Bio plex assay system

The scRNA-expressing and NRF2 shRNA-expressing stable U937 cell lines were incubated with PMA for 24 h, and culture media were collected. Levels of IL-1β, IL-6, TNFα, and monocyte chemotactic protein 1 (MCP-1) within the collected media were analyzed with the Bio-Plex assay system (Bio-Rad) according to the manufacturer’s protocol.

To evaluate the effects of iso-α-acids on Alzheimer’s-like disease, 6-month-old transgenic 5 × FAD and WT mice were orally administered daily 1 mg/kg iso-α-acids for 7 days. The mice were subjected to a novel object recognition test after 7 days of administration, and their brains were then removed for quantification of Aβ and cytokines. The hippocampus and cerebral cortex were homogenized in TBS buffer (Wako) with a multibeads shocker (Yasui Kikai, Osaka, Japan). After centrifugation at 50,000 × g for 20 min, the supernatant was collected. Pellets were re-homogenized in TBS containing 1% Triton X-100 (Wako) and the supernatant was collected after centrifugation. The total protein concentration of the supernatant was measured with the BCA protein assay kit (ThermoScientific, Yokohama, Japan). The first supernatant was assayed for quantification of soluble Aβ1-42 (Wako) by enzyme-linked immunosorbent assay (ELISA). To quantify cytokines and chemokines, supernatants were evaluated by the Bio-Plex assay system (Bio-Rad). The second supernatant was used for quantification of insoluble Aβ1-42 (Wako) by ELISA.

Homogenate samples of the left hippocampus and the cortex were prepared as described in a previous study [20 (link)]. The hippocampus or cortex was homogenized in tris-buffer solution (TBS) containing a protease inhibitor cocktail (BioVision, CA, USA) using a multi-beads shocker (Yasui Kikai, Osaka, Japan). The supernatant (first) was collected after centrifugation at 50,000×g for 20 min, and the pellet was homogenized again in TBS containing 1% Triton X-100 (Wako, Osaka Japan), and the supernatant (second) was collected after centrifugation at 50,000×g for 20 min. The total protein concentration of each supernatant was measured using the BCA Protein Assay Kit (Thermo-Scientific, Yokohama, Japan). The first supernatant was used to quantify soluble Aβ_1-42 (Wako), phosphorylated tau (pS199, ThermoFisher Scientific, Yokohama, Japan), total tau (ThermoFisher Scientific), synaptophysin (LSBio, Seattle, WA, USA), BDNF (Promega, Madison, WI, USA), and IGF-1 (R&D Systems, Minneapolis, MN, USA) by ELISA and cytokines and chemokines by a Bio-Plex assay system (Bio-Rad, Hercules, CA, USA). The second supernatant was used to quantify insoluble Aβ_1-42 (Wako) by ELISA.

To evaluate the effect of iso-α-acids on the brain function of HFD loaded mice, the left hippocampus was collected and weighed. Samples were homogenized using a multi-beads shocker (Yasui Kikai, Osaka, Japan) in TBS buffer containing a protease inhibitor cocktail (BioVision, Mountain View, USA). The homogenates were centrifuged (50,000 × g, 30 min) and the supernatants were used in the following assays: total protein content of the supernatant was measured using the BCA protein assay kit (Thermo Scientific, Rockford, USA). Inflammatory cytokines and chemokines were evaluated using the Bio-Plex assay system (Bio-Rad, Richmond, USA). Lipid peroxidation was assessed by measuring malondialdehyde (MDA) using a MDA adduct competitive ELISA kit (Cell Biolabs, San Diego, USA). As an indicator of neuronal cells, the amounts of total CREB and phosphorylated CREB were measured using a CREB (total) ELISA kit (Invitrogen, Carlsbad, USA) and Phospho-CREB (S133) ELISA kit (R&D Systems, Minneapolis, USA), respectively. Brain derived neurotropic factor (BDNF) levels were evaluated using a BDNF ELISA kit (Promega, Madison, USA).

To measure cytokines and tau in the brain, the hippocampus and frontal cortex were homogenized in TBS buffer (Wako, Monza, Monza and Brianza, Lombardy, Italy) containing protease inhibitor cocktail (Biovision, CA, USA) and phosphatase inhibitor cocktail l and II (Wako) with a multi-bead shocker (Yasui Kikai, Osaka, Japan). After centrifugation at 50,000× g for 20 min (MX-107, Tommy, Tokyo, Japan), the supernatant was collected. The pellets were sonicated in sarkosyl solution (1% N-lauroylsarcosine (Sarkosyl) in 1 mM Tris, 1 mM EGTA, 1 mM DTT, and 10% sucrose, pH 7.5), and the supernatant was collected after centrifugation at 386,000× g for 20 min at 4 °C. The pellets were treated with formic acid and dried. The samples were dissolved in an assay buffer (0.2 g/L KCl, 0.2 g/L KH₂PO₄, 8.0 g/L NaCl, 1.150 g/L Na₂HPO₄, 5% BSA, 0.03% Tween 20, and 1× protease inhibitor cocktail (Calbiochem) in ultrapure water, pH 7.4). The total protein concentration of each supernatant was measured using a BCA protein assay kit (ThermoScientific, Yokohama, Japan). Each supernatant was assayed for quantifying total tau and phosphorylated tau (pTau) of pS199, pS396, and pT231 (Thermo Scientific, Waltham, MA, USA) by ELISA. For quantifying cytokines and chemokines, the first supernatant was evaluated by a Bio-Plex assay system (Bio-Rad, Hercules, CA, USA).

To measure levels of cytokines, Aβ, and Tau, we homogenized the hippocampus of the left hemisphere in TBS buffer (Wako) with a multi-beads shocker (Yasui Kikai, Osaka, Japan). After centrifugation at 50,000× g for 20 min, we collected the supernatant. We measured the total protein concentration of each supernatant with a BCA protein assay kit (ThermoScientific, Yokohama, Japan). We assayed the supernatant to quantify soluble Aβ_1–42 (Wako), Tau (ThermoScientific), and phosphorylated Tau (pTau, pS199, ThermoScientific) by ELISA and cytokines by a Bio-Plex assay system (Bio-Rad, Hercules, CA, USA).

Microglial cells were isolated from brains of newborn C57BL/6J mice (<7 days old) via magnetic cell sorting (MACS) after conjugation with anti-CD11b antibody, as described previously (Ano et al., 2015 (link)). Briefly, isolated CD11b-positive cells (>90% pure, evaluated by flow cytometer) were plated into poly-D-lysine (PDL)–coated 96-well plates (BD Biosciences, Billerica, MA, United States) and cultured in a DMEM/F-12 (Gibco, Carlsbad, CA, United States) medium supplemented with 10% fetal calf serum (Gibco) and 100 U/ml penicillium/streptomycin (Sigma-Aldrich, St. Louis, MO, United States). Microglia at a density of 30,000 were treated with each iso-α-acids for 12 h, and then with lipopolysaccharide (LPS, 5 ng/ml, Sigma-Aldrich, St. Louis, MO, United States) and interferon-γ (IFN-γ, 0.5 ng/ml, R&D systems, Minneapolis, MN, United States) for 12 h. After stimulation, the supernatant was used for the TNF-α production assay. Concentrations of cytokines sand chemokines in the supernatant was measured by the Bio-Plex assay system (Bio-Rad, Hercules, CA, United States).