Ultrafree mc

The methanolic solutions of chiral-separated (R)- and (S)-CypL were filtered through a centrifugal filter Ultrafree-MC (0.22 μm; Millipore, Billerica, MA, USA) and diluted 2 fold with methanol or mixed together in a 1:1 ratio. The methanolic extract of dried luminous ostracods was prepared as follows: ten dried luminous ostracods in a commercially available kit for observation of bioluminescence of sea-firefly (Hatenouruma, Tokyo) were homogenized in 200 μL of ice-cold methanol on ice and centrifuged at 14,000× g for 3 min at 4 °C followed by filtration through an Ultrafree-MC centrifugal filter (0.22 μm; Millipore). Ten microliter aliquots of these prepared solutions were subjected to chiral HPLC analysis. Chiral HPLC analysis was performed on a Waters ACQUITY UPLC H-Class system (Waters) equipped with a CHIRALCEL OZ-RH chiral column (ϕ4.6 × 150 mm, 5 μm; Daicel Chemical Industry), a multiwavelength detector (ACQUITY UPLC PDA eλ detector; Waters), and a fluorescence detector (ACQUITY FLR detector; Waters). The HPLC conditions were as follows: mobile phase, 30% (v/v) acetonitrile in a 100 mM solution of potassium hexafluorophosphate in H₂O; flow rate, 0.8 mL min⁻¹; fluorescence detection, excitation/emission, 430/570 nm.

To localize INA in the subcellular fractions, bark tissues were excised from current-year stems of high-bush blueberry (cv. Weymouth). Two hundred grams of bark tissues were homogenized with mortar and pestle in 400 mL of 0.5 M mannitol, 20 mM glycylglycine–NaOH (pH 7.5), 5 mM MgCl₂, 1 mM EDTA and 1 mM DTT. The homogenate was vacuum-filtrated through a 100-μm nylon mesh and two layers of filter paper. The filtrate was sequentially centrifuged at 200g for 10 min, 11 000 g for 10 min and 100 000 g for 1 h to obtain pellet fractions and soluble fraction (supernatant of 100 000 g). The soluble fraction was concentrated 50-fold using a centrifugal filter unit (10 000 MW cut off) (Ultrafree-MC, Millipore) followed by dialysis against 20 mM glycylglycine–NaOH (pH 7.5). The debris remaining on the filter papers was rinsed with 1000 mL of the homogenizing medium, followed by 1000 mL of 200 mM NaCl and finally with 1000 mL of Milli-Q water to give the cell wall fraction. For INT determination of each subcellular fraction, ∼20 mg of the cell wall fraction, 50 μL of the homogenate fraction (equivalent to ∼20 mg bark) or 1/200 volume fractions (equivalent to 1 g bark tissues) of the 200 g pellet, 11 000 g pellet, 100 000 g pellet and concentrated soluble fraction were dispensed into each tube (containing 0.5 mL water).