecum) were washed several times in phosphate buffer solution (PBS), weighed, homogenized and centrifuged for the determination of ferric ion in supernatant as the representative of tissue iron accumulation by iron assay (Ab83366, Abcam, Cambridge, UK). Of note, cecum is an intestinal part with the highest abundance of fungi,35 endogenous sources of (1→3)-β-D-glucan (BG), an organismal molecule of interest.