The entire dissection procedure was performed in a biological safety cabinet. Before dissection, crabs were washed twice with sterile seawater. All dissection instruments were sterilized over an open flame to eliminate residual DNA and washed with 75% ETOH to prevent cross-contamination. After removing upper carapaces, the digestive gland, gill, stomach, heart and mid-gut from each crab were excised for DNA extraction (Fig 1 ). Total genomic DNA was extracted according to a modified standard phenol–chloroform procedure incorporating a grinding step in liquid nitrogen to mechanically lyse cells [15 (link)]. After extraction, DNA samples of various body parts were denoted as D (digestive gland), S (stomach), H (heart), G (gill), and M (mid-gut). Bacterioplankton from seawater samples were filtered on cellulose acetate membranes (pore size, 0.2 μm; ADVANTEC, Tokyo, Japan). Microbial biomass was removed from membranes by washing with TE buffer (50 mM Tris-HCl, 1 mM EDTA, pH 8.0). Suspensions were collected in microtubes and total DNA was extracted by the same method as described. The DNA from seawater samples was denoted as SW. All DNA samples were stored at -20°C.
Cellulose acetate membrane
Manufactured by Advantec
Sourced in Japan
The Cellulose Acetate Membrane is a porous membrane made from cellulose acetate. It is used for filtration and separation applications in various laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Tff system, by Merck Group (2 mentions)
Ods am 301 column, by YMC (2 mentions)
No 42 filter paper, by Cytiva (1 mentions)
Waters 2996, by Waters Corporation (1 mentions)
Sodium chloride (nacl), by Fujifilm (1 mentions)
Dulbecco s phosphate buffered saline pbs, by Merck Group (1 mentions)
Tween 80, by Merck Group (1 mentions)
Milli q, by Merck Group (1 mentions)
Toc l tnm l, by Shimadzu (1 mentions)
Dr2800, by HACH (1 mentions)
Drb 200, by HACH (1 mentions)
Filter paper number 42, by Cytiva (1 mentions)
Bio dot sf, by Bio-Rad (1 mentions)
11 protocols using cellulose acetate membrane
1
Crab Tissue Sampling and DNA Extraction
2
Concentration of Waterborne Pathogens
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To concentrate pathogenic bacteria (Salmonella) and protozoa (Cryptosporidium), 10 L samples were filtered through 0.45µm pore size nitrate-cellulose filter membranes (Sartorious) and cellulose-acetate membranes with a pore size of 3 µm (Advantec), respectively as described by Ahmed et al. (2008) (link). The filters were immediately placed into 15 ml screw cap centrifuge tubes containing 10 ml Phosphate Buffered Saline (PBS) and vortexed vigorously for 5 min to detach the organisms from the membranes. Samples were then centrifuged at 10,000 rpm for 15 min at 4°C. The supernatant was discarded and the pellet re-suspended in 2 ml of sterile distilled water.
3
Profiling Aquatic Microbial Communities
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Water samples at 3.0, 5.0, and 5.5 m depth were collected from Lake Shunet (54° 25’N, 90° 13’E) on July 21, 2010. The collection procedure was described in our previous research14 . 20 L of water were pumped from each depth into four sterile 5-L polypropylene bottles. Water was filtered through 10-μm plankton net to remove large particles. The water was stored for 3 h in the bottles, and part of the water was then transferred into sterile 2.0-ml screw tubes (SSIbio®) and stored at −80 °C until DNA extraction. The rest of water was concentrated using tangential flow filtration (TFF) system (Millipore) with 0.22-μm polycarbonate membrane filters. The bacteria in the retentate were then retained on cellulose acetate membranes (0.2 μm pore size; ADVANTEC, Tokyo, Japan) and stored at −80 °C until DNA extraction. The physicochemical properties of samples were mentioned in a previous study14 . The pH was 8.1, 7.6, and 6.7; water temperature was 15.5, 9.5, and 7.5 °C; and salinity was 26, 40, and 71 g/L in the 3.0-, 5.0-, and 5.5-m samples, respectively.
4
Freshwater Microbiome Sampling Protocol
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Water samples at 3.0, 5.0, and 5.5 m deep were collected from Lake Shunet (54° 25'N, 90° 13'E) on July 21, 2010. The collection procedure was described in our previous research [12] . Briefly, water was pumped from each depth into sterile containers. Part of the water was transferred into sterile 2.0-ml screw tubes (SSIbio ® ) and stored at -80°C until DNA extraction. The rest of the water was filtered through 10-μm plankton and concentrated using tangential flow filtration (TFF) system (Millipore) with 0.22-μm polycarbonate membrane filters. The bacteria in the retentate were then retained on cellulose acetate membranes (0.2 μm pore size; ADVANTEC, Tokyo, Japan) and stored at -80°C until DNA extraction.
5
Detecting Endozoicomonas in Reef-building Coral Samples
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Permits for coral sampling were received from local governments. In order to detect Endozoicomonas in various reef-building coral samples, 30 healthy coral samples were collected from 3 locations (Table S1 and Fig. S1 ): Kenting (Taiwan, tropical region, 21°56′58.3″N, 120°45′11.9″E), Hemei (Taiwan, subtropical region, 25°05′34.45″N, 121°55′2.06″E), and Kochi (Japan, near temperate region, 32°46′42.95″N, 132°43′56.06″E). The taxonomic affiliations of the collected coral samples belonged to 9 genera and consisted of a robust clade in Hexacorallia (Stylophora and Favia), a complex clade in Hexacorallia (Porites, Euphyllia, Acropora, Isopora, and Montipora), Octocorallia (Heliopora), Zoantharia (Palythoa), and Hydrozoa (Millepora).
