All oligonucleotides (HPLC purified) were synthesized and modified by Sangon Biotechnology Co., Ltd. (Shanghai, China). D-PBS buffer, 20× PBS buffer, 5× TBE buffer, RNase-free ddH2O, Tween-20, Bovine Serum Albumin (BSA), HRP-labeled Streptavidin (SA-HRP), and EL-TMB Chromogenic Reagent kit were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). ASFV p30 recombinant protein, p30 monoclonal antibody, porcine reproductive and respiratory syndrome virus (PRRSV) recombinant N protein, staphylococcus aureus enterotoxin type A recombinant protein (SEA), and pig immunoglobulin G (IgG) were purchased from Aiyi Biotechnology. (Nanjing, China). 6× His tag peptides were bought from Synpeptide Biotechnology Co., Ltd. (Nanjing, China). Swine serum was sourced from Solarbio Science & Technology Co., Ltd. (Beijing, China). NHS (N-hydroxysuccinimide)-Activated Magnetic Beads (NHS-MB) were purchased from Beaver Biosciences Inc. (Suzhou, China). Taq PCR Master Mix and ChamQ Universal SYBR qPCR Master Mix were purchased from Vazyme Biotechnology Co., Ltd. (Nanjing, China). The 96-well polystyrene microtiter plate (MaxiSorp) was bought from Thermo Fisher Scientific (Shanghai, China). ASFV-positive freeze-dried serum, porcine negative serum national reference was purchased from the National Center of Veterinary Culture collection (CVCC) (Beijing, China).
Pbs buffer
Manufactured by Sangon
Sourced in China
PBS buffer is a widely used aqueous solution commonly employed in various laboratory applications. It serves as a standard buffer solution, maintaining a stable pH to support the optimal functioning of biological samples and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
Tween 20, by Sangon (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Tbe buffer, by Sangon (1 mentions)
Rnase free ddh2o, by Sangon (1 mentions)
El tmb chromogenic reagent kit, by Sangon (1 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (1 mentions)
Taq pcr master mix, by Vazyme (1 mentions)
Dopamine hydrochloride, by Merck Group (1 mentions)
Ammonia solution, by Sinopharm (1 mentions)
Absolute ethanol, by Sinopharm (1 mentions)
Type 70 ti rotor, by Beckman Coulter (1 mentions)
Creatinine urinary assay kit, by Cayman Chemical (1 mentions)
Optima xpn 100, by Beckman Coulter (1 mentions)
Dnase 1, by Transgene (1 mentions)
Ezup column bacteria genomic dna purification kit, by Sangon (1 mentions)
Myd88, by Proteintech (1 mentions)
Runx2, by Abcam (1 mentions)
P ikb, by Abcam (1 mentions)
Gapdh primary antibody, by Cell Signaling Technology (1 mentions)
19 protocols using pbs buffer
1
ASFV p30 Sandwich ELISA Protocol
2
Dopamine Hydrochloride-Loaded EPL Synthesis
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Dopamine hydrochloride was supplied by Sigma‐Aldrich (Shanghai, China). EPL (Mw 3000–5000) was supplied by Zhengzhou Bainafo Bioengineering Co., Ltd (Zhengzhou, China). Ammonia solution and ethanol absolute were provided by Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). The 20× PBS buffer was purchased from Sangon Biotech Co., Ltd (Shanghai, China). Deionized water (DI, Millipore Milli‐Q grade) was used in all experiments. Other solvents and reagents were analytical grade and directly used without further purification.
