Combining the Benefits of EdU and BrdU
How Click-iT® EdU Works
Highly Specific Detection of EdU and BrdU
The new anti-BrdU antibody clone MoBU-1 displays no cross-reactivity with EdU used in the Click-iT® EdU assay (Figure 1). Detection with this dual pulse method is highly specific for each modified nucleoside and does not require media replacement or removal of EdU before the addition of BrdU (Figure 2).
Figure 1. The anti-BrdU antibody clone MoBU-1 does not react with EdU. Jurkat T cells were treated with 10 μM EdU for 1 hr, the cells were fixed in ethanol, and an acid denaturation method was used before labeling with FITC conjugates of the most commonly used anti-BrdU clones: 3D4, PRB-1, and MoBU-1. Cells were analyzed with a BD™ LSR II flow cytometer using a 488 nm excitation laser, and fluorescence was collected with a 530/30 bandpass filter. Of the clones tested, only clone MoBU-1 did not react with EdU.
|Figure 2. Dual pulse labeling of cell proliferation with BrdU and Click-iT® EdU.TF-1 erythroblast cells were pulsed with 20 μM EdU for 1 hr followed by 10 μM BrdU for 1 hr. The cells were fixed in ethanol, and an acid denaturation method was used before labeling with anti-BrdU (clone MoBU-1)–Alexa Fluor® 488 conjugate and Click-iT® EdU–Alexa Fluor® 647 azide. Data were collected with a BD™ LSR II flow cytometer using 488 nm excitation with a 530/30 bandpass filter, and 633 nm excitation with a 660/20 bandpass filter. Cells colored blue are negative for both EdU and BrdU; cells colored dark green are positive for both EdU and BrdU; cells colored red are positive for EdU but negative for BrdU; cells colored light green are negative for EdU but positive for BrdU.|
A Simple Assay for a Diverse Set of Applications
Figure 3. Dual pulse labeling of cell proliferation using high-content imaging and analysis. The effect of various drugs on DNA replication was assessed using dual pulse labeling. U2OS cells were pulsed with 10 μM EdU for 60 min, washed, then treated with chloroquine, rosiglitazone, acetaminophen, or etoposide, or left untreated for 23 hr. Cells were pulsed with 10 μM BrdU for 60 min, then fixed and denatured before performing the Click-iT® EdU Alexa Fluor® 488 Imaging Assay to detect EdU (green). BrdU incorporation was detected using anti-BrdU (clone MoBU-1)–Alexa Fluor® 594 conjugate (red). Nuclei were counterstained with HCS NuclearMask™ Blue stain. (Left) Control cells dual-labeled with EdU and BrdU, imaged with a Zeiss Axiovert® 200M microscope. (Right) DNA replication was decreased by several drugs, as indicated by a decrease in BrdU-positive cells compared to EdU-positive cells. Data were acquired using the Arrayscan® VTI platform (Thermo Scientific Cellomics®).
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