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Anti human cd63

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Anti-human CD63 is a laboratory reagent used to detect and measure the expression of the CD63 protein, a marker found on the surface of various cell types, including platelets and exosomes. This product is intended for research use only and its core function is to facilitate the identification and analysis of CD63-expressing cells or vesicles.

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Lab products found in correlation

Annexin 5, by BD (2 mentions) Facscanto analyzer, by BD (2 mentions) Pac 1, by BD (2 mentions) Anti human cd62p, by BD (2 mentions) Anti human cd41b, by BD (2 mentions) Hrp conjugated secondary antibody, by GE Healthcare (1 mentions) Autoradiographic film, by GE Healthcare (1 mentions) Anti mouse igg fitc, by Thermo Fisher Scientific (1 mentions) Cytoflex flow cytometry, by Beckman Coulter (1 mentions) Anti mouse alexa647, by Thermo Fisher Scientific (1 mentions) Thrombin, by Merck Group (1 mentions)

Close spelling variants of this product

Anti-CD63 (45 citations) Anti-human CD63-FITC (clone H5C6, (5 citations) Mouse anti-human CD63 (5 citations) PE mouse anti-human CD63 (4 citations) Anti-human CD63 antibody (3 citations) PE-Cy7-anti-human CD63 (3 citations) Anti-human CD63 (clone H5C6, (2 citations) Mouse-anti-human CD63 primary antibody (2 citations)

6 protocols using anti human cd63

1

Ultrastructural Localization of CD63 and GFP

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Immuno-EM was carried out in the Harvard EM facility. Adult mice (P60) were perfused with 4% paraformaldehyde (PFA) and 0.1% glutaraldehyde. The brain was dissected out and post-fixed in 4% PFA for 2 h and then brain slices (100 μm) were prepared using vibratome. The slices were then quenched, permeabilized, and blocked in blocking buffer (3% bovine serum albumin, 5% normal donkey serum, and 0.1% Triton-X-100) at 4 °C. Anti-human CD63 (BD Pharmingen, #556019), anti-mouse CD63 (MBL, # D263-3) or GFP (Abcam, #6556) antibody was then added and incubated overnight at 4 °C. After wash, slices were incubated with Protein A-gold for 1 h at 25 °C. The images were taken using the JEOL 1200EX transmission electron microscope.
Men Y., Yelick J., Jin S., Tian Y., Chiang M.S., Higashimori H., Brown E., Jarvis R, & Yang Y. (2019). Exosome reporter mice reveal the involvement of exosomes in mediating neuron to astroglia communication in the CNS. Nature Communications, 10, 4136.
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2

Quantification and Western Blot Analysis of Extracellular Vesicle Proteins

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Protein content of DC lysates, EV pellets and 100 K supernatants was quantified as above and the same amount of protein (7 μg) prepared in non-reducing (for CD63) or reducing (for calnexin and osteopontin) conditions. Samples were denaturated at 65 °C for 15 min, resolved on SDS-PAGE 10% polyacrylamide gels (Bio-Rad), and then transferred to nitrocellulose membranes. Membranes were blocked and probed overnight with the primary antibody anti-human CD63 (BD Pharmingen™), anti-human calnexin (Abcam) or anti-human osteopontin (Rockland), followed by HRP-conjugated secondary antibody (GE Healthcare). Membranes were incubated with ECL substrate and chemiluminescent signal detected upon exposure to autoradiographic films (all from GE Healthcare). Full scans of the films are available in Supplementary Fig. S5.
Silva A.M., Almeida M.I., Teixeira J.H., Maia A.F., Calin G.A., Barbosa M.A, & Santos S.G. (2017). Dendritic Cell-derived Extracellular Vesicles mediate Mesenchymal Stem/Stromal Cell recruitment. Scientific Reports, 7, 1667.
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3

