The toxic strain of the freshwater cyanobacterium A. circinalis used in this study is a nonaxenic strain TA04. The field sample of A. circinalis was collected at the Tullaroop reservoir, Victoria, Australia, and the TA04 strain was one of single-trichome isolates prepared by Negri et al. [33 (link)]. A. circinalis (TA04) was provided by Dr. Susan Blackburn, CSIRO, Australia, and cultured in CB’ medium [see, ref.12, SI] (30 mL). The cells were harvested by filtration using a glass fiber filter (GA100, 1.0 μm, Advantec, Tokyo, Japan), suspended with 2.0 mL of 0.5 M AcOH and sonicated three times for 30 s on ice. Then, the resulting solutions were centrifuged at 20,000× g for 5 min at 4 °C. The supernatants were filtered through a Cosmospin filter H (0.45 μm, Nacalai Tesque, Kyoto, Japan). A part (200 μL) of the filtrate was adjusted to pH 7–8 using 2 M NH3 aqueous, and loaded on an activated charcoal column (100 μL vol.) (for chromatography, 63–300 µm >40%, Wako Pure Chemical Industries, Ltd.). After the column was washed with water (0.2 mL), PST were eluted with AcOH/EtOH/H2O (5:50:45, v/v/v, 1 mL). The solvent was removed using N2 gas, and the resulting residue was resuspended with 100 μL of 0.05 M AcOH.
Ga 100
Manufactured by Advantec
Sourced in Japan
The GA-100 is a laboratory instrument designed for gas analysis. It is capable of detecting and measuring various gas compositions with high precision. The core function of the GA-100 is to provide accurate and reliable gas analysis data for research, quality control, and environmental monitoring applications.
Automatically generated - may contain errors
Lab products found in correlation
C020a047a, by Advantec (4 mentions)
P42a angle rotor, by Hitachi (3 mentions)
C045a047a, by Advantec (3 mentions)
Himac cp80wx ultracentrifuge, by Hitachi (2 mentions)
13pet tube, by Hitachi (2 mentions)
P40st swing rotor, by Hitachi (2 mentions)
Cosmospin filter h, by Nacalai Tesque (1 mentions)
Himac cp80wx, by Hitachi (1 mentions)
Mx 307, by TOMY (1 mentions)
8 protocols using ga 100
1
Extraction and Purification of Paralytic Shellfish Toxins from Cyanobacterium
2
Cellular DNA Synthesis Assay with SE and SCC
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To examine cellular DNA synthesis (cell proliferation), we used the [3H]-thymidine incorporation assay. MCF-7 cells (3 × 103 cells/well) were seeded onto 96-well plates. After 24 h, cells were cultured in the presence of various concentrations of SE (0.1–1000 μg/mL) or SCC (0.25–250 μg/mL) for 24 h. Each culture was pulsed with 1.85 kBq of [3H]-thymidine for 2 h. To measure radioactivity, cell lysates were prepared by the addition of 100 μL of deionized water for 5 min. The lysates were added dropwise onto a glass filter (GA-100, Advantec, Tokyo, Japan) to adsorb cellular DNA, and they were then vacuumed and washed with 5 mL deionized water. Radioactivity of the glass filter was quantified using a liquid scintillation counter.
3
Milk Serum Extraction by Ultracentrifugation
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Defatted milk (500 mL) was subjected to UC using a P42A angle rotor (Hitachi Koki, Tokyo, Japan) in a Himac CP80WX ultracentrifuge (Hitachi Koki) at 12,000×g for 1 h at 4°C, after which the middle layer was collected, and the lower slush portion along with the pellet was discarded. This fraction was again subjected to UC at 35,000×g for 1 h, after which the middle layer was collected and subjected to another round of UC at 75,000×g for 3 h at 4°C. The supernatant was collected and filtered using 1.0-, 0.45-, and 0.2-μm filters (GA-100, C045A0474, and C020A047A, respectively; Advantec, Tokyo, Japan) to remove remaining MFGs and debris and obtain milk serum (whey).
