A novel method based on click chemistry, which overcomes limitations of cell cycle analysis by classical determination of BrdU incorporation, allowing multiplex antibody staining

Abstract

Quantification of BrdU incorporation into DNA is a widely used technique to assess the cell cycle status of cells. DNA denaturation is required for BrdU detection with the drawback that most protein epitopes are destroyed and classical antibody staining techniques for multiplex analysis are not possible. To address this issue we have developed a novel method that overcomes the DNA denaturation step but still allows detection of BrdU. Cells were pulsed for a short time by 5-ethynyl-2'-deoxyuridine, which is incorporated into DNA. The exposed nucleotide alkyne group of DNA was then derivatized in physiologic conditions by the copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) using BrdU azides. The resulting DNA-bound bromouracil moiety was subsequently detected by commercial anti-BrdU mAb without the need for a denaturation step. Continuous labeling with EdU showed a slightly increased anti-proliferative activity compared to BrdU. However, using a lower concentration of EdU for labeling can compensate for this. Alkynyl tags could be detected quickly by a highly specific reaction using BrdU azides. Fluorescence quenching by the DNA dye PI using both BrdU azides was negligible. Our labeling method is suitable for FCM and HCA and shows a higher signal to noise ratio than other methods. This method also allowed multiplex analysis by simultaneous detection of EdU-BrdU, caspase-3, and phospho-histone 3 mAbs, proving sensitivity and feasibility of this new technique. In addition, it has the potential for use in vivo, as exemplified for bone marrow studies. We have established a new method to determine the position of cells in the cell cycle. This is superior when compared to traditional BrdU detection since it allows multiplex analysis, is more sensitive and shows less quenching with PI. The method provides new opportunities to investigate changes in protein expression at different cell cycle stages using pulse labeling experiments.

Chemokine Receptor 2-Mediated Accumulation of Fungicidal Exudate Macrophages in Mice That Clear Cryptococcal Lung Infection

“…We used an established assay that measures EdU incorporation into replicating cells using flow cytometric analysis (per the Materials and Methods section Evaluation of Macrophage Proliferation In Vivo). [43][44][45][46] Within bone marrow, we clearly identified a population of proliferating leukocytes, including polymorphonuclear neutrophils (positive control), whereas among lung leukocyte populations, AMs, ExMs, and polymorphonuclear neutrophils (negative control 47 ) were not proliferating (Table 1). Collectively, these data indicate that the substantial accumulation of ExMs (and AMs) during cryptococcal infection is not attributable to macrophage proliferation within the lung.…”

“…After 2 hours, mice were sacrificed and bone marrow cells (as positive control) and specific lung leukocyte populations identified by flow cytometric analysis were evaluated for EdU uptake following manufacturer's protocols and as previously described. [43][44][45][46] Gates depicting positive uptake were set based on the staining characteristics of control leukocytes obtained from infected mice not receiving EdU. The assay was further validated by measuring EdU incorporation in polymorphonuclear neutrophils (defined as SSC moderate-high Ly-6C moderate CD11b ϩ ), which proliferate in bone marrow but not on arrival in the lung.…”

Ethanol Alters the Balance of Sox2, Oct4, and Nanog Expression in Distinct Subpopulations During Differentiation of Embryonic Stem Cells

“…Cell proliferation, cell cycle, and apoptosis assays Cell cycle kinetics were determined by incorporation of a fluorescent uridine analog conjugated to Alexa Fluor 647, using a Click-iT EdU flow cytometry assay kit (Invitrogen) [46][47][48]. Briefly, cells were incubated with 10 mM EdU (5-ethynyl-2¢-deoxyuridine) at 37°C for 1 h. At the end of the incubation period, cells were disaggregated into a single-cell suspension, fixed, permeabilized, and incubated with the Click-iT reaction cocktail for 30 min in the dark.…”

Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification

“…The mild labeling of EdU in mtDNA allows for direct comparisons with additional cellular markers [9][10][11] and enhances the utility of this technique over other thymidine analogs, such as 5-bromo-2-deoxyuridine (BrdU), which requires a harsh treatment to recover its epitope within DNA. Our laboratory [11][12] and others [13][14][15] have successfully used BrdU to label mtDNA.…”

“…EdU is superior to other thymidine analogs, such as 5-bromo-2-deoxyuridine (BrdU), because the initial click reaction to label EdU [5][6][7] does not require the harsh acid treatments or enzyme digests that are required for exposing the BrdU epitope. The milder labeling of EdU allows for direct comparison of its incorporation with other cellular markers [9][10] . The ability to visualize and quantify mtDNA biogenesis provides an essential tool for investigating the mechanisms used to regulate mitochondrial biogenesis and would provide insight into the pathogenesis associated with drug toxicity, aging, cancer and neurodegenerative diseases.…”