Mouse anti cd9

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Mouse anti-CD9 is a primary antibody that recognizes the CD9 cell surface antigen. CD9 is a member of the tetraspanin family and is expressed on various cell types, including hematopoietic cells, endothelial cells, and epithelial cells. This antibody can be used in applications such as flow cytometry and immunohistochemistry to detect and analyze CD9-expressing cells.

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Lab products found in correlation

Mouse anti cd81, by Santa Cruz Biotechnology (5 mentions) Mouse anti tsg101, by Santa Cruz Biotechnology (3 mentions) Mouse anti β actin, by Merck Group (2 mentions) Mouse anti cd63, by BD (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Mouse anti α sma, by Merck Group (1 mentions) Ibind western system, by Thermo Fisher Scientific (1 mentions) Chemi lumi one, by Nacalai Tesque (1 mentions) Mouse anti cd63, by Abcam (1 mentions) Supersep ace gel, by Fujifilm (1 mentions) Rabbit anti e cad, by Cell Signaling Technology (1 mentions) Amersham imager 600, by GE Healthcare (1 mentions) Trans blot turbo transfer system, by Bio-Rad (1 mentions) Mouse anti cd63, by Santa Cruz Biotechnology (1 mentions) Bca assay, by Thermo Fisher Scientific (1 mentions) Mouse anti alix, by Santa Cruz Biotechnology (1 mentions) Anti e cadherin, by Santa Cruz Biotechnology (1 mentions) Anti n cadherin, by Santa Cruz Biotechnology (1 mentions) Mouse anti gapdh, by Santa Cruz Biotechnology (1 mentions) Macsplex human exosome kit, by Miltenyi Biotec (1 mentions)

7 protocols using mouse anti cd9

Comprehensive Proteomic Analysis of Extracellular Vesicles

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Cells and EV proteins were separated on 10%–15% SuperSep™ Ace gels (Fujifilm) and transferred onto polyvinylidene difluoride membranes using a Trans‐Blot® Turbo™ Transfer System (Bio‐Rad). Western blotting was performed on iBind™ Western Systems (Thermo Fisher Scientific) according to the manufacture's instructions¹⁴ using the following primary antibodies: mouse anti‐αSMA (Sigma, A5228), rabbit anti‐FAPα (Abcam, ab137549), rabbit anti‐E‐cad (Cell Signaling Technologies, 24E10), mouse anti‐CD9 (Santa Cruz, sc‐59,140), mouse anti‐CD63 (Abcam, ab59479), and mouse anti‐CD81 (Santa Cruz, sc‐166,029). Protein bands were visualized using HRP‐conjugated secondary antibodies and Chemi‐Lumi One (Nacalai Tesque) on an Amersham Imager 600 (GE Healthcare).

Toshida K., Itoh S., Harada N., Morinaga A., Yugawa K., Tomiyama T., Kosai‐Fujimoto Y., Tomino T., Kurihara T., Nagao Y., Morita K., Oda Y, & Yoshizumi T. (2022). Cancer‐associated fibroblasts promote tumor cell growth via miR‐493‐5p in intrahepatic cholangiocarcinoma. Cancer Science, 114(3), 937-947.

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Exosomal Protein Quantification and Characterization

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The concentration of exosomal total protein was quantified by the bicinchoninic acid (BCA) assay (Thermo Fisher Inc.) using bovine serum albumin (BSA) as standard. The pellets were also dissolved in 200 μL RIPA buffer for protein assay. The samples were individually homogenized in 5 mM Tris–HCI (4 mM EDTA, pH 7.4, containing 1 M pepstatin, 100 M leupeptin, 100 M phenylmethyl sulfonylfluoride, and 10 g/mL aprotinin) and cleared by centrifugation at 14,000g for 10 min at 4 °C. Approximately 100 μg of protein were run on a discontinuous SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5% skim milk in TBS containing 0.05% Tween 20 and were incubated with the following primary antibodies: (1) Mouse anti-CD9 (sc-13118, Santa Cruz), (2) Mouse anti-CD63 (sc-5275, Santa Cruz), (3) Mouse anti-TSG101 (sc-7964, Santa Cruz), (4) Mouse anti-Alix (sc-53540, Santa Cruz), (5) Mouse anti-calnexin (10,427–2-AP, Promega, Madison), (6) Mouse anti-PD-L1 (sc-293425, Santa Cruz), (7) Rabbit monoclonal anti-E-cadherin (ab194982), (8) Rabbit polyclonal anti-N-cadherin (ab18203) and (9) Mouse anti-GAPDH (sc-47724, Santa Cruz, CA, USA). The optical density (OD) of the signals was quantified and expressed as the ratio of the tested proteins to GAPDH for analysis of protein in cells and to ALIX for analysis of protein in exosomes.

