Serum samples were collected into serum separator tubes (SST) and centrifuged at 3,000 rpm for 15 min at 4 °C and mixed with 1x PBS (pH 7.4, Welgene, ML008-01). Stool samples were filtered by a cell strainer after dilution with 10 mL of PBS for 24 hours. After the preparation, samples from serum, stool, and urine were centrifuged at 10,000 × g for 30 min at 4 °C to separate supernatant and pellet. As a result, this sample divided into the pellet contained bacterial cells and the supernatant contained EV. The supernatant was sterilized through a 0.22 μm filter to eliminate bacteria and other foreign particles from sample supernatants. The sterilized supernatants were boiled at 100 °C for 40 minutes to extract DNA from bacterial and EV. Again, remaining floating particles and waste were eliminated by centrifugation at 13,000 rpm at 4 °C for 30 minutes. DNA were extracted using a DNA isolation kit (PowerSoil DNA Isolation Kit, MO BIO, USA) from all sample supernatants plus stool sample pellet according to standard protocol.
Ml008 01
Manufactured by Welgene
ML008-01 is a laboratory equipment designed for scientific research and analysis. It is a versatile instrument capable of performing various tasks within a controlled laboratory environment. The core function of this product is to facilitate the measurement and analysis of samples, data, or other relevant materials in a laboratory setting. However, a more detailed and unbiased description cannot be provided without the risk of extrapolation or interpretation.
Automatically generated - may contain errors
Lab products found in correlation
Qiaxpert system, by Qiagen (6 mentions)
Powersoil dna isolation kit, by Qiagen (2 mentions)
45 ti rotor, by Beckman Coulter (2 mentions)
Mobio powersoil dna isolation kit, by Qiagen (2 mentions)
Dneasy powersoil kit, by Qiagen (1 mentions)
Dual luciferase assay system, by Promega (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Nanog, by Abcam (1 mentions)
Alexa fluor 488 or 568, by Abcam (1 mentions)
10 protocols using ml008 01
1
Comprehensive Microbial DNA Extraction
2
Isolation and Characterization of Bacterial Extracellular Vesicles
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Bacterial EVs were isolated from the sera as described previously [21 (link)22 (link)23 (link)]. Male Tg-APPswe/PS1dE9 mice were sacrificed at the age of 8 months and blood was collected from the hearts of sacrificed mice. Collected blood was centrifuged at 1,500 × g for 15 min at 4℃, and sera were separated, frozen and stored at −70℃ until use.
The sera were diluted 1 in 3 with 1x PBS (pH 7.4; ML008-01, Welgene Inc., Gyeongsan, Korea) and centrifuged at 10,000 × g for 1 min at 4℃. Then, the supernatants were collected and filtered through a 0.22-µm filter to remove bacteria and foreign particles. The separated bacterial EVs were boiled at 100℃ for 40 min. They were then centrifuged at 13,000 rpm for 30 min at 4℃, and the supernatants were collected. Bacterial DNA was extracted from the boiled EVs with a PowerSoil DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA USA) in accordance with the manufacturer's instructions. The DNA from the EVs in each sample was quantified with a QIAxpert system (QIAGEN, Hilden, Germany).
The sera were diluted 1 in 3 with 1x PBS (pH 7.4; ML008-01, Welgene Inc., Gyeongsan, Korea) and centrifuged at 10,000 × g for 1 min at 4℃. Then, the supernatants were collected and filtered through a 0.22-µm filter to remove bacteria and foreign particles. The separated bacterial EVs were boiled at 100℃ for 40 min. They were then centrifuged at 13,000 rpm for 30 min at 4℃, and the supernatants were collected. Bacterial DNA was extracted from the boiled EVs with a PowerSoil DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA USA) in accordance with the manufacturer's instructions. The DNA from the EVs in each sample was quantified with a QIAxpert system (QIAGEN, Hilden, Germany).
3
Isolating Mouse Embryonic Fibroblasts
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Mouse embryonic fibroblasts (MEFs) were obtained from wild type C57BL/6J mouse and Disc1-LI C57BL/6J mouse line. Embryos with embryonic day 14.5 were used. Body part of embryos without red blood organs was washed with PBS (Welgene, ML 008-01) and minced with blades. Samples were dissociated further with trypsin-EDTA (HyClone, SH30042.02) and pipetting for 10 min. Complete media (DMEM with 10% FBS and 1% penicillin/streptomycin) was applied to block trypsin activity. Collected cells were centrifuged for 3 min at 1000 rpm and pellets were resuspended with complete media. To dissociate into single cells, cell strainer was applied. Dissociated cells were seeded into culture dishes and media change was conducted after 6 h.
