Micro smash ms 100

Neovascularized murine corneas were enucleated and homogenized using a Micro Smash MS-100 homogenizer (Tomy Seiko co., LTD, Japan), for 30 s power at 3,500 rpm, two times, and centrifuged at 5,000 × g, 5 min to remove tissue debris. Supernatants were collected and quantified for ELISA according to the manufacturer’s instructions using ELISA kits for mouse VEGF (DY493-05), IFN-γ (DY485), TNF-α (DY410-05), and IL1β (DY401-05) (R&D Systems Minneapolis, MN, USA).

For immunoprecipitation, cells were collected by centrifugation and disrupted in lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.5% or 1% NP-40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and complete protease inhibitors [Roche]), with glass beads using Micro Smash MS-100 (Tomy). Anti-FLAG M2 beads (Sigma) and GFP-Trap beads (Chromotek) were used to immunoprecipitate FLAG- and GFP-tagged proteins, respectively.

Intracellular proteins were extracted from cell pellets after separating the supernatants for extra-cellular protein extraction. The pellets were washed once and resuspended in extraction buffer (50mM Tris, 1mM EDTA, 20mM DTT) containing complete Mini Protease Inhibitor Cocktail (Roche). Homogenization of cells was carried out in a Micro Smash MS-100 (Tomy Seiko Co., Ltd., Japan) with 0.1mm glass beads in screw cap tubes at a pulse of 4,000rpm for 20 seconds in 8 cycles. The cells were then centrifuged to get the intracellular proteins in the supernatant.

Before dissection, tweezers were sterilized with 70% ethanol and UV light irradiation for 10 min. Insects were dissected under a binocular microscope. The gut was roughly homogenized using tweezers in 978 µL of sodium phosphate buffer included in FastDNA Spin Kit for Soil (MP-Biomedicals), and transferred into Lysing Matrix E (MP-Biomedicals). DNA extraction was performed according to the manufacturer’s instruction with beads-beating for 1 sec at 5,500 rpm, using Micro Smash MS-100 (TOMY). For V. mandarinia samples collected at AIST, beads-beating was performed twice. Extracted DNA samples were quantified using Qubit dsDNA HS Assay Kit (Molecular Probes), and their integrity were verified by agarose electrophoresis.

The dissected pieces of human tissue for enzyme activity assay were kept at −80°C without fixation until use. C57BL/6J male mice at 8 weeks of age were euthanized and maintained at 4°C. Mouse tissue was collected at 0, 12, 24, or 48 h after death and kept at −80°C until use. Tissue was homogenized in 7 mM sodium pyrophosphate (pH 8.3) at 3500 rpm for 2 min using Micro Smash MS-100 (Tomy, Tokyo, Japan) and centrifuged at 5500 × g for 10 min. The activity of DAO was determined as described previously (Sasabe et al., 2012 (link), 2014 (link)). Briefly, 50 μ l tissue lysate was added to a mixture [150 μ l of 100 mM D-Ala, 100 μ l of 0.1 mM flavin adenine dinucleotide (FAD), 150 μ l of 700 units/ml catalase in 133 mM sodium pyrophosphate (pH 8.3), and 50 μ l of 70% v/v MeOH], processed with constant agitation at 37°C for 30–60 min, and terminated by adding 500 μ l of 10% trichloroacetic acid. To 250 μ l of the supernatant solution were added 250 μ l of 5 M KOH and 250 μ l of 0.5% 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole in 0.5 M HCl. After 15 min incubation at room temperature, 250 μ l of 0.75% KIO₄ in 0.2 M KOH was added to the mixture with vigorous shaking, and absorbance at 550 nm was measured. DAO activity was calculated as described by Watanabe et al. (1978 (link)) and expressed as the amount of D-alanine oxidized per minute per milligram of protein.

To evaluate the tissue distribution of TUS-007 in vivo, the organs of ICR mice in the PK analysis were used. A total of 40–80 mg of each organ was homogenized in 0.6 mL of tissue lysis buffer (50 mM Tris-HCl pH 8.0, 20 mM EDTA, 10 mM NaCl, 1% SDS) by Micro Smash MS-100 using stainless (5.5φ) and zirconia (1.0φ) beads (TOMY SEIKO CO., LTD., Tokyo, Japan). Lysates were centrifuged at 18,000× g for 10 min, and 2.25 mL of methanol/chloroform (ratio of 2/1) was added to the supernatants, which were then left to stand for 30 min at room temperature. Then, 0.75 mL of chloroform and 0.75 mL of distilled water were added and then centrifuged at 1500× g for 15 min. The layer of chloroform was dried under reduced pressure and redissolved with the mobile phase. TUS-007 was analyzed by HPLC (Alliance e2695, Waters Corporation., MA, USA) using a column of COSMOSIL^® C18-AR-II (150 × 4.6 mm, Nacalai Tesque., Kyoto, Japan) with the isocratic elution mode with 0.1% trifluoroacetic acid (TFA) in 50:50 (v/v) acetonitrile/water mobile phase at a flow rate of 1.0 mL/min.