Wang J., Zhang H., Sun X., Wang X., Ren T., Huang Y., Zhang R., Zheng B, & Guo W. (2020). Exosomal PD-L1 and N-cadherin predict pulmonary metastasis progression for osteosarcoma patients. Journal of Nanobiotechnology, 18, 151.

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Exosome Protein Expression Analysis

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The following antibodies were used: mouse anti-CD9 (Santa Cruz Biotechnology, sc-59140), mouse anti-CD63 (BD Pharmingen, 556019), mouse anti-CD81 (Santa Cruz Biotechnology, sc-555675), mouse anti-β-actin (Millipore, MAB1501), mouse anti-MHC-2 (Cell Signaling Technology, 68258), mouse anti–NK-κB (Cell Signaling Technology, 8242), mouse anti–phosphor-NK-κB (Cell Signaling Technology, 3033), rabbit anti-ANXA1 (Thermo Fisher Scientific, 71-3400), rabbit anti-FPR2 (Novusbio, NLS1878), mouse anti-HT2-280 (Terrace, TB-27AHT2-280), and rabbit anti-SFTPC (Millipore, AB3786). Additionally, the following reagents were used: the Human MACSPlex Exosome Kit (Miltenyi Biotec, 130-108-813), Phorbol 12-myristiate-12 acetate (PMA Sigma-Aldrich, P8139), poly (I:C) (InvivoGen, tlrl-pic), lipopolysaccharide LPS (Sigma‐Aldrich, L2630), dexamethasone (Fujifilm, 041-18861), recombinant ANXA1 (American Research products, 01-2062), and WRW4 (R&D Systems, 2262/1).

Fujita Y., Kadota T., Kaneko R., Hirano Y., Fujimoto S., Watanabe N., Kizawa R., Ohtsuka T., Kuwano K., Ochiya T, & Araya J. (2024). Mitigation of acute lung injury by human bronchial epithelial cell-derived extracellular vesicles via ANXA1-mediated FPR signaling. Communications Biology, 7, 514.

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Antibodies for Cellular Analysis

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Antibodies used were rabbit anti‐p21 (Cell Signaling Technology, 2947), rabbit anti‐p16 (Santa Cruz Biotechnology, sc‐468), mouse anti‐β‐actin (Millipore, Billerica, MAB1501), mouse anti–α smooth muscle actin (Sigma‐Aldrich, A2547), goat anti‐type I collagen (SouthernBiotech, 1310‐01), rabbit anti‐β‐catenin (Cell Signaling Technology, 8480), rabbit anti‐non‐phospho (active) β‐catenin (Cell Signaling Technology, 8814), rabbit anti‐histone H3 (Cell Signaling Technology, 4499), mouse anti‐CD81 (Santa Cruz Biotechnology, 555675), mouse anti‐CD9 (Santa Cruz Biotechnology, sc‐59140), mouse anti‐CD63 (BD Pharmingen, 556019), rabbit anti‐Hsc70 antibody (Proteintech, 10654‐1‐AP) and mouse anti–Tom20 (Santa Cruz Biotechnology, sc‐17764). Hoechst 33242 (Sigma‐Aldrich, H342) and collagen type I solution from rat tail (Sigma‐Aldrich, C3867) were purchased reagents.

