Spin x centrifuge tube

Saliva was sampled to measure cortisol levels twice daily at 7:30 a.m. and at 6:30 p.m., as described in previous research (Kaplan et al., 2012 ). Animals were not captured by the experimenter and saliva was collected under free-ranging conditions in their home cages. A thin cotton swab (Matsumotokiyoshi Co., Ltd., Chiba, Japan) was used to collect saliva from the marmosets. The marmosets were fully trained to bite cotton swabs in their home cages before the experiments. The cotton swabs were dipped in powdered sugar to ensure that the bites would last sufficiently long (approximately 3–5 min) (Kaplan et al., 2012 ). A 2.0-mL Costar Spin-X centrifuge tube with a nylon filter (0.22 μm) was filled with the swabs and centrifuged at 10,000 rpm for 5 min to extract the liquid portion of the sample. The collected samples were stored in a −80°C freezer until further use. Saliva samples were collected three times per individual, with no two collections occurring in the same month. The duration of the experiment was 3 months, from June to August in 2019. Cortisol levels (μg/dl) were measured using an AIA-360 Automated Immunoassay Analyzer with AIA-pack cortisol test cups (Tosoh Corporation, Tokyo, Japan) and averaged per subject across all saliva collections either in the morning or the evening.

NAD⁺ and NAM extraction and subsequent quantitative analysis were performed as previously described (Yoshino and Imai, 2013 (link)). Briefly, tissue sample (typically 50–70 mg) was homogenized in 500 µL HClO₄ (0.4 M), incubated on ice for 5 min and precipitated by adding 80 µL KOH (0.2 M) with shaking for 2 min. The samples were then centrifuged at 3000 × g, 10 min, 4°C. Supernatant was filtered using Spin-X centrifuge tube (Costar, filter size 0.22 µM) and the samples were stored in −80°C until HPLC measurements. The samples from tissues were subjected to HPLC using a 20 mm × 3.9 mm Sentry Guard column (Nova-Pak C18 bonded silica) connected to a 150 mm × 4.6 mm Atlantis T3 silica-based, reversed-phase C18 columns (Waters Corporation). NAD⁺ and NAM were detected by UV detector and UV absorbance was monitored at 261 nm. Elution of NAD⁺ and NAM from samples was verified and quantified by co-elution with known amounts of NAD⁺ and NAM standards (Sigma-Aldrich).

Three dry N. benthamiana leaf disks (9 cm diameter) were ground and saponified by mixture of ethanol/H₂O/KOH pellets in 9 : 1 : 1 (v/v/w) (1 ml) at 70°C for 1 h. The ethanol was removed by evaporation (1 h, 70°C), and the samples were extracted with 1 ml ethyl acetate/H₂O in 1 : 1 (v/v). The suspensions were centrifuged at 16 000 g for 1 min and the supernatants collected. The supernatants were dried under N₂ and resuspended in 50 μl derivatizing reagent, 1‐(Trimethylsilyl)imidazole‐Pyridine mixture (Sigma‐Aldrich). The samples were incubated at 70°C for 30 min before analysis by GC‐MS analysis. For C. rubella metabolites extraction, c. 30 mg of fresh tissues (leaves or flowers) were ground. Samples were extracted and prepared for GC‐MS as described above. Ground samples used for LC‐MS analysis were extracted with 1 ml ethyl acetate (shaken overnight). The samples were then extracted with 500 μl of water. The supernatants were dried under N₂ and resuspended in 100 μl methanol. The samples were all cleaned via 0.22‐μm nylon filter tube (Spin‐x centrifuge tube; Costar, Cole‐Pamer, St Neots, UK) prior to LC‐MS analysis.

Membranes from 2 to 4 l of photosynthetically grown cells were harvested from discontinuous sucrose gradients, diluted in 20 mM HEPES, pH 7.5 and pelleted at 45 000 rpm (180000 × g) for 2.5 h using a Beckman Type 50.2 Ti rotor at 4°C. Membranes were resuspended to an OD₈₇₅ of 30 and incubated in 0.1–3.0% β‐DDM for 30 min at 4°C. Insoluble material was removed by centrifugation at 20 000 rpm (32 000 × g) in a Beckman JA‐25.50 rotor at 4°C. The supernatant was loaded onto a pre‐equilibrated 150 μl anti‐FLAG M2 affinity resin column. The column was washed with 15 volumes of wash buffer containing 0.04% β‐DDM. FLAG‐tagged and associated proteins were desorbed by the addition to the plugged column of 100 μg FLAG peptide dissolved in 500 μl wash buffer. The resin was transferred to a cryovial and rotated for 1 h at room temperature prior to a 1500 × g centrifugation for 5 min in a Costar Spin‐X centrifuge tube containing a cellulose acetate membrane with 0.22 μm pores to separate proteins from the resin.

M. tuberculosis cells in 30 ml cultures were pelleted by centrifugation, washed once in PBS (Phosphate Buffered Saline) supplemented with 0.05% Tween 80 and the pellets stored at -80°C until further use. Total lysates were obtained by sonication in TBS-T (25 mM Tris pH 7.5, 150 mM NaCl, 0.05% Tween 20), followed by filtration through 0.22 μm filters (Pall Life Sciences). Fifty microliters of Monoclonal Anti-HA Agarose Antibody beads (Sigma-Aldrich) were incubated with approximately 1 mg of bacterial extract in Spin-X centrifuge tubes (Costar) for 4 h at 4°C on an orbital shaker. The resin was washed four times in PBS and the immunoprecipitated material was eluted from the beads in PBS-SDS sample buffer (100 mM Tris HCl pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, 20% glycerol) during a 5 min incubation at 95°C. Immunoprecipitated proteins were analyzed either by mass spectrometry as described [24 (link)] or by immunoblot. A mock (no antibody) control was run in parallel with agarose beads only.