For the immunoprecipitation of exosomes and their subsequent analysis by fluorescence-activated cell sorting (FACS), 4 μm-diameter aldehyde/sulphate latex beads (Interfacial Dynamics) were incubated with purified anti-CD9 or anti-CD24 (BD Biosciences) or anti-ADAM-10 (Abcam, UK) antibodies at 22°C overnight at gentle agitation, as previously described. For FACS analysis, 30 μg of exosomes were incubated with 3 × 105 anti-CD9, anti-CD24 or anti-ADAM-10 beads in 150 μl of PBS at 4°C overnight at gentle agitation. The reaction was stopped by incubation of exosome-bead complexes in 0.2 M glycine for 30 min. The exosome-bead complexes were washed twice with FACS buffer (3% exosome depleted FBS in PBS). The beads-bound exosomes were then incubated with human IgG (BD Biosciences) at 4°C for 30 min with subsequent washing with FACS buffer and incubation with fluorescein-conjugated anti-CD9, anti-CD24, anti-CD63, anti-CD81 or isotype antibodies (all from BD Biosciences) for 40 min at room temperature at gentle agitation. The complexes were washed twice, suspended in 300 μl of FACS buffer and analyzed by flow cytometry using FACS Canto II (BD Biosciences) and FACS Diva 6.1 Software. The median fluorescence intensity (MFI) of the stained exosomes was analyzed as compared to isotype control.
Isotype antibodies
Manufactured by BD
Sourced in United States
Isotype antibodies are a class of immunoglobulin molecules that are used for various laboratory and research applications. They function as specific binding agents for target antigens, enabling detection, isolation, and analysis of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 percp cy5, by BD (2 mentions)
Cd8 fitc, by BD (2 mentions)
Cd16 bv421, by BD (2 mentions)
Cd14 pe, by BD (2 mentions)
Facsaria 2 flow cytometer, by BD (2 mentions)
Ultra low attachment 6 well plate, by Corning (2 mentions)
Facscalibur ow cytometer, by BD (2 mentions)
Cd13 pe, by BioLegend (2 mentions)
Hla g, by Abcam (2 mentions)
Cd105 pe, by R&D Systems (2 mentions)
Hla dr, by BD (2 mentions)
Hla abc, by BD (2 mentions)
Anti cd24, by BD (1 mentions)
Anti adam10, by Abcam (1 mentions)
Anti cd63, by BD (1 mentions)
Anti cd9, by BD (1 mentions)
Anti cd81, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Efv drug powder, by Merck Group (1 mentions)
Pepsin, by Merck Group (1 mentions)
8 protocols using isotype antibodies
1
Exosome Immunoprofiling by FACS
2
Breg Cells Suppress CD8+ T Cells
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The sorted Breg cells and non-Breg cells were cultured in vitro with CpG (bacteria-derived oligodeoxynucleotides; eBioscience, Waltham, MA, USA; 10 μg/ml) for 48 h to stimulate the production of IL-10 [13 (link)]. Then, they were, respectively, cocultured with CD8+ T cells at a 1 : 3 ratio for 72 hours. The anti-IL-10 blocking antibody was added to the coculture of Breg cells and CD8+ T cells. For the control, CD8+ T cells were incubated with anti-IL-10 blocking antibody or PBS. And at the last 5 h of culture, PMA, ionomycin, and BFA were added. For intracellular staining of perforin and Granzyme B, cells were first incubated with anti-CD8-PE-CY7 monoclonal antibody at 4°C for 20 minutes and then with anti-human perforin-PE and Granzyme B-APC antibody (BD) at 4°C for 30 min in the dark. Simultaneously, the incubation with isotype antibodies (BD) was performed. After that, the cells were collected and analyzed with flow cytometry.
