Reactive oxygen species (ROS) were detected using 1 M 2,7-dichlorodihydrofluorescein diacetate (DCFDA) in HMEECs. The cells were then incubated in a humidified 5% CO2 incubator for 30min at 37C after treatment with DCFDA. In contrast, CD63, an EV marker, was observed when the MSCs were incubated in 5% normal goat serum for 1h at RT to prevent non-specific labeling. Anti-CD63 (1:200, ab 118,307, Abcam, MA, USA) was used as the primary antibody for 1h at 4C. The samples were washed with PBS three times for 5min each time, followed by incubation with a secondary antibody, goat anti-rabbit IgG H&L (Alexa Fluor 488; 1:1000, ab150077, Abcam, MA, USA), for 1h at RT. After washing the samples three times for 5min with PBS, they were immobilized with a mounting solution containing DAPI (4,6-diamidino-2-phenylindole). All the samples were observed by confocal microscopy (Carl Zeiss Microscopy GmbH, Jena, Germany), and the images were analyzed using ZEN lite ver. 2.3.
Ab118307
Manufactured by Abcam
Sourced in United States
Ab118307 is a lab equipment product. It is a single-use item designed for use in scientific research. The core function of this product is to facilitate specific experimental procedures. No further details on the intended use or application of this product can be provided in an unbiased and factual manner.
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Lab products found in correlation
Ab205718, by Abcam (3 mentions)
Ab30871, by Abcam (3 mentions)
Confocal microscopy, by Zeiss (2 mentions)
Ab150077, by Abcam (2 mentions)
Goat anti rabbit igg h l alexa fluor 488, by Abcam (2 mentions)
Ab223052, by Abcam (2 mentions)
Ab37168, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Enhanced chemiluminescence reagent, by Solarbio (1 mentions)
Ab155296, by Abcam (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Non fat milk, by Thermo Fisher Scientific (1 mentions)
Sds page, by Beyotime (1 mentions)
Ab92726, by Abcam (1 mentions)
Ab109201, by Abcam (1 mentions)
Mouse anti rabbit igg hrp sc 2357, by Santa Cruz Biotechnology (1 mentions)
Immobilon crescendo western hrp substrate, by Merck Group (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Mini gel apparatus, by Bio-Rad (1 mentions)
Polyvinylidene fluoride (pvdf), by Thermo Fisher Scientific (1 mentions)
9 protocols using ab118307
1
Detecting ROS and EVs in MSCs
2
Extracellular Vesicle Protein Analysis
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RIPA lysis buffer (Beyotime) was applied for extracting the total protein. After measurement of protein concentration, protein (about 40 µg/lane) was subjected to SDS-PAGE (Beyotime). Subsequently, the proteins were transferred to nitrocellulose membranes (Invitrogen), which were then blocked with 5% non-fat milk (Yili, Beijing, China) and incubated with primary antibody overnight at 4°C. The primary antibodies including cluster of differentiation 63 (CD63; 1:1000, ab118307), cluster of differentiation 9 (CD9; 1:500, ab223052), TPD52 (1:5000, ab155296), and GAPDH (1:2000, ab37168) were bought from Abcam (Cambridge, UK). After that, the corresponding secondary antibody (1:4000, ab205718, Abcam) was used for the combination with the primary antibody. At last, the visualization of protein blots was achieved by an enhanced chemiluminescence reagent (Solarbio, Beijing, China).
3
Extracellular Vesicle Protein Analysis
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EVs derived from MSCs were isolated through centrifugation, and the amount of total protein was quantified using the Bradford assay. Protein samples were separated using 10% SDS-PAGE with a mini gel apparatus (Bio-Rad, Hercules, CA, USA) and transferred onto PVDF membranes (T831.1, Merck Millipore, MA, USA). Each membrane was blocked with 3% skim milk in Tris-buffered saline containing 0.05% Tween 20. The primary antibodies anti-CD9 (1:1000, ab92726, Abcam, MA, USA), CD63 (1:1000, ab118307, Abcam, MA, USA), and anti-CD81 (1:1000, ab109201, Abcam, MA, USA) were used as primary antibodies and were incubated overnight at 4C. Mouse anti-rabbit IgG-HRP (sc-2357, Santa Cruz, CA, USA) was used as a secondary antibody for 1h. The bands were visualized using enhanced chemiluminescence according to the manufacturers instructions (Immobilon Crescendo Western HRP substrate, Millipore, Darmstadt, Germany). The band intensities were quantified using the ChemiDoc XRS+System (Bio-Rad). All the samples were developed within 10min to obtain a band.
4
Characterization of Exosomes using TEM and Flow Cytometry
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For exosomes characterization, the exosome samples were subjected to transmission electron microscopy (TEM) and flow cytometry analysis. In TEM procedure, exosome suspension samples were fixed with glutaraldehyde 1%, and then loaded on the carbon grids at room temperature. After drying process, grids were washed twice and stained with uranyl acetate 1% for 10 min at room temperature. Exosomes were imaged using TEM system (Philips BioTwin, CM100, Amsterdam The Netherlands) at 80 kV as described previously [56 (link)]. In flow cytometry analysis, primary anti-CD63 antibody (ab118307, Abcam) was added to 100 μL of each exosome samples and incubated at the 4 °C refrigerator for 2 h. Afterwards, CD63-PE secondary antibody (12-0639-42, eBioscience, San Diego, CA, USA) was added and maintained at 37 °C for 1 h. Data were evaluated using BD FACSCalibur instrument and FlowJo software (version 7.6.1).
