Enhanced paper-based ELISA for simultaneous EVs/exosome isolation and detection using streptavidin agarose-based immobilization

Lee, Junwoo; Kim, Hyerin; Heo, Youhee; Yoo, Yong Kyoung; Han, Sung Il; Kim, Cheonjung; Hur, Don; Kim, Hyungsuk; Kang, Ji Yoon; Lee, Jeong Hoon

doi:10.1039/c9an01140d

Cited by 69 publications

(46 citation statements)

References 45 publications

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“…To isolate exosomes from the M0, M1 and M2 Mφs, exosome-depleted FBS was generated by ultracentrifugation of FBS at 100,000×g for 70 min with an L-80 ultracentrifuge (45 Ti rotor) from Beckman Coulter (Brea, CA, USA) (Hoshino et al, 2015), which removed the bovine exosomes from the FBS. Although we do not have nanoparticle tracking data of FBS before and after ultracentrifugation, we trust that FBS was effectively depleted of exosomes since other studies used similar methods (Kobayashi et al, 2014;Lee et al, 2019). Following a 24-h incubation with or without M1/M2 induction, the culture medium was discarded, and the cells were washed twice with PBS to remove remaining cytokines.…”

Section: Isolation and Characterization Of M0 M1 And M2 Mφ-derived Ementioning

confidence: 99%

Exosomes derived from M0, M1 and M2 macrophages exert distinct influences on the proliferation and differentiation of mesenchymal stem cells

Xia

et al. 2020

PeerJ

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Background Different phenotypes of macrophages (M0, M1 and M2 Mφs) have been demonstrated to play distinct roles in regulating mesenchymal stem cells in various in vitro and in vivo systems. Our previous study also found that cell-conditioned medium (CM) derived from M1 Mφs supported the proliferation and adipogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs), whereas CM derived from either M0 or M2 Mφs showed an enhanced effect on cell osteogenic differentiation. However, the underlying mechanism remains incompletely elucidated. Exosomes, as key components of Mφ-derived CM, have received increasing attention. Therefore, it is possible that exosomes may modulate the effect of Mφ-derived CM on the property of BMMSCs. This hypothesis was tested in the present study. Methods In this study, RAW264.7 cells were induced toward M1 or M2 polarization with different cytokines, and exosomes were isolated from the unpolarized (M0) and polarized (M1 and M2) Mφs. Mouse BMMSCs were then cultured with normal complete medium or inductive medium supplemented with M0-Exos, M1-Exos or M2-Exos. Finally, the proliferation ability and the osteogenic, adipogenic and chondrogenic differentiation capacity of the BMMSCs were measured and analyzed. Results We found that only the medium containing M1-Exos, rather than M0-Exos or M2-Exos, supported cell proliferation and osteogenic and adipogenic differentiation. This was inconsistent with CM-based incubation. In addition, all three types of exosomes had a suppressive effect on chondrogenic differentiation. Conclusion Although our data demonstrated that exosomes and CM derived from the same phenotype of Mφs didn’t exert exactly the same cellular influences on the cocultured stem cells, it still confirmed the hypothesis that exosomes are key regulators during the modulation effect of Mφ-derived CM on BMMSC property.

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Section: Isolation and Characterization Of M0 M1 And M2 Mφ-derived Ementioning

confidence: 99%

Exosomes derived from M0, M1 and M2 macrophages exert distinct influences on the proliferation and differentiation of mesenchymal stem cells

Xia

et al. 2020

PeerJ

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“…With a sufficient volume, the same sample can be analyzed several times to study different targets of interest. In addition, the time-consuming process may be overcome in paper-based ELISA assays [ 15 ] or by pre-enrichment of the sample and subsequent detection using nanocubes with enzyme-like characteristics [ 16 ]. The development of reference materials may enhance ELISA-based approaches, since they may be designed according to the cell origin, surface density of the antigen, etc.…”

Section: Enzyme-linked Immunosorbent Assay (Elisa)mentioning

confidence: 99%

Extracellular Vesicles: Current Analytical Techniques for Detection and Quantification

et al. 2020

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Since their first observation, understanding the biology of extracellular vesicles (EV) has been an important and challenging field of study. They play a key role in the intercellular communication and are involved in important physiological and pathological functions. Therefore, EV are considered as potential biomarkers for diagnosis, prognosis, and monitoring the response to treatment in some diseases. In addition, due to their properties, EV may be used for therapeutic purposes. In the study of EV, three major points have to be addressed: 1. How to isolate EV from cell culture supernatant/biological fluids, 2. how to detect them, and 3. how to characterize and quantify. In this review, we focus on the last two questions and provide the main analytical techniques up-to-date for detection and profiling of EV. We critically analyze the advantages and disadvantages of each one, aimed to be of relevance for all researchers working on EV biology and their potential applications.

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“…The label-free EV microarray assay was used to characterize the EVs derived from the naïve, M1, and M2 macrophages. The concentration of the EVnaïve, EVM1, and EVM2 samples were 2×10 13 EVs/mL. Fig.…”

Section: Characterization Of Evs Release By Different Macrophages Phementioning

confidence: 93%

“…To characterize and calibrate the PC biosensor, we measured the macrophage-derived EVs at four concentrations ranging from 2×10 10 EVs/mL to 2×10 13 EVs/mL. The samples were consecutively diluted in phosphate buffered saline (PBS) by a factor of ten.…”

Section: Hyperspectral Imaging Microscopementioning

confidence: 99%

“…To fully exploit the diagnostic and therapeutic potential, EVs need to be quantified for their membrane proteins with high accuracy, throughput, and efficiency. However, existing EV analysis methods are usually based on time-consuming, expensive immunoblotting or enzyme-linked immunosorbent assay or Western Blotting assays 13,14 . The fluorescence-based EV detection methods using microbeads and magnetic particles have the multiplexing capability but require laborious assays 15 .…”

Section: Introductionmentioning

confidence: 99%

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Hyperspectral imaging-based exosome microarray for rapid molecular profiling of extracellular vesicles

et al. 2021

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Enhanced paper-based ELISA for simultaneous EVs/exosome isolation and detection using streptavidin agarose-based immobilization

Abstract: We suggested the paper-based ELISA using a streptavidin agarose resin-based immobilization. This method reduces assay times (∼2 h), provides strong binding, and retains good sensitivity and linearity.

Cited by 69 publications

References 45 publications

Exosomes derived from M0, M1 and M2 macrophages exert distinct influences on the proliferation and differentiation of mesenchymal stem cells

Exosomes derived from M0, M1 and M2 macrophages exert distinct influences on the proliferation and differentiation of mesenchymal stem cells

Extracellular Vesicles: Current Analytical Techniques for Detection and Quantification

Hyperspectral imaging-based exosome microarray for rapid molecular profiling of extracellular vesicles

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