Human UCB-MSCs were evaluated using surface marker detection at passage 5 (P5) to confirm the effect of oxygen concentration (hypoxia and normoxia) on MSC characterization. hUCB-MSCs at 80% confluence were harvested and suspended in FACS buffer (1×107 cells/ml). Antibody was then added to each samples: Anti-CD90 fluorescein isothiocyanate (FITC), Anti-CD44 phycoerythrin (PE), Anti-CD105-PerCP-Cy5.5, Anti-CD73-allophycocyanin (APC), MSC negative antibodies set (Anti-CD34/CD11b/CD19/CD45/HLA-DR-PE), positive isotype cocktail (mIgG-FITC, mIgG-PerCP-Cy5.5, mIgG-APC), negative isotype cocktail (mIgG1-PE, mIgG2a-PE) (562245, BD Biosciences) followed by incubation at 4°C for 30 min. For cell cycle analysis using propidium iodide, cells were fixed with cold 70% ethanol overnight at −20°C. They were then washed once with PBS and incubated in PBS containing 50 μg/ml propidium iodide and 1 mg/ml RNase A for 30 min at room temperature. After staining, cells were washing with PBS and measured by flow cytometry. Antibodies listed above are purchased from BD Biosciences.
Migg2a pe
Manufactured by BD
Sourced in United States
The MIgG2a-PE is a laboratory equipment product. It serves as a fluorescently-labeled secondary antibody for the detection and quantification of target proteins in various immunoassays and cell-based applications.
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Lab products found in correlation
2 protocols using migg2a pe
1
Evaluating hUCB-MSC Characterization under Hypoxia
2
Phenotyping of hUCB-MSCs and Macrophages
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Cells were washed and resuspended in staining buffer. Fluorochrome-conjugated monoclonal antibodies were added to cells and incubated for 30 min in the dark. An hUCB-MSC surface marker analysis with flow cytometry was performed using the following monoclonal antibodies: CD105, CD73, CD90, CD45, CD34, CD14, CD11b, CD19, HLA-DR, positive isotype control cocktail (mIgG1 FITC, mIgG1 PErCP-Cy5.5, and mIgG1 APC), and negative isotype control cocktail (mIgG1 PE and mIgG2a PE) purchased from BD Biosciences (San Jose, CA, USA). Phenotyping of macrophages with flow cytometry was performed using the antibodies of M2 macrophage differentiation markers (CD163, BD Biosciences; CD206, Abcam, Cambridge, UK) and M1 macrophage differentiation markers (CD80; Abcam). After staining of primary antibody, cells were washed and resuspended in 150 μL of the appropriate secondary antibody (Alexa Fluor® 488-conjugated donkey anti-rat, Alexa Fluor® 647-conjugated donkey anti-rabbit; Abcam). Data were acquired using BD LSRFortessa flow cytometers and analyzed with BD FACSDiva software (BD Biosciences, Franklin Lakes, NJ, USA).
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