A Faster, Simpler Way to Analyze DNA Replication
Simplifying Cell Proliferation Analysis
Compared to tedious BrdU assays, new, simplified Click-iT® EdU flow cytometry kits make it even easier to detect and quantify DNA synthesis. The new assay kits contain 50% fewer components and a streamlined experimental protocol to make direct S-phase DNA measurement in proliferating cells simpler than ever.
Evaluate Cell Proliferation
The proportion of cells in the DNA synthesis phase provides a reliable indication of the proliferative capacity of a cell population. Click-iT® EdU can be used to evaluate proliferation dynamics in primary human cells (an important experimental model). As shown in Figure 1, for Gibco® Human Skeletal Myoblasts (HSkM) plated at three different densities and grown in differentiation media for 48 hours, higher plating densities correlated with a lower proliferative fraction of cells.
Figure 1. Correlation between HSkM plating density and proliferative fraction. Gibco® Human Skeletal Myoblasts (HSkM) were thawed and plated in low-glucose DMEM plus 2% horse serum at (A) 10,000 cells/cm2, (B) 25,000 cells/cm 2 or (C) 50,000 cells/cm2. These plots show a correlation between cell plating density and proliferative fraction, with the percentage of EdU-positive cells decreasing as plating density increases. Cells were grown for 48 hr at 37°C/5% CO2, then fed a 10 μM pulse of EdU 2 hr prior to analysis. EdU incorporation into newly synthesized DNA was detected using a click reaction with Alexa Fluor® 488 dye azide. Cells were analyzed with the Attune® Acoustic Focusing Cytometer with a 488 nm laser using the standard BL1 (530/30) emission filter.
Detect Variation in Proliferative Cells
Click-iT® EdU can also be used to detect variations in the proliferative fraction of cells due to cell cycle disruption. Figure 2 shows how Click-iT® EdU assays were used with several cultured cell lines to directly measure the percentage of cells in S phase. Dual-parameter analysis of DNA content vs. Click-iT® EdU fluorescence clearly identifies cells in different phases of the cell cycle. The percentage of cells in S phase provides an indication of the degree of cell proliferation.
Figure 2. Dual-parameter analysis of cell cycle perturbation. Gibco® Primary Adult Human Dermal Fibroblasts (HDFa), cervical carcinoma cells (HeLa), and human alveolar epithelial cells (A549) were grown in culture for 48 hr. Cells were either treated with a 500 nM pulse of the antimitotic drug Paclitaxel or left untreated. Cells were treated with 10 µM EdU for 2 hr prior to analysis. Incorporated EdU was detected using a reaction with Click-iT® EdU Alexa Fluor® 488 dye azide. Cells were analyzed using the Attune® Acoustic Focusing Cytometer with 405 nm and 488 nm lasers using standard VL1 (450/40) and BL1 (530/30) emission filters. Dual-parameter density plots of FxCycle™ Violet vs. Alexa Fluor® 488 Click-iT® EdU fluorescence clearly identified cells in the G0/G1, S, and G2/M phases of the cell cycle. Untreated A549 cells had a significantly higher percentage of cells in S phase and a lower percentage in G0/G1 than other cell types, reflecting a higher proliferative index. Upon treatment with paclitaxel, more than 90% of A549 and HeLa cells were arrested in G2 phase due to mitotic inhibition. A similar pattern of arrest was seen with HDFa, with the exception of approximately 26% of cells which remained in the quiescent G0 state.
An Easier Way to Analyze Cell Proliferation
The unique chemistry of assays based on Click-iT® EdU technology enables a simple and efficient method for evaluating cell proliferation. These new assays help researchers to accurately correlate proliferative dynamics with different plating densities, and clearly identify cells in the S phase of the cell cycle. Click-iT® EdU labeling is compatible with most fixation protocols.
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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