3
Evaluating mEV Cell Entry Dynamics
4
Optimized Caco-2 Cell Permeability Assay
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Pluronic F127 (F127), maleic anhydride, toluene, pyridine, diethyl ether, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, pepsin, and high-performance liquid chromatography (HPLC) water were used as received from Sigma-Aldrich Co. (St Louis, MO, USA). The tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] was purchased from Promega Corporation (Fitchburg, WI, USA). Transwell polycarbonated filter plate with insert (24 well with pore diameter 0.4 µm) was purchased from Corning Costar (New York, NY, USA). Anti-GP2 antibodies were purchased from MBL (Nagoya, Japan). Isotype antibodies were purchased from BD Biosciences (San Jose, CA, USA). Human colon carcinoma Caco-2 cell line (passages 20–30) and human Burkitt’s lymphoma Raji B line (passages 30–40) were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and used within the passage mentioned in the parentheses. EFV drug powder and all other chemicals were obtained from Sigma-Aldrich Co.
5
Evaluating mEV Cell Entry Dynamics
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To evaluate the propensity of mEVs to enter different cell types in the peripheral blood and particularly monocytes, we performed an mEV entrance experiment. Peripheral blood mononuclear cells (PBMCs) were enriched from a healthy donor as previously described[11 (link)]. In an ultra-low attachment 6-well plate (Corning, Kennebunk, ME, USA), 6 × 106 PBMCs were cultured in RPMI-1640 supplemented with 10% FBS at 2 × 106 /mL. Ten μg of mEVs transfected with Cy-5 labelled antagomir-155 were added. Cells were incubated for 24 h and sampled at 2 h, 8 h and 24 h. Cells were immediately stained with CD8-FITC, CD4-PerCP-Cy5.5, CD14-PE, CD16-BV421 (all from BD, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) for 30 min at room temperature (RT). Cells were then washed and fixed in 2% PFA for 10 min at RT, followed by washing. Flow cytometry analyses were performed on a FACSAria II flow cytometer (BD) and data were analyzed with FlowJo software (BD). At least 10,000 cells were collected for each sample. Gates were set using isotype antibodies (BD).
6
Flow Cytometric Analysis of T Cells
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Flow cytometric analysis was performed as previously described28 (link). In brief, T cells were harvested from transwell chambers and washed twice before incubating with fluorescein-conjoncted antibodies or isotype antibodies (BD Biosciences) for 20 min at 4 °C avoiding from light. Cells were then washed twice prior to perform on Accuri C6 flow cytometers (BD Biosciences) and analyzed by CFlow Plus software (BD Biosciences).
7
Phenotyping PD-MSCs Using Flow Cytometry
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PD-MSCs PRL-1 (5 passages) were stained with monoclonal antibodies speci c for the following proteins to phenotype cell-surface antigens: CD34 (PE), CD90 (PE), HLA-ABC (FITC), HLA-DR (FITC) (BD Bioscience, San Diego, CA, USA), CD13 (PE) (BioLegend, San Diego, CA, USA), CD105 (PE) (R&D Systems, Minneapolis, MN, USA), and HLA-G (FITC) (Abcam). After staining, cells were washed in PBS and treated with appropriate isotype antibodies (BD Biosciences, San Jose, CA, USA). Cells were analyzed by a FACSCalibur ow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). For each sample, at least 10,000 events were acquired.
8
Phenotyping PD-MSCs Using Flow Cytometry
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PD-MSCs PRL-1 (5 passages) were stained with monoclonal antibodies speci c for the following proteins to phenotype cell-surface antigens: CD34 (PE), CD90 (PE), HLA-ABC (FITC), HLA-DR (FITC) (BD Bioscience, San Diego, CA, USA), CD13 (PE) (BioLegend, San Diego, CA, USA), CD105 (PE) (R&D Systems, Minneapolis, MN, USA), and HLA-G (FITC) (Abcam). After staining, cells were washed in PBS and treated with appropriate isotype antibodies (BD Biosciences, San Jose, CA, USA). Cells were analyzed by a FACSCalibur ow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). For each sample, at least 10,000 events were acquired.
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