Cells were grown for 1 h in medium supplemented with 10 μm BrdU. Cells were fixed using ethanol and the DNA was denatured using 1 ml of 2 N HCl/Triton X-100 and then neutralized with 500 μl 0.1 M Na2B4O7. Cells were blocked using PBS/1% BSA/0.5% Tween-20 and incubated with mouse anti-BrdU primary antibody (clone BU20A, Dako) followed by goat anti-mouse FITC-conjugated secondary antibody (DAKO). Cells were suspended in 1 ml of PBS containing 5 μg/ml propidium iodide and 5 μl RNAse (25 mg/ml), incubated at room temperature for 30 min, and analyzed by FACS. Cell-cycle distribution was measured by flow cytometry in a Fortessa flow cytometer (Becton Dickinson) and analyzed using FlowJo software (FlowJo).
Goat anti mouse fitc conjugated secondary antibody
Manufactured by Agilent Technologies
Sourced in Denmark
The Goat anti-mouse FITC conjugated secondary antibody is a laboratory reagent used to detect the presence of mouse primary antibodies in various immunoassays and imaging techniques. The FITC (Fluorescein Isothiocyanate) conjugation allows for fluorescent detection of the bound secondary antibody, enabling visualization and quantification of the target mouse antibody.
Automatically generated - may contain errors
3 protocols using goat anti mouse fitc conjugated secondary antibody
1
Quantifying Cell Proliferation by BrdU Incorporation
2
Flow Cytometric Analysis of CXCR4 Expression
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To determine surface CXCR4, AGS cells were stained as previously reported [19 (link)]. Cells were incubated with 10 μg/mL of mouse anti-human CXCR4 (Clone 12G5, Santa Cruz, Heidelberg, Germany) or isotype antibody (Dako, Tehran, Iran) for 45 min on ice, then washed with FCM buffer (PBS containing 1% BSA), and incubated with goat anti-mouse FITC conjugated secondary antibody (Dako) for 30 min. Then cells were washed three times, fixed in 1% paraformaldehyde, and subjected to flow cytometric analysis (FACS Calibur, Beckman Dickinson, San Jose, CA). To detect intercellular CXCR4, surface CXCR4 was blocked by incubating the cells with 10 μg/mL of mouse anti-human CXCR4 (Santa Cruz) for 45 min, and then cells were washed three times with FCM buffer. Cells were then permeabilized with 0.1% Triton X100 (Sigma) for 10 min, washed three times with cold PBS, and stained with either mouse anti-human CXCR4-PE/Cy5 or isotype antibody for 30 min at 4°C. Cells were next washed and ran by flow cytometry. Ultimately, CXCR4 expression was analyzed by FCS Express software (Los Angeles, CA).
3
Characterization of Bone Marrow-Derived Mesenchymal Stem Cells Exposed to Helicobacter pylori
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BMD-MSCs were cocultured with H. pylori for 24 h, harvested, and washed with FCM buffer (PBS containing 1% BSA) three times. Cells were stained as described previously.[23 (link)] Briefly, cells were incubated with 10 μg/mL of mouse anti-human CXCR4 (Santa Cruz, Dallas, TX) or isotype antibody (Dako, Glostrup, Denmark) for 45 min on ice, then washed and incubated with goat anti-mouse FITC conjugated secondary antibody (Dako) for 30 min. Cells then were washed three times, fixed in 1% paraformaldehyde, and subjected to FACS analysis (FACS Calibur, Beckman Dickinson, San Jose, CA). The purity of BMD-MSCs was determined using stained anti-CD45-FITC, CD73-FITC, CD34-FITC, and Stro-1. Cells were incubated with 10 μL of either anti-CD45-FITC, CD73-FITC, or CD34 FITC for 30 min at room temperature, washed three times with FCM buffer, and subjected to FACS analysis. To stain BMD-MSCs for Stro-1 (R&D, Minneapolis, MN) cells were incubated with anti-Stro-1 monoclonal antibody for 45 min on ice, then washed and incubated with secondary antibody as mentioned above. FACS data were analyzed by FCS Express software (De Novo Software, Los Angeles, CA).
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