Purification of exosomes was confirmed by PS Capture Exosome ELISA Kit (Wako) according to the manufacturer’s instructions. Antibodies used as capture reagents were as follows: anti-CD63 contained in the above kit, and anti-CD81, anti-CD9 and anti-Hsp70 contained in the EXOAB-KIT-1 (System Biosciences). For detection of the latter three antigens, we used a goat anti-rabbit IgG HRP-conjugated secondary antibody.
Ps capture exosome elisa kit
Manufactured by Fujifilm
Sourced in Japan
The PS Capture Exosome ELISA Kit is a laboratory product designed for the detection and quantification of exosomes. It utilizes a sandwich ELISA method to capture and measure exosomes in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Exoab kit 1, by System Biosciences (3 mentions)
Sandwich elisa, by Fujifilm (1 mentions)
Multi analyte elisarray, by Qiagen (1 mentions)
Moc87, by Abcam (1 mentions)
Xfluor4geniospro, by Tecan (1 mentions)
Biotin labeling kit nh2, by Dojindo Laboratories (1 mentions)
Imark microplate absorbance spectrophotometer, by Bio-Rad (1 mentions)
Ps capture exosome elisa kit streptavidin hrp, by Fujifilm (1 mentions)
Softmax program, by Molecular Devices (1 mentions)
7 protocols using ps capture exosome elisa kit
1
Exosome Purification and Characterization
2
Quantifying Alzheimer's Biomarkers in Samples
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The levels of Aβ in Transwell culture medium or tissue supernatants were determined with a sandwich ELISA (Wako, Osaka Japan). For tissues, hippocampi or cortices were homogenised in 4 M guanidine-HCl buffer (pH 8.0) with an ultrasonic homogeniser (Taitec, Saitama, Japan) and incubated at room temperature for 3 h. The homogenates were diluted with 0.1% bovine serum albumin in PBS and centrifuged at 16,000 × g for 20 min. The resulting supernatants were then applied to the ELISA to measure Aβ levels or to Multi-Analyte ELISArray (Qiagen, Hilden, Germany) to measure levels of the proinflammatory cytokines TNF-α, IL-6 and IL-1β according to the manufacturer’s instructions.
EV-associated NCAM-1 and L1CAM were measured by ELISA from MyBiosource (San Diego, CA) and LifeSpan BioSciences (Seatle, WA). Total exosome levels were analysed using PS Capture Exosome ELISA Kit with anti-CD9 antibody (Wako) according to the manufacture’s instruction. For detection of exosomal amyloid fibrils, conformational specific anti-amyloid fibril antibody (mOC87, abcam) and HRP-conjugated anti-rabbit IgG were used as detection antibodies in PS Capture Exosome ELISA Kit.
EV-associated NCAM-1 and L1CAM were measured by ELISA from MyBiosource (San Diego, CA) and LifeSpan BioSciences (Seatle, WA). Total exosome levels were analysed using PS Capture Exosome ELISA Kit with anti-CD9 antibody (Wako) according to the manufacture’s instruction. For detection of exosomal amyloid fibrils, conformational specific anti-amyloid fibril antibody (mOC87, abcam) and HRP-conjugated anti-rabbit IgG were used as detection antibodies in PS Capture Exosome ELISA Kit.
3
Exosome Quantification via Tim-4 Binding
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T-cell immunoglobulin domain and mucin domain-containing protein 4 (Tim-4), which specifically binds to phosphatidylserine, was immobilized in a 96-well plate using a PS Capture™ Exosome ELISA Kit (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The EVs prepared above (1.9 × 108 in 10 μL) were added to the plate and allowed to stand for 1 h at room temperature. After washing 3 times with 300 μL of washing buffer from the PS Capture™ Exosome ELISA Kit, 50 ng of rabbit anti-ESAT-6 antibody in 100 μL of the washing buffer was added to each well. After standing for 1 h at room temperature, the wells were washed three times with 300 μL of the washing buffer, followed by the addition of FITC-labeled goat anti-rabbit IgG antibody (50 ng, in 100 μL washing buffer). The mixture was incubated for 1 h, and the wells were again washed three times with 300 μL of the washing buffer. The washing buffer (100 μL) was added to the wells, and the fluorescence intensity was measured using a microplate reader (XFluor4GENiosPro; TECAN, Zürich, Switzerland) with an excitation wavelength of 485 nm and an emission wavelength of 520 nm.