3
Isolation and Preservation of Urinary Extracellular Vesicles
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Briefly, approximately 50 ml first-void morning urine sample was collected during the day of the saline infusion test. Urine sample (10 ml of urine sample was directly used for the measurement of creatinine content using a creatinine [urinary] Assay Kit, 500701-96, Cayman, USA), and the remaining sample was treated with 3 ml PMSF (10 mM) and 50 μl leupeptin (1 mg/ml) before freezing at -80°C (16 (link), 17 (link)). Urinary EVs were isolated via ultracentrifugation, as previously described by Salih et al. (18 (link)). All centrifugations were performed in an ultracentrifuge (Optima XPN-100, Beckman Coulter, USA) with an angle rotor (Type 70 Ti Rotor, Beckman Coulter, USA) at 4°C. Briefly, frozen urine was quickly thawed at 32°C and vortexed for 90 s. The urine was first centrifuged at 17,000×g for 15 min to remove high-density particles. The supernatant obtained above was ultracentrifuged at 200,000×g for 90 min to obtain a gel-like precipitate and the new supernatant was discarded. This gel-like pellet was resuspended in 1× PBS buffer (Sangon Biotech, China), and then ultracentrifuged at 200,000 × g for 90 min to obtain a new pellet containing EVs. The pellet was resuspended in 160 μL 1× PBS buffer and froze at -80°C after aliquoting.
4
Sugarcane Leaf DNA Extraction
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Approximately 5 g of sugarcane leaves were cut into small pieces using scissors and ground to obtain homogenate in 1 × PBS buffer (Sangon Biotech Co., Ltd., Shanghai, China). The homogenates were placed in 50 ml centrifuge tubes and centrifuged at 12,000 rpm for 5 min. The supernatant was discarded, and the pellet was resuspended in 50 ml of 1 × PBS buffer; this step was repeated thrice. The suspension was sequentially filtered through 100, 70, 40, 10, and 5 μm filters (Erwu Industrial Co., Ltd., Shanghai, China). The filtrate was centrifuged at 12,000 rpm for 5 min, and the supernatant was discarded; 20 μl of DNase I (3 units/μl) (TransGen Biotech Co., LTD, Beijing, China), 20 μl of 10 × DNase I Reaction Buffer, and 200 μl of ddH2O were added to the pellet and mixed well. Next, the pellet was incubated at 37°C for 10 min, and then 40 μl of EDTA (25 mmol/L) was added and incubated at 65°C for 10 min. The obtained solution was centrifuged at 12,000 rpm for 5 min, the supernatant was discarded, and the pellet was used to extract DNA using an Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech Co., Ltd., Shanghai, China), according to the manufacturer's instructions. Three biological replicates were performed.
5
Nanoparticle Tracking Analysis of OMVs
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The size distribution and concentration of outer membrane vesicles were measured by nanoparticle tracking analysis (NTA) (Particle Metrix, Meerbusch, Germany). The vesicle samples were appropriately diluted using 1× PBS buffer (Sangon Biotech) to measure the particle size and concentration. NTA measurements were recorded and analyzed at 11 positions. A polystyrene particle of 100 nm was used to calibrate the ZetaView system.
6
Protein Expression Analysis in Cells
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Cell extracts were cleaned with cold PBS buffer (Sangon Biotech, Shanghai, China), prepared with RIPA buffer (Beyotime, Shanghai, China), and supplemented with the protease inhibitor (Cocktail, MCE, USA), and protein concentrations were quantified using the BCA protein assay kit (Beyotime) according to the manufacturer's protocols. The protein lysate was separated via 10% SDS-PAGE (Solarbio, Beijing, China). Protein samples were transferred to PVDF membranes (Millipore, USA) and blocked with 5% nonfat milk (Solarbio, Beijing, China). Then, protein samples were incubated overnight at 4°C with EZH2 (CST, #5246), H3K27me3 (CST, #9733), p-p65 (CST, 3033s), p-IKB (Abcam, ab133462), TLR4 (Proteintech, 66350-1g), MyD88 (Proteintech, 23230-1-AP), Runx2 (Abcam, ab236639), OCN (Abcam, ab133612), and GAPDH primary antibodies (Cell Signaling Technology, Danvers, MA, USA). After washing, protein samples were incubated with secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) for 1 h. Finally, ECL kit (Beyotime) was used to assess protein bands.
