Cell Proliferation Method: Click Chemistry Based on BrdU Coupling for Multiplex Antibody Staining

Abstract

Determination of incorporation of the thymidine analog 5‐bromo‐2′‐deoxyuridine (BrdU) into DNA is a widely used method to analyze the cell cycle. However, DNA denaturation is required for BrdU detection with the consequence that most protein epitopes are destroyed and their immunocytochemical detection for multiplex analysis is not possible. A novel assay is presented for identifying cells in active S‐phase that does not require the DNA denaturation step but nevertheless detects BrdU. For this purpose, cells were pulsed for a short time by 5‐ethynyl‐2′‐deoxyuridine (EdU) which is incorporated into DNA. The nucleotide‐exposed ethynyl residue was then derivatized by a copper‐catalyzed cycloaddition reaction (“click chemistry” coupling) using a BrdU azide probe. The resulting DNA‐bound bromouracil moieties were then detected by commercial anti‐BrdU monoclonal antibodies without the need for a denaturation step. This method has been tested using several cell lines and is more sensitive than traditional BrdU and allows multicolor and multiplex analysis in flow cytometry (FCM) and image‐based cytometry. © 2015 by John Wiley & Sons, Inc.

FOXM1 expression is significantly associated with chemotherapy resistance and adverse prognosis in non-serous epithelial ovarian cancer patients

“…Nuclei were isolated using pepsin/0.1 M HCl mixture and subsequently treated with 2 N HCl for 20 min; BrdU detection was performed after DNA denaturation using FITC Anti-BrdU mAb (BD Biosciences) for 1 h and DNA-content staining using PI (2 μg/ml) for 30 min at room temperature [29]. BrdU analysis and cell-cycle determination were performed using BD CellQuest Pro™ on BD FACSCalibur flow cytometer (BD Biosciences, New Jersey, USA) by acquiring at least 5000 single nuclei, with S-Phase estimation based on DNA-active (BrdU+ S cells) and -inactive (BrdU- S cells) synthesis.…”

Assaying Cell Cycle Status Using Flow Cytometry

“…A disadvantage of BrdU incorporation method is that both membrane permeabilization and harsh DNA denaturation processes are required for antibody penetration to the incorporated BrdU. As an alternative of BrdU, 5-ethynyl-2'-deoxyuridine (EdU) has been developed to overcome the limitations of BrdU method (Cappella et al, 2008; Cavanagh et al, 2011; Salic and Mitchison, 2008). After EdU treatment during cell proliferation, incorporation of EdU can be subsequently detected by a fluorescent azide molecule through a copper (I) catalyzed reaction which results in a stable triazole ring formation between EdU and fluorescent dye (called "Click reaction").…”

“…Since the small-sized fluorescent dye readily penetrates the cell and it easily reacts with EdU even in intact DNA double strand, EdU method is highly sensitive and much faster than a classical BrdU incorporation method. Also, EdU incorporation assay can be combined with multiplex cell surface/intracellular staining, which is very useful for many applications (Cappella et al, 2008; Diermeier-Daucher and Brockhoff, 2010). The original version of the Click reaction cannot be used for multiplex detection of some fluorophores such as GFP and R-PE which are easily damaged by high concentration of copper and reactive oxygen species.…”

Use of Fluorescence Lifetime Imaging Microscopy (FLIM) as a Timer of Cell Cycle S Phase

“…Classical methods of analysis of cell proliferation are based on incorporation of thymidine analogues during DNA replication and/ or labeling with a suitable tracers such as 3 H-thymidine, fluorescent antibody or dye reacting with 5-bromo-2’-deoxyuridine (BrdU) or 5-ethynyl-2’-deoxyuridine, respectively [1–3]. Fluorescence-based microscopy and flow cytometry platforms have replaced the unsafe autoradiography [4, 5], but they still remain tedious, mostly end-point, suffer from antibody variability, the need of epitope unmasking, limited in-depth staining and toxicity of click-reaction products.…”