The Click-iT Plus EdU Flow Cytometry Assay Kit (Sigma) was used to quantify cell proliferation following the manufacturer's instructions. Spleen leukocytes were seeded at a concentration of 2 × 106 cells/ml in 96-well plates and incubated with the different stimuli for 3 days at 20 °C. Subsequently, 5-ethynyl-2′-deoxyuridine (EdU) was added to the cultures at a final concentration of 1 μM and the cells were incubated for an additional 24 h. After that time, treated and untreated cells were collected and stained with anti-IgM (1.14) conjugated to allophycocyanin (1 μg/ml) for 30 min at 4 °C. The incorporation of EdU to the DNA was determined following the manufacturer's instructions and then analyzed by flow cytometry in a FACS Celesta flow cytometer. Flow cytometry analysis was performed with FlowJo® v.10.
Click it plus edu flow cytometry assay kit
Manufactured by Merck Group
The Click-iT Plus EdU Flow Cytometry Assay Kit is a laboratory equipment product. It is designed for the detection and quantification of cellular proliferation using flow cytometry. The kit employs a chemical reaction to label dividing cells, which can then be analyzed using a flow cytometer.
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2 protocols using click it plus edu flow cytometry assay kit
1
Quantifying Cell Proliferation by Flow Cytometry
2
Measuring B Cell Proliferation by EdU Flow Cytometry
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The Click-iT Plus EdU Flow Cytometry Assay Kit (Sigma) was used to measure the proliferation of IgM+ B cells following manufacturer's instructions. Briefly, blood and spleen leukocyte suspensions at a concentration of 2 × 106 cells per ml were incubated in 96-well plates for 3 days at 20°C with different stimuli depending on the specific experiment as described above. Thereafter, 5-ethynyl-2′-deoxyuridine (EdU) was added to the cultures at a final concentration of 1 μM and the cells were incubated for an additional 24 h. After that time, stimulated and unstimulated cells were collected and stained with anti-IgM (1.14) coupled to allophycocyanin (1 μg/ml) for 20 min at 4°C. Whenever cells had been stimulated with anti-IgM, the cells were only labeled with EdU (1 μM) as described above. The incorporation of EdU to the DNA was determined following the manufacturer's instructions and then analyzed by flow cytometry in a FACS Calibur flow cytometer (BD Biosciences) equipped with CellQuest Pro software (BD Biosciences). Flow cytometry analysis was performed with FlowJo V10 (TreeStar).
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