E701, E703, E709, E403, SCS15, SCS17, and SCS19 are sampling stations in the South China Sea. Water samples (E701, E703, E709, and E403) were collected in September 2011. Samples of SCS15, SCS17, and SCS19 were collected in May 2013 (Figure 1 ). Water samples at each station were collected and 1000 mL of seawater was filtered with 0.22 µm pore size filters (47 mm in diameter, Millipore Corp., Bedford, USA) at low vacuum pressure to collect prokaryotic cells. Each sample was prepared in three replicates. After filtration was performed, the membranes were immediately frozen in liquid nitrogen and then stored at −20°C until DNA extraction was conducted in our laboratory.
Pore size filter
Manufactured by Merck Group
Sourced in United States
Pore size filters are laboratory equipment designed to separate and filter particles based on their size. These filters provide a physical barrier that allows the passage of liquids or gases while retaining particles larger than the specified pore size. The pore size can be adjusted to meet the specific needs of the application.
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Lab products found in correlation
Polybrene, by Merck Group (2 mentions)
0.22 μm filter, by Merck Group (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Fluostar optima microplate reader, by BMG Labtech (1 mentions)
Liposome 2000, by Thermo Fisher Scientific (1 mentions)
Dansyl chloride, by Merck Group (1 mentions)
Saturated sodium bicarbonate, by Merck Group (1 mentions)
2 m sodium hydroxide, by Merck Group (1 mentions)
Murine cxcl9, by Thermo Fisher Scientific (1 mentions)
Pspax2, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Dsred rab9 wt, by Addgene (1 mentions)
Pgl3 basic plasmid, by Promega (1 mentions)
E2695, by Waters Corporation (1 mentions)
Ca no3 2 4h2o, by Sinopharm (1 mentions)
17 protocols using pore size filter
1
Microbial Diversity in South China Sea
2
Sampling and Filtration of A. sanguinea Bloom
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Samples were collected along the Xiamen coast at sites within (A1; N 24°35′53.40′′, E 118°9′29.67′′) or near (H1; N 24°36′56.31′′, E 118°9′15.92′′) the A. sanguinea bloom (Fig. 1 ), during the summer of 2011. Samples were collected each day from 31 July to 5 August as described previously25 (link). Briefly, three replicate water samples (20 l) were collected from each site and filtered through 5-μm diameter pore-size filters (Millipore, US) to remove A. sanguinea and other large algae. The total free-living bacteria in the filtrate were then collected with 0.22-μm filters (Millipore, US) and stored at −70 °C until analysis.
3
Isolation of Cyanophage S-SRP02 from Freshwater
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Cyanophage S-SRP02 were isolated from viral concentrates collected from freshwater lake as described above. Briefly, 450 ml of sample water was filtered through 0.2 μm (Nucleopore) pore size filters and then concentrated through ultrafiltration at 5,000 g for 15 min with centrifugal unit cut-off of 100-KDa-MW (Amicon® Ultra-15 Centrifugal Filter Units; Millipore). Viral concentrates were serial-diluted before adding to exponentially growing cultures of host cyanobacteria in a 96-well plate and incubating at 28°C under low irradiance (20 μmol photons m–2s–1) with a 12-h/12-h light/dark cycle for 14 days. A more than 50% decrease in host population, as measured by flow cytometry using a CytoFLEX flow cytometer (Beckman Coulter Inc., Brea, CA, United States), indicated culture lysis. The discriminator was set on Forward scatter height (FSC-H) and Allophycocyanin height (APC-H), and samples (1 mL) were analyzed at a rate of 30 μL/min. 1.0 mm FluoSpheres microsphere beads (Thermo Fisher Scientific Inc., United States) were added for absolute counting. After three rounds of extinction dilution (Nagasaki and Yamaguchi, 1997 ) in 96-well microtiter plates, a clonal cyanophage isolate was obtained in the well of highest dilution rate.
4
Double-Layer Plaque Assay for Phage Detection
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As previously described [26 (link)], double-layer plaque assays were conducted. The cell cultures (OD600 = 0.3) were mixed with 4 mL of melted 0.7% LB agar at 50 °C and overlaid on nutrient agar plates. Following the solidification of the upper layer, 10 μL of the samples, pre-filtered through 0.22 μM pore size filters (Millipore Corporation, Bedford, MA, USA), was dropped onto the plate and incubated at 37 °C overnight for the detection of phages. The presence of plaques indicates that the strain is sensitive to phages. Plaques were counted to estimate the amount of phage particles in the sample.