4
Exosome Biomarker Profiling in Urine
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Before ELISA, thawed urine samples were centrifuged at 1,200 × g for 20minat 4°C to remove cell debris and urine salts. ELISA was performed using the PS Capture Exosome ELISA Kit (Cat# 298-80601 FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) according to the manufacturer’s instructions. Absorbance at 450 nm was measured using the iMark microplate Absorbance Spectrophotometer (Bio-Rad). Antibodies used as capture reagents were as follows: anti-CD63 antibody contained in the abovementioned kit, and mouse monoclonal anti-CD9 antibody (clone12A12, Shionogi, Osaka, Japan), rabbit polyclonal anti-MGAM antibody (Cat# 22195-1-AP, Proteintech, Chicago, IL, USA), mouse monoclonal anti-MUC1 antibody (Cat# sc-7313, Santa Cruz, Dallas, TX, USA), mouse monoclonal anti-Poliovirus receptor (PVR) antibody (Cat# 66913-1-Ig, Proteintech), mouse monoclonal anti-PKD2 antibody (Cat# H00005311-M01, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD133 (PROM1) antibody (Cat# 66666-1-lg, Proteintech), and rabbit monoclonal anti-CD90/Thy1 antibody (Cat# ab92574, Abcam, Cambridge, UK). For the latter three antibodies, biotin was conjugated to them using the biotin labeling kit-NH2 (Dojindo, Kumamoto, Japan), according to the manufacturer’s instructions.
5
Quantification of Exosomal Markers in Serum
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Serum levels of EVs expressing either platelet markers (CD41, CD61) or EV marker (CD63) were quantified using the PS Capture Exosome ELISA Kit (Streptavidin HRP) (Wako Pure Chemical Industries, Ltd) as described previously [9 (link)]. Briefly, 100-fold diluted sera (100 μl) were incubated with Tim4-coated plates for 2 h at room temperature (RT). After washing, a biotinylated antibody for each marker protein was incubated for 1 h at RT. Then, horseradish peroxidase (HRP)-labeled streptavidin was incubated for 2 h at RT followed by washing. After incubation with 3,3’,5,5’-tetramethylbenzidine solutions for 30 min, a stop solution was applied. An optical density (OD) was measured at 450 nm as the dominant wavelength and 620 nm as the secondary wavelength. The following antibodies were biotinylated and used for sandwich assays: anti-CD41 mAb (1 μg/mL, clone No. 745201; R&D Systems), anti-CD61 mAb (1 μg/mL, clone No. 256809; R&D Systems), anti-CD63 (component of PS Capture Exosome ELISA Kit; Wako).
CA19-9 was measured using Accuraseed CA19-9 reagents (FUJIFILM Wako Pure Chemical Co., Osaka, Japan).
CA19-9 was measured using Accuraseed CA19-9 reagents (FUJIFILM Wako Pure Chemical Co., Osaka, Japan).
6
Quantifying Exosome Levels via ELISA
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The amounts of exosomes in supernatants were measured using the PS Capture™ Exosome ELISA Kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan) according to the manufacturer's instructions. Briefly, supernatants were added to 96-well plates coated with a mouse anti-human CD63 antibody and incubated at room temperature for 1 h. After washing, the wells were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG Ab at room temperature for 1 h. The optical density at 450 nm was determined with the Softmax program (Molecular Devices).
7
Isolation and CKAP4 Quantification in SEVs
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Fifty milliliters of CM from cultured cells was subjected to sequential centrifugation steps of 2,000 Â g and 10,000 Â g; the supernatant was concentrated to 1 mL using an ultrafiltration unit (Vivaspin 20). CKAP4 in SEVs was measured using a PS Capture Exosome ELISA Kit (Wako) according to the manufacturer's recommendations. One-hundred microliters of biotin-labeled anti-CKAP4 mAb (5A6-17A11; 100 ng/mL) was used as the detection antibody.
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