Human CD9 Antibody

Detection of CD9 in Human Platelets by Flow Cytometry.

Human peripheral blood platelets were stained with Mouse Anti-Human CD9 Monoclonal Antibody (Catalog # MAB1880, filled histogram) or isotype control antibody (Catalog # MAB0041, open histogram) followed by anti-Mouse IgG PE-conjugated Secondary Antibody (Catalog # F0102B). View our protocol for Staining Membrane-associated Proteins.

Detection of Human CD9 by Western Blot

Validation of protein expression by Western blotting.All four exosome samples and their corresponding cell lines were used for validation. Furthermore, supernatant from the pelleted exosomes was used as a control. The selected proteins FASN, XPO1, CD9 and PDCD6IP, were tested with ENO1 and GAPDH as controls. PSA was tested to confirm it is secreted through alternative secretion pathway and therefore not present within exosomes. The nearest protein marker (kDa) is indicated for each blot. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/24391718), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of CD9 by Immunocytochemistry/ Immunofluorescence

Expression of EGFP & associated EV markers M1-CD9truc-EGFP & their conditioned medium. (A) Illustration of our EV reporter gene & protein. The CD9truc-EGFP coding sequence is driven by the CMV promoter & encodes a fusion protein consisting of the first 3 transmembrane domains of CD9 & EGFP, enabling genetic labeling & EV surface display of EGFP. (B) M1 stable transfected with CD9truc-EGFP are green fluorescent in contrast to non-transfected M1 . Nuclei (blue) & GFP (green). n = 3, ×200 magnification (C) Immunofluorescence labeling of CD9 in M1-CD9truc-EGFP using Anti-hCD9 AB shows colocalization between CD9 (red), EGFP (green) & nuclei (blue). ×200 magnification (n = 3) (D) M1 stable transfected with CD9truc-EGFP expressing a band reacting with an anti-GFP antibody in & PEG-precipitated conditioned medium. Actin & Lamin A/C were detected in cell lysates only, & EVs markers ALIX & Flotillin were detected in conditioned medium from transfected & non-transfected M1 (n = 3). Full-length blots are shown in Supplementary Figure S2A-F. (E) Tunable resistive pulse sensing on conditioned medium from non-transfected M1 & stable transfected CD9truc-EGFP indicated that the size distribution of EVs is not affected the expression reporter proteins (n = 3). (F) Size-exclusion chromatography fractions of CD9truc-EGFP cell-conditioned medium showed full-length CD9truc-EGFP in fractions 1–3, while shedded EGFP was present in fractions 10–12 (n = 3). Full-length blots are shown in Supplementary Figure S2G. Below the WB, the protein concentration in each fraction is shown (G) Anti-GFP immunoprecipitation of conditioned medium co-isolates CD9truc-EGFP & EV markers ALIX & TSG101 only in M1 stable transfected with CD9truc-EGFP (n = 3). Full-length blots are shown in Supplementary Figure S3A-C. Image collected & cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35017633), licensed under a CC-BY license. Not internally tested by R&D Systems.