Kadota T., Fujita Y., Araya J., Watanabe N., Fujimoto S., Kawamoto H., Minagawa S., Hara H., Ohtsuka T., Yamamoto Y., Kuwano K, & Ochiya T. (2021). Human bronchial epithelial cell‐derived extracellular vesicle therapy for pulmonary fibrosis via inhibition of TGF‐β‐WNT crosstalk. Journal of Extracellular Vesicles, 10(10), e12124.

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Quantifying Protein Levels via Western Blot

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In total, we isolated 30 μg of protein from each sample and separated this protein via 8% SDS-PAGE, after which it was transferred onto PVDF membranes (Millipore). After being blocked for 2 h, these membranes were then probed using mouse anti-CD9 or mouse anti-TSG101 (both 1:1,000, Santa Cruz Biotechnology). Blots were then probed with secondary HRP-conjugated anti-mouse IgG (1:5,000). A densitometer (GS-700; Bio-Rad Laboratories) was used to visualize protein bands in the resultant stained blots, with the Quantity One 4.4.0 software being used for quantification purposes.

Meng L., Xing Z., Guo Z., Qiu Y, & Liu Z. (2021). Hypoxia-induced microRNA-155 overexpression in extracellular vesicles promotes renal cell carcinoma progression by targeting FOXO3. Aging (Albany NY), 13(7), 9613-9626.

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Exosome Protein Detection by Western Blot

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Exosome protein samples for western blot were extracted by 10xRIPA buffer with protease inhibitors. Antibodies were purchased from the following sources: rabbit anti-CD63 (sc-15363, Santa Cruz, TX, USA); mouse anti-TSG101 (sc-7964, Santa Cruz, TX, USA); mouse anti-CD81 (sc-166029, Santa Cruz, TX, USA); mouse anti-CD9 (sc-13118, Santa Cruz, TX, USA); mouse anti-GM130 (sc-55590, Santa Cruz, TX, USA); anti-rabbit IgG HRP-linked (7074S, Cell Signaling, MA, USA); anti-mouse IgG HRP-linked (7076 S, Cell Signaling, MA, USA). 1 µg ml⁻¹ primary antibodies, and 50 ng ml⁻¹ secondary antibodies were used for blotting.

Luo W., Dai Y., Chen Z., Yue X., Andrade-Powell K.C, & Chang J. (2020). Spatial and temporal tracking of cardiac exosomes in mouse using a nano-luciferase-CD63 fusion protein. Communications Biology, 3, 114.

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Exosome Protein Extraction and Western Blot

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Proteins were extracted from exosomes and cells using RIPA lysis buffer with 100× protease inhibitor and 50× phosphatase inhibitor added. Total protein concentration was quantified using Pierce BCA Protein Assay Kit (catalog no. 23225, Thermo Fisher Scientific). Protein sample was mixed with sample buffer and loaded into a 10% sodium dodecyl-sulfate polyacrylamide (SDS-PAGE) gel to perform gel electrophoresis. Western blotting was performed using a protocol previously described in our research [51] . The primary antibodies used were mouse anti-CD9 (catalog no. sc-59140, Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-CD63 (catalog no. 10628D, Invitrogen, Thermo Fisher Scientific), mouse anti-CD81 (catalog no. sc-7637, Santa Cruz Biotechnology), mouse anti-HSP70 (catalog no. sc-32239, Santa Cruz Biotechnology), and rabbit anti-β-actin (catalog no. GTX109639, GeneTex, Hsinchu, Taiwan) using 1:500~1:1000 dilution. After incubation with appropriate secondary antibody at 1:5000 dilution, target proteins were detected using a T-Pro LumiFast Plus Chemiluminescence Detection Kit (catalog no. JT96-K002M, T-Pro Biotechnology, New Taipei City, Taiwan), and signals were visualized using Amersham Imager 600 (catalog no. 29083461, Cytiva Life Sciences).

Kurniawati I., Liu M.C., Hsieh C.L., Do A.D, & Sung S.Y. (2022). Targeting Castration-Resistant Prostate Cancer Using Mesenchymal Stem Cell Exosomes for Therapeutic MicroRNA-let-7c Delivery. Frontiers in bioscience (Landmark edition), 27(9).

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