5
Western Blot Analysis of Extracellular Vesicle Markers
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Total protein was extracted by RIPA lysis buffer (Beyotime). The protein samples were denatured by heating at 100°C for 3–5 min, and then quantified by BCA protein assay kit (Abcam, Cambridge, UK). After that, protein samples (about 30 μg/lane) were loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Next, the gels were transferred to polyvinylidene fluoride membranes (PVDF; Invitrogen). After blocking with 5% non-fat milk (Sangon Biotech), these membranes were then incubated for 12 h at 4°C with primary antibodies. The primary antibodies including TSG101 (1:500, ab30871), CD63 (1:1000, ab118307), CD9 (1:500, ab223052) B-cell lymphoma-2 (Bcl-2; 1:1000, ab59348), BCL2-associated X protein (Bax; 1:500, ab53154), MSI1 (1:1000, ab154497), and GAPGH (1:2000, ab37168) were purchased from Abcam. Subsequently, these membranes were continuously probed with secondary antibody (ab205718, 1:4000, Abcam). At last, protein bands were detected using enhanced chemiluminescence reagent (Tanon, Shanghai, China).
6
Protein Extraction and Western Blot
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Lysates of irradiated cells were collected using lysis compound (NaCl, Tris–HCl, NP-40 supplemented with protease inhibitor cocktails) for 1 h on ice. Samples were centrifuged at 14,000 rpm for 20 min. Then, the supernatants of all groups were collected, and the total protein concentration was determined applying the Nanodrop system (BioTek). Immunoblots were performed as described previously [48 (link)]. Briefly, 100 µg of total proteins was loaded into each well of SDS-gel electrophoresis and then transferred to PVDF membrane (Polyvinylidene Difluoride; 249 Millipore, Burlington, VT, USA). Next, the PVDF membrane was exposed to primary antibody against human CD63 (ab118307, Abcam) according to manufacturer’s recommendation. HRP-conjugated goat-anti-rabbit IgG antibody (Cell Signaling) was added to samples and kept for 1 h at room temperature. Using ECL system, the relative intensity of immunospecific bands were analyzed via ImageJ software ver.1.44p. β-actin protein (Cell Signaling) was used as endogenous control for western blot normalization.
7
Protein Extraction and Immunoblotting Protocol
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RIPA lysis buffer (KeyGene) was used for extracting total proteins. After measurement of protein concentration, proteins were subjected to SDS-PAGE, followed by transferring onto the PVDF membranes (Millipore, Billerica, MA, USA). After blockage using 5% skim milk (Beyotime, Shanghai, China), these membranes were immunoblotted with primary antibodies at 4°C for 10–14 h. Lastly, the combined signals were detected by an enhanced chemiluminescence kit (KeyGene) after incubation of corresponding secondary antibody. The antibodies including proliferating cell nuclear antigen (PCNA; 1:500, ab18197), Bcl-2 (1:500, ab196495), Bax (1:500, ab32503), Cleaved Caspase 3 (1:500, ab32042), IGFBP3 (1:1000, ab193910), β-actin (1:5000, ab227387), CD63 (1:1000, ab118307), TSG101 (1:500, ab30871), and HRP-conjugated IgG anti-rabbit (1:4000, ab205718) were commercially acquired from Abcam (Cambridge, MA, USA). All experiments were repeated three times.
8
Protein Isolation and Western Blot Analysis of MSCs
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Total protein was isolated from MSCs using radioimmunoprecipitation assay lysis buffer (R0010, Solarbio, Beijing, China) encompassing phenylmethylsulfonyl. Following dissolving in 2× sodium dodecyl sulfate (SDS) loading buffer, 50 μg protein received 5-min boiling at 100 °C. These samples were separated using 10% SDS–polyacrylamide gel electrophoresis and electroblotted to polyvinylidene fluoride membranes. Next, the membranes were incubated with diluted rabbit monoclonal antibody (Abcam) to TSG101 (ab30871, 1:2000), and rabbit polyclonal antibodies (Abcam) to CD63 (ab118307, 1:50), calnexin (ab75801, 1:2000), SALL4 (ab31968, 1:50), MMP-2 (ab37150, 1:100), MMP-9 (ab73734, 1:100), Cle-caspase-3 (ab2302, 1:1000), caspase-3 (ab4051, 1:1000), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, ab8245, 1:10,000) overnight at 4 °C. Subsequently, the membranes underwent 1-h incubation with horseradish peroxidase-tagged secondary antibody to IgG (1:1000). The immunocomplexes on the membrane received visualization with enhanced chemiluminescence reagent (BB-3501, Amersham, Little Chalfont, UK) before quantification of the band intensities with a IS gel image analysis system and an Image J software.
9
Quantifying Cellular Reactive Oxygen Species
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Reactive oxygen species (ROS) were detected using 1 μM 2',7'-dichlorodihydro uorescein diacetate (DCFDA) in HMEECs. The cells were then incubated in a humidi ed 5% CO 2 incubator for 30 min at 37°C after treatment with DCFDA. In contrast, CD63, an EV marker, was observed when the MSCs were incubated in 5% normal goat serum for 1 h at RT to prevent non-speci c labeling. Anti-CD63 (1:200, ab 118307, Abcam, MA, USA) was used as the primary antibody for 1 h at 4°C. The samples were washed with PBS three times for 5 min each time, followed by incubation with a secondary antibody, goat anti-rabbit IgG H&L (Alexa Fluor 488; 1:1000, ab150077, Abcam, MA, USA), for 1 h at RT. After washing the samples three times for 5 min with PBS, they were immobilized with a mounting solution containing DAPI (4,6-diamidino-2-phenylindole). All the samples were observed by confocal microscopy (Carl Zeiss Microscopy GmbH, Jena, Germany), and the images were analyzed using ZEN lite ver. 2.3.
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