Microfuge 18 centrifuge

Unlabeled and fluorescently labeled (80% unlabeled and 20% Alexa Fluor 647 NHS ester-labeled; Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) microtubules were polymerized from 4 mg ml⁻¹ porcine tubulin for 2 h at 37 °C in BRB80 (80 mM PIPES, 1 mM EGTA, 1 mM MgCl₂, pH 6.9) supplemented with 1 mM MgCl₂ and 1 mM GMPCPP (Jena Bioscience, Jena, Germany). The polymerized microtubules were centrifuged for 30 min at 18,000 × g in a Microfuge 18 Centrifuge (Beckman Coulter, Brea, CA) and the pellet was resuspended in BRB80 supplemented with 10 µM taxol (BRB80T). For microtubules used in experiments involving cohesive tau islands, a polymerization mixture of 25% DMSO, 20 mM MgCl l₂ and 5 mM GTP in BRB80 was prepared on ice and 1.25 µl of the mixture was added to 5 µl of 4 mg ml⁻¹ porcine tubulin. Microtubules were polymerized for 30 min at 37 °C. Subsequently, 100 µl BRB80T was added prior to centrifugation and resuspension as described above.

Blood was sampled terminally by severing the caudal peduncle and collecting blood directly into three heparinized haematocrit tubes. Two of the haematocrit tubes were centrifuged (micro-haematocrit centrifuge; Hawksley, Sussex, UK) for 3 min at 5000 g and haematocrit (H_CT) was measured as the proportion of red blood cells in whole blood. Blood from the remaining haematocrit tube was aliquoted into two 1.5 mL Eppendorf tubes for haemoglobin and methaemoglobin concentration determination. Haemoglobin concentration ([H_B]) was determined spectrophotometrically at 405 nm and quantified against a standard curve of known [H_B] using a Sigma-Aldrich haemoglobin assay kit (MAK115; St Louis, USA). Mean corpuscular haemoglobin concentration (MCHC) was calculated as [H_B] × 10/H_CT. Methaemoglobin concentration was determined by diluting 20 μL of whole blood in 1 mL of 34-mM phosphate buffer at pH 7.3. The haemolysate was centrifuged (microfuge^®18 centrifuge, Beckman Coulter, Brea, USA) for 3 min at 12 000 g, and the absorbance (DU800 spectrophotometer, Beckman Coulter, Brea, USA) was measured at 560, 576 and 630 nm, following published protocols (Benesch et al., 1973 ). Plasma N and N were quantified using a colorimetric assay kit (kit number 780001; Cayman Chemicals, Ann Arbor, USA) following the manufacturer’s instructions. All assays were run in duplicate.

Samples taken from the dissolution vessel were centrifuged at 13,000 rpm (14,300 rcf) for 10 min using a Microfuge^®18 Centrifuge (Beckman Coulter, Palo Alto, CA, USA). Then, the supernatant was measured using a Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK) with the Dip cell ZEN1002. The dispersant was water, and the samples were equilibrated at 37 °C before being measured using the 173° backscatter with automatic measurement duration in triplicate.

To detect the presence of Mycoplasma spp. or Chlamydia spp., one swab from each eye was placed in a 2 ml tube containing 400 μl of PBS and 1 mM EDTA, thoroughly vortexed and stored at −80°C until DNA extraction. For DNA extraction, each sample was thawed and thoroughly vortexed, swab was removed and the solution was transferred to a 1.5 ml tube containing Phase Lock Gel Light (Eppendorf, Hamburg, Germany). Four-hundred μl of phenol/chlorophorm (Invitrogen) were added and mixed by repeated inversion. After 10 min of incubation at RT, the tube was centrifuged (Microfuge 18 Centrifuge, Beckman Coulter) at 14000 rpm for 10 min at room temperature (RT). The acqueous phase was removed and transferred to a new eppendorf tube. Four-hundred μl of isopropanol and 40 μl of 3 M, pH 4.8, potassium acetate were added and mixed by repeated inversion. After 10 min of incubation at RT, the tube was centrifuged at 14000 rpm for 10 min, the surnatant was removed and the pellet gently washed with 500 μl of 70% ethanol. After centrifugation at 14000 rpm for 5 min the ethanol was completely removed and the pellet was resuspended in 50 μl of sterile deionized water. A 5 μl aliquot of this DNA suspension was used for PCR amplification.

The freeze-dried green tea extract (see Materials) was suspended in ultrapure water (50 mg in 1.6 mL) and centrifuged for 30 min at 13,000× g (Beckman Microfuge 18 Centrifuge) at room temperature. The sediment was washed twice by the addition of 200 µL of water, followed by centrifugation as above for 10 min. The three supernatants were then collected, thus generating the “water-soluble fraction”. In order to determine the relative content of the water-insoluble fraction, the sediment was washed twice by the addition of 200 µL of water, followed by centrifugation as above for 10 min. The residual water on the pellet was removed by freeze/vacuum rotary evaporation followed by a drying step at 60 °C for 1 h. The actual concentration (mg/mL) of the water-soluble fraction was determined by subtracting the weight of the dried water insoluble pellet from the weight of the starting material.