The sampling was carried out monthly from July to December 2016 (except for November) in the southern coastal area of Korea (detailed description in Supplementary Methods) on board an R/V Jangmok I or a fishing boat. Seawater from the bottom layer was collected approximately 1 m above the seafloor. The middle-layer sample was collected at half depth of each sampling site. The surface-water samples were collected with a clean bucket, while samples from the middle and bottom layers were collected with a 5-L PVC Niskin sampler (General Oceanics, Miami, FL, USA). Once collected, approximately 1–2 L of water samples were immediately filtered through 20-µm-pore-size Nuclepore® filters (Whatman®, Clifton, NJ, USA), then through 3.0-µm-pore-size Nuclepore® filters (Whatman®, Clifton, NJ, USA), and finally through 0.22-µm-pore-size SterivexTM filter units (MilliporeTM, Bedford, MA, USA) to collect MP (>20 µm), NP (3 to 20 µm), and FL (0.22 to 3 µm) bacteria, respectively. The filtration time was no longer than 30 minutes. We replaced the filters when the filtration speed slowed down. The filters were stored at −80 °C until DNA extraction. A total of 267 samples were collected over half a year.
Nuclepore filter
Manufactured by Cytiva
Sourced in United Kingdom, United States
Nuclepore filters are high-performance membrane filters used for a variety of laboratory applications. They feature uniform, precisely defined pore sizes and a smooth, flat surface. Nuclepore filters are made of polycarbonate and are inert, resistant to most chemicals, and suitable for a wide range of filtration and separation tasks.
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Lab products found in correlation
Malvern zeta sizer 4 spectrometer, by Malvern Panalytical (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
E myco plus kit, by iNtRON Biotechnology (1 mentions)
Nuclepore membrane filter, by Cytiva (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Chems, by Avanti Polar Lipids (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Tla100 rotor, by Beckman Coulter (1 mentions)
Falcon tube, by BD (1 mentions)
Nuclepore membrane, by Cytiva (1 mentions)
Tgfb3, by R&D Systems (1 mentions)
Affi gel beads, by Bio-Rad (1 mentions)
Dialyzed fbs, by Thermo Fisher Scientific (1 mentions)
Synergy 2 plate reader, by Agilent Technologies (1 mentions)
Hiseq 2000 system, by Illumina (1 mentions)
Experion, by Bio-Rad (1 mentions)
20 protocols using nuclepore filter
1
Coastal Microbial Community Profiling
2
Propagation and Culture of T. gondii
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Type I (GT1, ATCC 50853) and type III (CTG, ATCC 50842) strains of T. gondii were grown as tachyzoites in human foreskin fibroblasts (HFFs; obtained from the laboratory of John Boothroyd, Stanford University) as described previously (70 (link)). Parasites for all experiments were harvested shortly after natural egress, purified by passage through a 20-gauge needle, and separated from host cell debris using 3.0-µm polycarbonate Nuclepore filters (Whatman). RAW 264.7 macrophage cells (ATCC TIB-71) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies) with 10% fetal bovine serum (FBS; Life Technologies) and cultured at 37°C and 5% CO2. Bone marrow-derived macrophages (BMDMs) were isolated from the femurs of adult mice, as described previously (71 (link)). BMDMs were harvested in DMEM containing 20% L929 conditioned medium, 10% FBS, and 5% horse serum in a 100-mm by 20-mm untreated polystyrene culture dish (Corning). After a week of culture, the cells were maintained in DMEM containing 10% L929 conditioned medium, 10% FBS, and 5% horse serum. For experiments, BMDMs were rinsed in calcium-magnesium-free phosphate-buffered saline (PBS), harvested by incubation with 1.25 mM trypsin for 20 min, and seeded in DMEM containing 10% FBS. All strains and host cell lines were determined to be mycoplasma negative using the e-Myco Plus kit (Intron Biotechnology).
3
Quantifying cAMP Levels in Dictyostelium
4
Diatom Gene Expression Profiling
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Samples for DNA and RNA analyses were collected on days 0, 2 and 3. DNA samples (300–400 mL) were collected onto 0.2 μm pore size polycarbonate Nuclepore membrane filters (Whatman) with gentle vacuum (< 0.013 MPa), flash frozen in liquid nitrogen and then stored in a deep freezer at −80°C until analysis. Seawater samples (300–400 mL) for RNA analysis were filtered onto 0.2 μm pore size polycarbonate Nuclepore filters (Whatman) with gentle vacuum (< 0.013 MPa) and then stored in 1.5-mL cryotubes previously filled with 0.2 g of muffled 0.1-mm glass beads and 600 μL of RLT buffer (Qiagen) with 10 μL mL−1 β-mercaptoethanol (Sigma-Aldrich). The RNA samples were flash-frozen in liquid nitrogen and stored in a deep freezer at −80°C until analysis. The detailed methodologies of total DNA and RNA extractions were described in Endo et al. [16 ] and Endo et al. [27 ], respectively. The extracted RNA was reverse-transcribed into cDNA using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara). To quantify the diatom-specific rbcL gene (DNA) and its transcripts (cDNA), qPCR and qRT-PCR were performed according to the method of Endo et al. [27 ].
