Ab124527

Manufactured by Abcam

Sourced in United States

Ab124527 is a primary antibody that recognizes a specific target protein. It is designed for use in various laboratory techniques, such as immunoassays and immunohistochemistry. The antibody has been validated for its specificity and sensitivity, but its exact intended use or performance characteristics are not provided in this factual description.

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5 protocols using ab124527

Quantification of Mesenchymal Stem Cell Markers

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Cells (2 × 10⁵) were incubated with a specific monoclonal antibody conjugated with either fluorescein isothiocyanate (FITC) or phycoerythrin (PE) in 200 μL PBS (SH30256.01; HyClone) for 30 min in the dark at 4°C. The cell surface antigens were then analyzed by flow cytometry. Antibodies against CD34 (PE) (ab187284; Abcam), CD45 (PE) (ab134202; Abcam), CD73 (FITC) (ab239246; Abcam), CD90 (FITC) (ab124527; Abcam), and CD146 (FITC) (ab78451; Abcam) were used. Mouse monoclonal IgG1 (ab170190; Abcam) isotype was used as control.

Rao F., Zhang D., Fang T., Lu C., Wang B., Ding X., Wei S., Zhang Y., Pi W., Xu H., Wang Y., Jiang B, & Zhang P. (2019). Exosomes from Human Gingiva-Derived Mesenchymal Stem Cells Combined with Biodegradable Chitin Conduits Promote Rat Sciatic Nerve Regeneration. Stem Cells International, 2019, 2546367.

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Characterization of Lung Cell Markers

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Cells were washed with MACS flow buffer (MACS, 130-091-222) and permeabilized with BD Cytofix/Cytoperm (BD, 554714) prior to incubation with antibodies. Cells were labeled for antibodies against CD90 (Abcam, ab3105; Abcam, ab124527; Abcam, ab23894; BD, 555595), CD105 (Abcam, ab107595; Abcam, ab2529; Abcam, ab11414; R&D Systems, Fab10971p), Pro-SPC (Bioss, bs 10067R; Abcam, ab40879), CCSP (Abcam, ab171957), Epcam (Abcam, ab71916, Abcam, ab168828; Life Technologies, a15755), and Aqp5 (Abcam, ab78486; Abcam, ab85905) and detected by Alexa Fluor 488 (Abcam, ab150117, ab150077) or fluorescein isothiocyanate (FitC) (Abcam, ab6840) secondary antibodies. Both unstained and isotype controls (Abcam, ab18419; BD, 559320; Abcam, ab125938) were utilized as controls. Human adipose-derived mesenchymal stem cells (AD-MSCs) were obtained from Lonza. Flow Cytometry was performed on the CytoFlex (Beckman Coulter, Indianapolis, IN) and analyzed using FCS Express (De Novo Software, Glendale, CA) or CytExpert ((Beckman Coulter, Indianapolis, IN).

Dinh P.U., Cores J., Hensley M.T., Vandergriff A.C., Tang J., Allen T.A., Caranasos T.G., Adler K.B., Lobo L.J, & Cheng K. (2017). Derivation of therapeutic lung spheroid cells from minimally invasive transbronchial pulmonary biopsies. Respiratory Research, 18, 132.

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Isolation and Characterization of Adipose-Derived MSCs

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Cells were collected from subcutaneous abdominal fat (5–10g) using collagenase-H, and cultured for 3 weeks in advanced MEM medium (Gibco/Invitrogen) supplemented with 5% platelet lysate PLTmax, Mill Creek Life Sciences, Rochester, MN). MSCs were characterized by fluorescence-activated cell sorting analysis to determine cellular phenotype for the MSC markers CD90 (abcam, ab124527) and CD105 (abcam, ab53321), as previously shown [13 (link)]. In addition, we tested that our adipose MSCs were negative for the progenitor cell marker CD34 (BD Biosciences, 340441) and the common leukocyte marker CD45 (Biolegend, 304014). The third passage was collected and kept in Gibco Cell Culture Freezing Medium for subsequent studies.

Nargesi A.A., Zhu X.Y., Hickson L.J., Conley S.M., van Wijnen A.J., Lerman L.O, & Eirin A. (2019). Metabolic syndrome modulates protein import into the mitochondria of porcine mesenchymal stem cells. Stem cell reviews, 15(3), 427-438.

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Immunostaining and Flow Cytometry of Cardiac Cells

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Cells were fixed 14 days after transduction in 2% PFA, permeabilized with 0.2% Triton, and blocked with donkey serum (Sigma). Cells were then stained overnight at 4°C with different primary antibodies. Antibody dilutions were the following: anti-Vimentin (Sigma; V6630) 1 : 200, anti-Ki67 (sigma; ab15580) 1 : 200; anti-GFP (Abcam; ab545) 1 : 500; anti-sarcomeric α-actinin (αSA; Abcam; ab9465) 1 : 200; anti-connexin 43 (Santa Cruz; sc-9059) 1 : 300; MyHC (MF20; Hybridoma) 1 : 3; cardiac troponin T (Abcam; ab10214) 1 : 200.
Secondary fluorescents antibodies (Alexa-Fluor; 1 : 500) were used for detection. Nuclei were stained with Hoechst 33342 (Sigma).
Flow cytometry analyses were performed according to standard procedures with the following antibodies: anti-CD90/Thy1 antibody (FITC) (Abcam; ab124527); anti-cardiac troponin T (Abcam; ab10214).
Cardiac troponin T expression was analyzed by intracellular staining; cells were fixed with 2% PFA, blocked with donkey serum (Sigma), and permeabilized by fixation/permeabilization (BD Cytofix/Cytoperm).

Palazzolo G., Quattrocelli M., Toelen J., Dominici R., Anastasia L., Tettamenti G., Barthelemy I., Blot S., Gijsbers R., Cassano M, & Sampaolesi M. (2015). Cardiac Niche Influences the Direct Reprogramming of Canine Fibroblasts into Cardiomyocyte-Like Cells. Stem Cells International, 2016, 4969430.

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Comprehensive MSC Characterization by Flow Cytometry

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All MSCs were characterized by imaging flow cytometry (FlowSight, Amnis, Seattle, WA, USA) to confirm expression of CD73 (Abcam, #ab106677), CD90 (Abcam, #ab124527), and CD105 (Abcam, #ab53321), and to rule out expression of CD45 (Abcam, #ab51482) and CD34 (BD Biosciences, Cat. #340441). Gating was performed in a manner previously described^{26 (link)} and data analyzed using Amnis® Image Data Exploration Analysis Software (IDEAS version 6.2).
A MSC functional identification kit (R&D Systems, #SC006) was used to verify trilineage differentiation capacity of hESC-MSCs and MSC(AT)s in accordance with the manufacturer’s instructions. Images (20x or 40x, at least 6 per marker) were quantified as the ratio of positive cells/total cells per field.

Siddiqi S., Klomjit N., Jiang K., Conley S.M., Zhu X., Saadiq I.M., Ferguson C.M., Tang H., Lerman A, & Lerman L.O. (2022). Efficacy of Human Embryonic Stem Cells Compared to Adipose Tissue-Derived Human Mesenchymal Stem/Stromal Cells for Repair of Murine Post-Stenotic Kidneys. Stem cell reviews and reports, 19(2), 491-502.

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