Exosome isolation reagent (from cell culture media) was purchased from Thermo Fisher scientific (4478359, China). PKH67 Green Fluorescent Cell Linker Kit for General Cell Membrane Labeling (MINI67-1KT, China) and PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling (PKH26GL-1KT, China) were purchased from Sigma. Lipopolysaccharide (LPS, Escherichia coli 0111: B4, L4391), β‐glycerophosphate (G9422), L‐ascorbic acid 2‐phosphate (49752), and dexamethasone (D1756) were from Sigma, China. Antibodies used in this study were rabbit polyclonal antibody against alkaline phosphatase (ALP) (ab108337, Abcam), CD63 Antibody (sc-5275, SANTA CRUZ Biotechnology), CD81 Antibody (sc-166029, SANTA CRUZ Biotechnology), calnexin ( 2679 T, cell Signaling Technology), Grp94 (20292 T, cell Signaling Technology), and Lamin A/C (4777 T, cell Signaling Technology). Secondary antibody used for immunofluorescence staining was fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (H + L) Secondary Antibody (31635, Thermo Fisher Scientific, China).
Cd63 antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The CD63 antibody is a laboratory reagent used to detect the CD63 protein, which is a marker of exosome and lysosomal membranes. The antibody can be used in various experimental techniques, such as Western blotting, flow cytometry, and immunohistochemistry, to study the expression and localization of the CD63 protein.
Automatically generated - may contain errors
Lab products found in correlation
Cd81 antibody, by Santa Cruz Biotechnology (2 mentions)
Dexamethasone d1756, by Merck Group (1 mentions)
Pkh26 red fluorescent cell linker kit for general cell membrane labeling, by Merck Group (1 mentions)
Ab108337, by Abcam (1 mentions)
Calnexin, by Cell Signaling Technology (1 mentions)
Pkh67 green fluorescent cell linker kit for general cell membrane labeling, by Merck Group (1 mentions)
Fitc conjugated goat anti rabbit igg h l secondary antibody, by Thermo Fisher Scientific (1 mentions)
Grp94, by Cell Signaling Technology (1 mentions)
Lamin a c, by Cell Signaling Technology (1 mentions)
Facscalibur cytometer, by BD (1 mentions)
Cd9 antibody, by Abcam (1 mentions)
Tsg101, by Abcam (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Vimentin, by Proteintech (1 mentions)
Hsp70, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Cd133, by Proteintech (1 mentions)
Nanog, by Proteintech (1 mentions)
Gm130, by Cell Signaling Technology (1 mentions)
α sma, by Cell Signaling Technology (1 mentions)
8 protocols using cd63 antibody
1
Exosome Isolation and Membrane Labeling
2
Exosome-Bound MV Isolation and Characterization
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Pre-enriched MVs were incubated with exosome – Human CD63 Isolation Dynabeads (Life Technologies, Cat. No. 10606D) overnight at 4°C. Bead-bound MVs were isolated by magnetic separator. CD63 antibody (Santa Cruz, Cat. No. sc-31214) and Alexa Fluor 488 conjugated second antibody were added for incubation in 1 hour at room temperature one after another. Labeled MVs were analyzed with a FACSCalibur cytometer (Becton-Dickinson) using the FCS Express V3 software.
3
Exosome Characterization by TEM and Immunoblotting
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The size and morphology feature of exosomes were examined by transmission electron microscopy (TEM) (TEM, Phillip CM120, 60 kV). Protein were extracted from exosomes resuspension solution with RIPA lysis buffer and separated on 8% polyacrylamide gel before transferring to a nitrocellulose filter membrane. The blotting membrane was blocked with skim milk and incubated with CD9 antibody (1:1,000 dilution, Abcam, Cambridge, UK)/CD63 antibody (1:200 dilution, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA)/TSG101 (1:500 dilution, Abcam, Cambridge, UK) overnight. After TBST washing, secondary HRP-conjugated antibody was added for 1 h (1:5,000 dilution, HuaAn Biotechnology Inc., Hangzhou, China). The proteins were finally detected using chemiluminescence (Thermo Scientific, Rockford, IL, USA).
4
Serglycin-knockdown in Myeloma Cells
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RPMI 8226, OCIMy5, and CAG human myeloma cells were cultured as described previously [18 (link)]. SRGN-KD and control myeloma cells were engineered as described previously [18 (link)]. Serglycin and clathrin antibodies were obtained from Sigma and Abcam, respectively. CD63 antibody from Santa Cruz.
5
Immunoblotting of Exosomal Proteins
6
Immunofluorescence Staining of PD-L1 in Prostate Cancer Cells
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For Immunofluorescence staining of PC3 or DU145 cells, cells transfected with pcDNA3.1‐eGFP‐PD‐L1 were seeded and cultured overnight, then fixed with 4% paraformaldehyde for 30 min. After blocking with 2% BSA, cells were incubated with CD63 antibody (1:100; Santa Cruz; sc5275) overnight at 4°C, followed by goat anti‐mouse Alexa Fluor 594 (1:500; Invitrogen; A11005) secondary antibody incubation at room temperature for 1 h. The nuclei were stained with DAPI. Samples were imaged using an Olympus microscope at 60× magnification.
LNCaP cells were co‐cultured with exosomes‐PD‐L1eGFP derived from DU145 or PC3 cells and fixed with 4% paraformaldehyde for 30 min. The nuclei were stained with DAPI. Samples were imaged using an Olympus microscope at 60× magnification.
LNCaP cells were co‐cultured with exosomes‐PD‐L1eGFP derived from DU145 or PC3 cells and fixed with 4% paraformaldehyde for 30 min. The nuclei were stained with DAPI. Samples were imaged using an Olympus microscope at 60× magnification.
7
Protein Expression Analysis by Western Blot
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Western blot assays to detect the protein level in cells were performed according to reported protocols 41 (link). The protein concentrations were tested by BCA Protein Assay Kit (Pierce, USA). β-catenin, vimentin, α-SMA, HSP70, CD63, GM130, Sox2, Oct4, Nanog, CD44, CD133 and GAPDH antibodies were used in the study. β-catenin, α-SMA, HSP70, CD63, and GM130 antibodies (1:1000) were purchased from Cell Signaling Technology (MA, USA). CD63 antibody (1:1000) was purchased from Santa Cruz (USA), and vimentin, Sox2, Oct4, Nanog, CD44 and CD133 antibodies (1:500) were purchased from Proteintech Group (Rosemont, USA). The enhanced chemiluminescence reaction was used to detect the protein bands.
8
Exosome Protein Profiling by Western Blot
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After isolating the protein with radioimmunoprecipitaion assay (RIPA) lysis buffer (Beyotime), separation with a 4-20% precast gel (Willget, Shanghai, China) was completed. The protein was then transferred to a nitrocellulose membrane (PALL, New York, NY, USA). Then, the sections were incubated with CD9 antibody (1:1,000, Santa Cruz, Santa Cruz City, CA, USA), CD63 antibody (1:500, Santa Cruz) and CD81 antibody (1:500, Santa Cruz) at 4℃ overnight. After washing, the membrane was incubated with goat anti-mouse IgG (H+L) secondary antibody (1:20,000, Santa Cruz) for 2 hours (h). Finally, the protein band was visualized with ECL Star (Beyotime).
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