All corals and seawater were collected at depths of 5 to 7 m. Coral samples were collected using either bone scissors or a hammer and chisel, rinsed with sterilized seawater, and placed in 99% ethanol for transportation. Duplicate coral sampling was performed on 2 separate colonies of each selected coral genus, owing to limited financial support and sampling times. At each sampling site, 1 L of seawater was collected just before coral sampling, and then filtered through a cellulose acetate membrane with 0.2-μm pores (Advantec, Tokyo, Japan). Sample preparation, bench experiments, and bioinformatic analyses were summarized in a flowchart (Fig. S2 ).
All corals and seawater were collected at depths of 5 to 7 m. Coral samples were collected using either bone scissors or a hammer and chisel, rinsed with sterilized seawater, and placed in 99% ethanol for transportation. Duplicate coral sampling was performed on 2 separate colonies of each selected coral genus, owing to limited financial support and sampling times. At each sampling site, 1 L of seawater was collected just before coral sampling, and then filtered through a cellulose acetate membrane with 0.2-μm pores (Advantec, Tokyo, Japan). Sample preparation, bench experiments, and bioinformatic analyses were summarized in a flowchart (
6
HPLC Analysis of Polyphenol Profiles
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For high-performance liquid chromatography (HPLC) analysis, 1 g of freeze-dried materials was mixed with 20 mL of 800 g kg−1 methanol. The mixture was shaken for 24 hours at room temperature, filtered through a Whatman No. 42 filter paper, and was freeze-dried at −40°C until use. Polyphenols profile of SPLE and GCBE samples was determined according to the method of Jeng et al [15 ]. The polyphenols sample was filtered through a cellulose acetate membrane (0.20 μm, Advantec, Tokyo, Japan), and 10 μL of the filtrate was injected into HPLC (Waters 2996, Waters, Milford, MA, USA) using a ODS (Output Delivery System) column (ODS-AM 301 column, 4.6 × 150 mm, 5-μm particles; YMC, Kyoto, Japan). The temperature was set at 40°C. The mobile phase consisted of water containing 0.2% (v/v) formic acid (A) and methanol (B). Elution was performed with the linear gradient as follows: 2% B from 0 minutes to 15 minutes, 2% to 45% from 15 minutes to 50 minutes, and 45% B from 50 minutes to 65 minutes. The flow rate was 1 mL min−1. Polyphenolic compounds were then compared with the obtained standards.
7
TiO2 Nanoparticle Dispersion Protocols
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P25 TiO 2 nanopowder, which is a mixture of anatase and rutile phases in an approximate 8:2 ratio (Lot#: MKBJ8961V, purity > 99.5%, primary size ~21 nm, specific surface area ~50 m 2 /g), Dulbecco's phosphate-buffered saline (PBS), and Tween 80 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium chloride was purchased from Wako Pure Chemical Industries (Osaka, Japan). Ultrapure water (UPW; Milli-Q, Merck KGaA, Darmstadt, Germany) was autoclaved and filtered through a cellulose acetate membrane (0.20 µm pore size, Advantec, Tokyo, Japan). Disodium hydrogen phosphate (DSP) and Sodium chloride were dissolved in autoclaved UPW and filtered through the aforementioned cellulose acetate membrane. TiO 2 NPs were suspended in five different solutions, namely UPW, 0.2% DSP, PBS, 0.9% saline (S), or S containing 0.05% (w/v) Tween 80 (ST), and the procedure described in subsequent methods was followed. Vehicles used in the present study were selected based on published descriptions for the preparation of NPs (Bihari et al., 2008; Takeda et al., 2009; Kobayashi et al., 2009; Endoh, 2011; Horie et al., 2012; Wu et al., 2014) .
8
Membrane Bioreactor Performance Monitoring
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The mixed liquor suspended solid, temperature, DO, and pH in all three compartments of the MBR as well as the TMP of the membrane module were monitored throughout the operation. The liquid and solid phases of the activated sludges sampled from the first and second compartments were separated by centrifugation (15,300×g, 15 min, 4 °C) and the supernatants were filtered using a cellulose acetate membrane (ø0.20 μm, ADVANTEC, Tokyo, Japan). The TOC and total nitrogen (TN) concentrations in the supernatants and treated effluent were analyzed using a TOC-TN analyzer (TOC-L/TNM-L; Shimadzu, Kyoto, Japan). The COD value was measured with a COD analyzer (DR2800 and DRB200; Hach, CO, USA) and an appropriate kit (TNT820 or TNT821; Hach).
9
Polyphenol Extraction and Profiling
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For the preparation of polyphenol extracts, 1 g of ground materials was mixed with 20 mL of 80% methanol. The mixture was shaken for 24 hours at room temperature, filtered through a Whatman number 42 filter paper, freeze dried at −40°C until use. The polyphenol profile of tested extract was determined according to the method of Kurata et al [18 ]. The polyphenol extract was filtered through a cellulose acetate membrane (0.20 μm; Advantec, Tokyo, Japan), and 10 μL of the filtrate was injected into a high-performance liquid chromatography (HPLC) system (Waters 2996; Newark, Delaware, USA) using an ODS column (ODS-AM 301 column, 4.6 mm × 150 mm, 5 μm particles; YMC, Kyoto, Japan). The temperature was set at 40°C. The mobile phase consisted of water containing 0.2% (v/v) formic acid (A) and methanol (B). Elution was performed with the linear gradient as follows: 2% B from 0 minutes to 15 minutes, 2%–45% B from 15 minutes to 50 minutes, and 45% B from 50 minutes to 65 minutes. The flow rate was 1 mL/minute. Polyphenolic compounds were then compared with the obtained standards.
10
Detecting SOD1-YFP Aggregates
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