5
Bacteriocin Activity against Monophasic S. Typhimurium
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For this assay, the higher inhibitory LAB able to significantly inhibit monophasic S. Typhimurium in the co-culture method was used. A volume of 20 ml culture growing in MRS broth at 37 °C was obtained and centrifugated at 12,000 g for 10 min at 4 °C. Supernatant was neutralized to pH 6.5 with NaOH and filter-sterilised through 0.22 μm pore size filters (Millipore). The bacteriocinogenic activity of the cell-free supernatant was determined in Mueller-Hinton plate agar disk diffusion. The method was adapted from the previously described by Lee et al. [57 (link)]. Monophasic S. Typhimurium was inoculated on the surface and 50 μl of LAB supernatant was spotted onto the previously inoculated plate. To identify a dose-response effect, different volumes of the supernatant (50, 100 and 200 μl) were concentrated to 50 μl and were impregnated in a blank disk and placed also in the Mueller- Hinton agar plate and the diameter of inhibition was quantified. In all these expermients, filtered supernatant was obtained the same day as bacterial growth was made.
6
Hypoxia-conditioned media from cancer stem cells
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In order to obtain the CdM, CSCs were seeded as single cells at a density of 10,000 cells/cm2 in an ultra-low adherence 75 cm2 flasks with serum-free DMEM/F-12 and with supplements (mammospheres culture media detailed above). CSCs mammospheres were starved overnight by washing them twice with serum and supplement free DMEM/F-12 media, then centrifuged at 300 rpm for 10 min. Finally, the media was renewed without serum and without supplements for 8 h for intermittent and 72 h for the continuous hypoxic condition. The resultant CdM from where CSCs grown under hypoxia or normoxia were collected, centrifuged at 300 rpm for 10 min to remove cellular components and filtered through 0.2 µm pore-size filters (Millipore, Billerica, MA, USA). CdM from flasks under normoxic conditions were used as a control. Aliquots of the CdM were stored at −80 °C before being used. CdM were collected from eight sources, from CSCsNOR, and from CSCsHY P grown under the hypoxic shots (INTR.10, INTR.20, INTR.30, INTR.40, CONT.5, CONT.10, or CONT.15 ) and were utilized in successive experiments as discussed subsequently.
7
Microbial Community DNA Extraction
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Water samples (500 mL) were filtered through 5-μm diameter pore-size filters (Millipore, US) to remove particle attached cells. The filtrate was filtered again through 0.22-μm diameter pore-size filters as soon as the samples were taken to the laboratory. Filters were stored at −70°C until analysis (the 5-μm diameter pore-size filters were taken for a comparison study with DGGE). DNA extraction was performed as previously described2 . DNA quality was assessed using 260/280 nm and 260/230 nm ratios with a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, US). Final DNA concentrations were quantified with PicoGreen34 (link) using a FLUOstar Optima microplate reader (BMG Labtech, Germany).
8
GFP-Labeling of Rat Adipose Stem Cells
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HEK293T cells were cultured to 70% confluence in 10 cm dish and then transfected with 10 μg FG12 plasmid carrying green fluorescent protein (GFP) gene and 5 μg of each VSVG, REV, and PRRE packaging plasmids by liposome 2000 (Invitrogen). After 12 h, the medium was refreshed completely. After 60 h, the supernatants were collected and filtered through 0.45 μm pore-size filters (Millipore, Boston, MA). For labeling rASCs, 5 × 105 cells were seeded in a 10 cm culture dish for a day, and then, the medium was replaced with fresh medium plus virus-containing supernatant and 8 μg/mL polybrene (Sigma), followed by 12-hour incubation. GFP-labeled rASCs were sorted by flow cytometry and used for autotransplantation.
9
Dansylation Method for Bile Acid Analysis
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Derivatization of BAs was conducted according to the method described by Eerola, et al. [39 (link)]. One milliliter of extract or standard solution prepared as aforementioned was mixed with 200 μL of 2 M sodium hydroxide and 300 μL of saturated sodium bicarbonate (all from Sigma-Aldrich). Two milliliters of dansyl chloride (Sigma-Aldrich) solution (10 mg/mL) in acetone were added to the mixture and incubated at 40 °C for 45 min. The residual dansyl chloride was removed by adding 100 μL of 25% ammonium hydroxide and incubating for 30 min at 25 °C. Using acetonitrile, the mixture was adjusted to a final volume of 5 mL and centrifuged at 3000× g for 5 min. After filtration using 0.2 μm pore-size filters (Millipore), the filtered supernatant was kept at 4 °C until further analysis using HPLC.
10
Transwell Assay for T Cell Migration
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For transwell assays, the bottom wells were filled with 600 μl of T cell media with or without 100 ng/ml murine CXCL9 (Peprotech). 5 × 105 activated MigR control, Meso-CAR or FAP-CAR T cells were suspended in 100 μl T cell media and plated on the upper chamber of the transwell with 3 μm pore size filters (Millipore). After 4 hours of incubation at 37 °C, migrated T cells collected from the bottom wells were quantified using a cell counter.
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