7
Magna RIP Assay for Profiling NEAT1, miR-20b, and STAT3
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The Magna RNA-binding protein immunoprecipitation kit (Millipore, Sigma) was used to perform the RIP assay, according to the manufacturer's instructions. Briefly, cells (1×107) were centrifugated at 3,000 × g at 4°C for 10 min and resuspended with RIPA buffer (100 µl; Sigma-Aldrich; Merck KGaA) containing protease and RNase inhibitors. Cell lysates (100 µl) were incubated with human anti-Ago2 antibody (5 µg; Cell Signaling Technology, Inc.) or negative control IgG (5 µg; ProteinTech Group, Inc.) coated magnetic beads (50 µl; Thermo Fisher Scientific, Inc.) at 4°C overnight, while shaking to aid digestion of proteins and the precipitated RNA was isolated. Beads were washed twice using PBS buffer (Sangon Biotech Co., Ltd.), and the mixture was centrifuged at 2,500 × g for 10 min at 4°C. RNA was treated with proteinase K for 30 min at 55°C and extracted using TRIzol (Thermo Fisher Scientific, Inc.). The relative expression levels of NEAT1, miR-20b and STAT3 were determined via RT-qPCR analysis.
8
Immunofluorescence Staining of SF1 Protein
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Briefly, cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and then washed twice with PBS buffer (Sangon Biotech, Cat No. B548117), followed by permeabilization with 0.1% Triton X-100 (Sigma-Aldrich, Cat No. 9036-19-5) for 15min. Next, cells were washed twice with PBS and blocked using antibody diluent (DAKO, Cat No. s3022) for 1h at room temperature. Later, cells were incubated with primary antibody against SF1 (1:100, Proteintech, Cat No.18658-1-AP) at 4°C overnight, followed by YSFluor 594-conjugated secondary antibodies (1:500, Yeasen Biotechnology, Cat No. 34212ES60). Nuclei were stained with 4,6-diamidino-2 phenylindole (DAPI) and wells were mounted using DAPI Fluoromount-G (SouthernBiotech, Cat No. 0100-20).
9
Argonaute-2 RNA Immunoprecipitation
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The RIP assay was performed using an EZ-Magna RIP Kit (EMD Millipore) according to the manufacturer's instructions. NP cell lysates were lysed with RIPA buffer (Beyotime Institute of Biotechnology) for 5 min at 4°C. Antibodies including anti-Argonaute 2 (anti-Ago2; cat. no. ab186733; 1:50; Abcam) and anti-Immunoglobulin G (anti-IgG; cat. no. 12-370; 1:100; EMD Millipore) were incubated with protein A/G magnetic beads (Pierce; Thermo Fisher Scientific, Inc.) for 1 h at 4°C. Then, cell lysate was mixed with the beads to incubate for 4 h at 4°C. Beads were washed twice using PBS buffer (Sangon Biotech Co., Ltd.), and the mixture was centrifuged at 2,500 × g for 10 min at 4°C. RNA was purified with 150 µl proteinase K buffer (Roche Diagnostics) and extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), and the immunoprecipitated RNA was detected via RT-qPCR as aforementioned.
10
CTVT Tissue Biopsy and Molecular Diagnosis
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All experimental procedures were conducted in compliance with the ethical approvals (certificate numbers: SYDW-20130420-74 and SMKX-20170301-154) granted by the Kunming Institute of Zoology. In brief, CTVT tissue biopsies were collected during anesthesia-assisted surgery at veterinary animal hospitals in Kunming, China. The collected tissues were then preserved in RNAlater Stabilization Solution (Invitrogen, Thermo Fisher Scientific Inc.) containing CTVT-KM1, CTVT-KM2, CTVT-KM3, CTVT-SZ3, CTVT-KM23, CTVT-KM24, CTVT-KM25 and CTVT-KM26 tissues or PBS buffer (Sangon Biotech Shanghai Co., Ltd.) for CTVT-KM11 tissue as detailed in Table S1 . PCR was employed to confirm the CTVT-specific LINE-MYC genomic rearrangement for diagnostic purposes.11 (link),86 (link) Details of the primers and PCR programs can be found in Table S7 .
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