5
Liposome Preparation Methods
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The appropriate lipids were mixed in organic solution and the solvent was evaporated to dryness under a N2 stream. Then the sample was kept under vacuum for 1 h to remove solvent traces. The lipids were swollen in Assay Buffer in order to obtain multilamellar vesicles (MLVs). Large unilamellar vesicles (LUV) were produced from MLV according to the extrusion method described by Mayer et al. (1986) [60 ]. They were subjected to 10 freeze/thaw cycles, and then extruded using a LIPEX Liposome Extrusion System (Evonik Health Care, Essen, Germany) with a 0.1-μm pore size Nuclepore filters (Whatman, 110,605). Small unilamellar vesicles (SUV) were obtained by sonicating MLV with a probe tip sonicator (MSE Soniprep 150, MSE, UK) for 20 min (10 sec on, 10 sec off) on ice. Vesicle size was checked by quasi-elastic light scattering using a Malvern Zeta-Sizer 4 spectrometer (Malvern Instruments, Malvern, UK). LUV had an average diameter of ≈100 nm and SUV average diameter was ≈50 nm. Phospholipid concentration was determined by phosphate analysis [61 ].
6
Liposome Synthesis via Thin-Film Hydration
7
Preparation and Characterization of Liposomes
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The appropriate lipids were mixed in organic solution, and the solvent was evaporated to dryness under an N2 stream. The sample was then kept under N2 stream for an additional 30 min to remove solvent traces. The lipids were swollen in 25 mM Hepes (pH 7.4), 150 mM NaCl, and 1 mM DTT (Sigma-Aldrich) to obtain multilamellar vesicles (MLVs) at 1 mg/ml. LUVs were prepared from MLVs. They were subjected to 10 freeze/thaw cycles and then extruded using 0.2-μm pore size Nuclepore filters (Whatman). SUVs were obtained by sonicating MLVs with a probe tip sonicator (MSE Soniprep 150, MSE, UK) for 10 min (10 s on, 10 s off) on ice. For cosedimentation assays, 43 μg (unless otherwise specified) of liposomes was incubated with 5 or 0.2 μM of the indicated KLCCTD or tetrameric kinesin-1, respectively (100 μl, final volume), for 30 min and spun down at 170,000g in a Beckman TLA-100 rotor. Supernatant and pellet fractions were collected and analyzed by SDS-PAGE and Coomassie stain.
8
Determining Optimal 16S rRNA Concentration for Microbial Diversity Analysis
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Determination of the impact of the starting 16S rRNA concentration on recoverable reads and sample diversity was performed using six water samples collected from the same site at the same time as the gill samples from tilapia and gray mullet from the Nile Delta. Samples were double filtered through 0.4-μm- and 0.2-μm-pore-size Nuclepore filters (Whatman International Ltd., United Kingdom). Filters were stored in Longmire’s solution prior to DNA extraction directly on the filter. 16S rRNA copy number was assessed though qPCR, and a serial dilution was performed for a total count from 108 to 102 for each sample.
9
Preparation of Large Unilamellar Liposomes
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The appropriate lipids (ePC/DOPE/PI/DOG, 33:55:10:2 mol ratio) were mixed in organic solution, and the solvent was evaporated to dryness under a N2 stream. Then, the sample was kept under vacuum for 1 h to remove solvent traces. The lipids were swollen in System Buffer (150 mM NaCl, 50 mM Tris, pH 7.5) in order to obtain multilamellar vesicles (MLVs). Large unilamellar vesicles (LUV) were produced from MLV according to the extrusion method described by Mayer et al. [40 (link)]. They were subjected to 10 freeze/thaw cycles and then extruded through a LIPEX Liposome Extrusion System (Transferra Nanosciences, Burnaby, CA) using 0.05-μm pore size Nuclepore filters (Whatman, 110605). Vesicle size was checked by quasi-elastic light scattering using a Malvern Zeta-Sizer 4 spectrometer (Malvern Instruments, Malvern, UK). LUV had an average diameter of ≈ 80 nm. Phospholipid concentration was determined by phosphate analysis [41 (link)].
10
Saline Lake Water Characterization
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Six Qinghai–Tibetan lakes with different salinities were selected for this study: Erhai Lake (EHL) is a freshwater lake; Qinghai Lake (QHL) and Tuosu Lake (TSL) are saline lakes; Gahai Lake (GHL), Xiaochaidan Lake (XCDL), and Chaka Lake (CKL) are hypersaline lakes [28 (link)]. A sampling cruise was carried out in May 2017. At inshore sites (~10–20 m away from shoreline) of each lake, salinity, pH, and temperature of surface water (~0–10 cm) were measured with portable meters (SANXIN, Shanghai, China). Water samples (~40 ml each) for measurements of total dissolved nitrogen (TDN) and total dissolved phosphorus (TDP) were collected after filtration through 0.22-μm Nuclepore filters (Whatman, UK). Subsequently, about 6-l surface water was collected from each lake into acid-washed and sterilized 2-l polycarbonate bottles (Nalgene, USA). To obtain tDOM, onshore soils around each lake were taken from topsoil layer (0–10 cm) without plant. All samples were kept cold and in the dark during transportation to the laboratory. Samples were stored at 4 °C in the laboratory until further processing.
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