4
Isolation and Purification of Milk sEVs
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Isolation and purification of milk sEVs were carried out as described previously [15 (link),16 (link)] with slight modifications. Briefly, after removing the milk fat by centrifugation at 2000× g for 20 min using a centrifuge (MX-307, Tomy Seiko, Tokyo, Japan), defatted milk was preheated at 37 °C for 10 min. For efficient isolation of milk sEVs, acetic acid was added (finally 1%), and casein was removed by centrifugation at 5000× g for 20 min. The whey was filtrated by using 1.0, 0.45, and 0.2 μm pore-size filters (GA-100, C045A047A, and C020A047A, Advantec, Tokyo, Japan). Further, milk sEVs were concentrated from the whey by ultracentrifugation (UC) at 100,000× g at 4 °C for 1 h using a P42A angle rotor (Hitachi Koki, Tokyo, Japan) in a Himac CP80WX ultracentrifuge (Hitachi Koki). The supernatant was discarded and the bottom pellet was resuspended with phosphate-buffered saline (PBS) up to 10 mL into a 13PET tube (Hitachi Koki). The UC was carried out twice again at 100,000× g at 4 °C for 1 h using a P40ST swing rotor (Hitachi Koki) and the milk sEVs pellet was resuspended with 100 µL of PBS for further use.
5
Isolation of Milk-Derived Small Extracellular Vesicles
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For the isolation of milk sEV from BLV-infected and uninfected cattle, we followed the procedure previously described by Yamauchi et al.40 (link) and Rahman et al.41 (link) with slight modifications. Importantly, after defatting of milk, milk sEV were purified using acetic acid followed by sequential filtration through 1.0, 0.45, and 0.2 μm filters (GA-100, C045A047A, and C020A047A, Advantec, Tokyo, Japan). Subsequently, milk sEV were concentrated by ultracentrifugation (UC) at 100,000 × g at 4 °C for 1 h using a P42A angle rotor (Hitachi Koki, Tokyo, Japan) in a Himac CP80WX ultracentrifuge (Hitachi Koki). After the first UC, the supernatant was discarded and the pellet was resuspended with PBS up to 10 ml into a 13PET tube (Hitachi Koki) followed by another UC at 100,000 × g at 4 °C for 1 h using a P40ST swing rotor (Hitachi Koki). sEV pellet was collected and resuspended with 100 µl of PBS for further use.
6
Sperm DNA Synthesis Quantification
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Sperm suspensions (1 × 104/well) dispensed into a 96 well plate were treated with TiNP (as described above) and incubated with 1.85 kBq of [3H]-thymidine for 3 h. To measure the radioactivity in sperms, sperm lysate was prepared by addition of 5% SDS for 15 min on ice. The lysate was dripped onto the glass filter (GA-100, Advantec, Tokyo, Japan) to adsorb sperm DNA, vacuumed, and washed with water 3 times. Radioactivity of the glass filter was quantified by the liquid scintillation counter (Tri-Carb A5110TR, PerkinElmer Japan, Yokohama, Japan).
7
Quantification of Cyanotoxin Intermediates
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The preliminary culture (150 mL) was mixed with 150 mL of fresh CB’ medium to start the culture. A 30 mL culture of A. circinalis (TA04) was harvested every other day until day 10. The cells and the medium were separated by filtration using the glass fiber filter (GA100, 1.0 μm, Advantec, Tokyo, Japan). Analytical samples of the cells were prepared according to our previous report33 (link)34 (link). Analytical samples of the medium were prepared from the filtrates of GA100. A part of the filtrate (8 mL) was loaded on a Cosmosil 140C18-OPN (100 mg) packed in a glass pipette, which had been conditioned with MeOH and water, then washed with 2 mL of water. Int-A’ (3 ), Int-C’2 (4 ), and Cyclic-C’ (7 ) were eluted with 2 mL of MeOH/H2O/AcOH (50:45:5, v/v/v). The eluate was concentrated in vacuo and redissolved with 400 μL of 0.5 M AcOH. Compounds 3 , 4 , and 7 were quantified using the column-switching LC-MS/MS (MRM) method using API200 mass spectrometer33 (link)34 (link). The recoveries of 3 , 4 , and 7 were 48%, 37%, and 73%, respectively, for this procedure, determined by the spiked medium. The final concentrations of the intermediates were calculated by considering the above recovery rates.
8
Milk Extracellular Vesicle Isolation
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Milk sEV were isolated as described previously (Rahman et al. 2019 (link)(Rahman et al. , 2021b;; Yamauchi et al. 2019) (link). Following the defatting of milk, whey (milk serum) was purified using acetic acid followed by sequential filtration through 1.0, 0.45, and 0.2 µm pore-size filters (GA-100, C045A047A, and C020A047A, Advantec).
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