Platelet Activation Markers by Flow Cytometry

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Flow cytometry was used to measure the expression levels of CD62P (P-selectin), GPIIb/IIIa (integrin αIIbβ3), CD63 (lysosomal glycoprotein), and phosphatidylserine on perfused platelets.
Following blood perfusion through a flow channel with a stenotic region but with no capture region, a 5 μL aliquot of blood supernatant was incubated for 20 minutes with anti-human CD62P, PAC−1, anti-human CD63, or annexin V (BD Biosciences, San Jose, CA, USA) to label P-selectin, active GPIIb/IIIa, lysosomal glycoprotein, and phosphatidylserine, respectively. Two 5 μL blood aliquots were obtained and labeled prior to perfusion. Platelet activation in one aliquot was achieved by addition of thrombin immediately prior to labeling (c = 0.1 units/mL, EMD Millipore, Billerica, MA, USA) and other aliquot was left unstimulated to serve as positive and negative activation controls, respectively. Blood was not treated with PPACK for thrombin activated positive control. To locate platelets in flow cytometry analysis, another 5 μL blood aliquot was labeled with anti-human CD41b (BD Biosciences, San Jose, CA, USA), which binds to the GPIIb subunit of GPIIb/IIIa regardless of the activation state of the receptor. Following labeling, platelets were fixed in 1% paraformaldehyde and stored at 4 °C. Analysis of 100,000 events was conducted on a FACScanto analyzer (BD Biosciences, San Jose, CA, USA).
Rahman S, & Hlady V. (2019). Downstream Platelet Adhesion and Activation Under Highly Elevated Upstream Shear Forces. Acta biomaterialia, 91, 135-143.
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4

Ultrastructural Localization of CD63

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Immuno-EM was carried out in the Harvard EM facility. Adult mice (P60) were perfused with 4% paraformaldehyde (PFA) and 0.1% glutaraldehyde. The spinal cord was dissected out and post-fixed in 4% PFA for 2 hrs and then spinal cord slices (100 μm) were prepared using a vibratome. The slices were then quenched, permeabilized, and blocked in blocking buffer (3% bovine serum albumin, 5% normal donkey serum, and 0.1% Triton X-100) at 4°C. Anti-human CD63 (BD Pharmingen, #556019) antibody was then added and incubated overnight at 4°C. After wash, slices were incubated with Protein A-gold for 1 hour at 25°C. The images were taken using the JEOL 1200EX transmission electron microscope.
Yelick J., Men Y., Jin S., Seo S., Espejo-Porras F, & Yang Y. (2020). Elevated exosomal secretion of miR-124-3p from spinal neurons positively associates with disease severity in ALS. Experimental neurology, 333, 113414.
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5

EV Immunophenotyping by Flow Cytometry

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107 EVs derived from each cell line were diluted in PBS in a final volume of 50 µL. Anti-human CD9 (1:100; Cymbus Biotechnology, London, UK) and anti-human CD63 (1:100; clone H5C6, BD Pharmingen, California, USA) were added and samples were incubated for 1 h at RT and, after that, anti-mouse IgG FITC or anti-mouse Alexa 647 (1:200; ThermoFisher Scientific, Waltham, MA) were added and incubation proceeded for 1 h at RT. For staining control, EVs were incubated only with secondary antibody. 1 mL of filtered PBS was added to each sample and the fluorescent intensity were determined using CytoFLEX flow cytometry (Beckman Coulter, CA, USA). The equipment was calibrated using a mixture of fluorescent beads with size ranging from 100 nm to 1 µm for vesicles detection. Data analysis was performed with CytExpert 2.0 Software.
Andrade L.N., Otake A.H., Cardim S.G., da Silva F.I., Ikoma Sakamoto M.M., Furuya T.K., Uno M., Pasini F.S, & Chammas R. (2019). Extracellular Vesicles Shedding Promotes Melanoma Growth in Response to Chemotherapy. Scientific Reports, 9, 14482.
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6

Quantifying Platelet Activation Markers

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Flow cytometry was used to measure the expression levels of CD62P (P-selectin), integrin αIIbβ3, CD63 (lysosomal glycoprotein), and phosphatidylserine. Following blood perfusion through an albumin coated flow chamber in the absence of a capture region, a 5 μL aliquot of blood supernatant was collected and incubated for 20 minutes with anti-human CD62P, PAC-1, anti-human CD63, or annexin V (BD Biosciences, San Jose, CA, USA) to label P-selectin, active αIIbβ3, lysosomal glycoprotein, or phosphatidylserine, respectively. Two 5 μL blood aliquots were labeled prior to perfusion. One aliquot was stimulated by the addition of thrombin immediately prior to labeling (c = 0.1 units/mL, EMD Millipore, Billerica, MA, USA) and the other was left unstimulated to serve as positive and negative controls, respectively. A 5 μL aliquot was also labeled with anti-human CD41b (BD Biosciences, San Jose, CA, USA), which binds to the αIIb subunit of integrin αIIbβ3 regardless of the activation state of the receptor, to locate platelets during flow cytometry analysis. Following labeling, platelets were fixed in 1 % paraformaldehyde and stored at 4 °C. Analysis of 100,000 events was conducted on a FACScanto analyzer (BD Biosciences, San Jose, CA, USA).
Rahman S., Eichinger C, & Hlady V. (2018). Effects of Upstream Shear Forces on Priming of Platelets for Downstream Adhesion and Activation. Acta biomaterialia, 73, 228-235.
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