4
Extraction and Quantification of EV DNA
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DNA extraction from EVs was performed as follows4 (link). The serum from each participant was collected in serum separator tubes and centrifuged at 3000 rpm for 15 min at 4 °C. The supernatant was collected and stored at − 80 °C until analysis. Then, the supernatant was mixed with 1 × phosphate-buffered saline (pH 7.4, ML008-01, Welgene, Republic of Korea) and subsequently centrifuged at 10,000× g for 10 min at 4 °C. To isolate EVs, bacteria and foreign particles were thoroughly eliminated through sterilization of the supernatant using a 0.22 μm filter. The separated EVs were boiled for 40 min at 100 °C and centrifuged at 13,000 rpm for 30 min at 4 °C to eliminate the remaining floating particles and waste. EV DNA was extracted using the DNeasy Powersoil Kit (QIAGEN, Germany) and quantified using the QIAxpert system (QIAGEN).
5
Bacterial Extracellular Vesicle Isolation
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In this study, we used ultracentrifugation and microfiltration methods to isolate bacterial EVs46 (link)–49 (link). Blood samples in ethylenediaminetetraacetic acid tubes were collected at admission. The serum was separated by centrifugation (1500×g, 15 min) at 4 °C and stored at − 70 °C until analysis. The serum samples were diluted 1:3 with 1 × PBS (pH 7.4; ML008-01, Welgene Inc., Gyeongsan, Korea) and centrifuged at 10,000×g for one minute at 4 °C. The supernatants were acquired and filtered with a 0.22-μm size to remove foreign particles and bacteria. Separated bacterial extracellular membrane vesicles were boiled at 100 °C for 40 min and then centrifuged at 13,000 rpm for 30 min at 4 °C. The supernatants were then acquired. Bacterial DNA was extracted from the boiled extracellular membrane vesicles with a PowerSoil DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA USA) according to the manufacturer’s protocols. The DNA from the extracellular membrane vesicles in each sample was quantified with a QIAxpert system (QIAGEN, Hilden, Germany)31 (link),45 (link).
6
Extracellular Vesicle DNA Isolation
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Serum samples were collected from the participants using the same method as that used in the previous studies [20 (link),21 (link)]. Blood samples were collected into serum vacuum separator tubes. For each sample, the serum was isolated from the blood, and then the extracellular vesicles (EVs) were collected. To isolate the EVs, the serum was centrifuged at 1500× g for 15 min at 4 °C and diluted in 1× phosphate-buffered saline (PBS, pH 7.4, ML008-01; Welgene, Gyeongsan, Republic of Korea). Thereafter, centrifugation was performed at 10,000× g at 4 °C for 1 min. Then, the supernatant was filtered using a 0.22 μm filter and ultracentrifuged at 150,000× g for 3 h at 4 °C using a 45 Ti rotor (Beckman Instruments, Brea, CA, USA). The obtained pellet, containing the EVs, was diluted in PBS and stored at −80 °C. Thereafter, an isolation kit (MoBio PowerSoil DNA Isolation Kit; Qiagen, Hilden, Germany) was used to obtain the DNA. DNA was extracted from the EVs according to the manufacturer’s instructions. The extracted DNA was quantified using a QIAxpert system (Qiagen).
7
Extraction and Quantification of EV DNA
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We isolated EVs from the serum samples using the differential centrifugation method, as described previously [39 (link)]. In brief, serum samples were centrifuged at 3000 rpm for 15 min at 4 °C, and 100 uL of the supernatant was mixed with 1 × PBS, pH 7.4 (ML 008-01, Welgene, Republic of Korea). The floating particles were sunk through centrifugation at 10,000× g for 1 min at 4 °C. After centrifugation, bacteria and foreign particles were thoroughly eliminated by sterilizing the supernatant through a 0.22-um filter.
To extract the DNA from the EVs’ membranes, EVs separated from serum in the previous steps were boiled for 40 min at 100 °C. To eliminate the remaining floating particles and debris, the supernatant was collected after 13,000 rpm of centrifugation for 30 min at 4 °C. EVs’ DNA was extracted using a DNA isolation kit according to the standard protocol (PowerSoil DNA Isolation Kit, MO BIO, Carlsbad, CA, USA). The DNA from EVs in each sample was quantified by using the QIAxpert system (QIAGEN, Hilden, Germany).
To extract the DNA from the EVs’ membranes, EVs separated from serum in the previous steps were boiled for 40 min at 100 °C. To eliminate the remaining floating particles and debris, the supernatant was collected after 13,000 rpm of centrifugation for 30 min at 4 °C. EVs’ DNA was extracted using a DNA isolation kit according to the standard protocol (PowerSoil DNA Isolation Kit, MO BIO, Carlsbad, CA, USA). The DNA from EVs in each sample was quantified by using the QIAxpert system (QIAGEN, Hilden, Germany).
8
Dual Luciferase Assay Protocol
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Luciferase assay was conducted using Dual Luciferase Assay system (Promega, E1910), according to manufacturer’s instruction. Cells were cultured on 12-well plate and transfected with PEI. Prior to cell prep, cells were washed with PBS (Welgene, ML 008-01). 100 μl of passive lysis buffer (PLB) was applied to each well, and the plate was agitated for 10 min to detach cells from the plate. The lysate was collected and centrifuged for 30 s at 13,000 rpm. The 40 μl of supernatant was transferred to 96-well white plate (Nunc, 136101). 50 μl of luciferase assay buffer II was injected into a well of 96-well plate, and shaking was performed for 1 second. Thereafter, Firefly luciferase activity was measured for 10 s. 50 μl of Stop & Glo Buffer was injected into the same well of 96-well plate, and shaking was performed for 1 s. Thereafter, Renilla luciferase activity was measured for 10 s. Injection of substrates and luminescence detection were programmed and conducted by automatic injector and plate reader (TECAN, infinite M200 PRO).
9
Extracellular Vesicle DNA Extraction
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Subjects who consented to participate in the study were enrolled, and blood samples were collected into vacutainer serum separator tubes. Extracellular vesicles (EVs) were collected from the blood as follows. The serum was centrifuged at 1500× g at 4 °C for 15 min and diluted in 1× phosphate-buffered saline (PBS, pH 7.4, ML008-01; Welgene, Gyeongsan, Republic of Korea). Thereafter, centrifugation was done at 10,000× g at 4 °C for 1 min, followed by filtration using a 0.22 μm filter. Ultraculation was used to obtain EVs. The obtained supernatant was subjected to ultracentrifugation at 150,000× g for 3 h at 4 °C in a 45 Ti rotor (Beckman Instruments, Brea, CA, USA). The obtained EV pellet was diluted in PBS and stored at −80 °C. A DNA isolation kit (MoBio PowerSoil DNA Isolation Kit; Qiagen, Hilden, Germany) were used for DNA extraction from EVs [20 (link)]. The extracted DNA was quantified using the QIAxpert system (QIAGEN, Hilden, Germany).
10
Immunocytochemistry for Cell Characterization
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For immunocytochemistry, the cells were cultured in 4-well dishes until 70∼80% confluency. Thereafter, they were fixed with 4% paraformaldehyde for 30 min at 4℃, washed with phosphate-buffered saline (PBS) (Welgene, ML 008-01), and treated with 0.3% Triton X-100 in PBS for 10 min. Next, the cells were blocked with PBS containing 3% bovine serum albumin and 0.3% Triton X-100 for 30 min at 25℃. The blocked cells were then incubated overnight at 4℃ with the following primary antibodies: Oct4 (1：1,000; Santa Cruz Biotechnology), Nanog (1：1,000; Abcam), Tuj1 (1：1,000; Millipore), GFAP (1：1,000, Abcam), and O1 (1：1,000, eBioscience). The following day, incubated cells were washed thrice with PBS for 10 min and incubated with fluorescently labeled (Alexa Fluor 488 or 568; Abcam) secondary antibodies. The latter were used according to the manufacturer’s specifications. Cells were finally washed and stained with 10 μg/ml Hoechst (Hoechst 33342; Thermo Fisher Scie-ntific, H3570) and 0.3% Triton X-100 in PBS for 2 min at 25℃.
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