The APO-BrdU TUNEL Assay Kit includes complete protocols for use in flow cytometry applications, though it may also be adapted for use with fluorescence microscopy. Each kit contains:

\begin{itemize}
\item Terminal deoxynucleotidyl transferase (TdT), for catalyzing the addition of BrdUTP at the break sites
\item 5-Bromo-2'-deoxyuridine 5'-triphosphate (BrdUTP)
\item Alexa Fluor 488 dye–labeled anti-BrdU mouse monoclonal antibody PRB-1, for detecting BrdU labels
\item Propidium iodide/RNase staining buffer, for quantitating total cellular DNA
\item Reaction, wash and rinse buffers
\item Positive control cells (a fixed human lymphoma cell line)
\item Negative control cells (a fixed human lymphoma cell line)
\item Detailed protocols
\end{itemize}

Sufficient reagents are provided for approximately 60 assays of 1 mL samples, each containing approximately $1 \times 10^{6}$ cells/mL.

\subsection{Detecting DNA Strand Breaks with ChromaTide Nucleotides}

Break sites have traditionally been labeled with biotinylated dUTP, followed by subsequent detection with an avidin or streptavidin conjugate \cite{37,38,39,40} (Section 7.6, Table 7.9). However, a more direct approach for detecting DNA strand breaks in apoptotic cells is possible via the use of our ChromaTide BODIPY FL-14-dUTP (C7614) as a TdT substrate \cite{41,42} (Figure \ref{fig:figure_15_5_9}). The single-step BODIPY FL dye–based assay has several advantages over indirect detection of biotinylated or haptenylated nucleotides, including fewer protocol steps and increased cell yields. BODIPY FL dye–labeled nucleotides have also proven superior to fluorescein-labeled nucleotides for detection of DNA strand breaks in apoptotic cells because they provide stronger signals, a narrower emission spectrum and less photobleaching \cite{41} (Figure \ref{fig:figure_15_5_9}).

\textit{In situ} DNA modifications by labeled nucleotides have been used to detect DNA fragmentation in what may be apoptotic cells in autopsy brains of Huntingtons and Alzheimer disease patients \cite{43,44,45,46}. DNA fragmentation is also associated with amyotrophic lateral sclerosis \cite{47}.

Analogous to TdT's ability to label double-strand breaks, the \textit{E. coli} repair enzyme DNA polymerase I can be used to detect single-strand nicks \cite{48,49}, which appear as a relatively early step in some apoptotic processes \cite{50,51,52}. Because our ChromaTide BODIPY FL-14-dUTP (C7614) and ChromaTide fluorescein-12-dUTP \cite{53,54} (C7604) are incorporated into DNA by \textit{E. coli} DNA polymerase I, they are also effective for \textit{in situ} labeling with the nick translation method \cite{55}.

\subsection{High-Content Analysis of Genotoxicity and Cytotoxicity}

In mammalian cells, a double-strand break (DSB) in genomic DNA is a potentially lethal lesion. One of the earliest known responses to DSB formation is phosphorylation of H2A histones. Specifically, DNA damaging agents induce phosphorylation of histone variant H2AX at Ser139, leading to the formation of DNA foci at the site of nuclear DSBs \cite{56,57,58}.

The HCS DNA Damage Kit (H10292) was developed to enable simultaneous quantitation of two cell health parameters, genotoxicity and cytotoxicity, by high-content analysis in the same cell (Figure \ref{fig:figure_15_5_10}). DNA damage is measured as an indication of genotoxicity and accomplished by specific antibody-based detection of phosphorylated H2AX (Ser139) in the nucleus. Cytotoxicity is measured with the Image-iT DEAD Green viability stain (also available as a stand-alone reagent, I10291).

\begin{figure}
\centering
\includegraphics{figure_15_5_9.pdf}
\caption{HL-60 cells treated with camptothecin for 3 hours. The DNA strand nicks characteristic of apoptosis were detected with the TUNEL (terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling) assay using the fluorescently labeled nucleotide ChromaTide BODIPY FL-14-dUTP (C7614). Image contributed by Zbigniew Darzynkiewicz, Cancer Research Institute, New York Medical College.}
\label{figure_15_5_9}
\end{figure}

\begin{figure}
\centering
\includegraphics{figure_15_5_10_1.pdf}
\includegraphics{figure_15_5_10_2.pdf}
\includegraphics{figure_15_5_10_3.pdf}
\caption{Detection of genotoxicity and cytotoxicity in valinomycin-treated A549 cells using the HCS DNA Damage Kit (H10292). A549 cells were treated with 30 $\mu$M or 120 $\mu$M valinomycin for 24 hr before performing the assay. With increasing concentrations of valinomycin, cells showed genotoxic effects as indicated by detection with a pH2AX antibody in conjunction with Alexa Fluor 555 goat anti-mouse IgG labeled secondary antibody, and cytotoxic effects as indicated by staining with the Image-iT DEAD Green viability stain (green fluorescence). Blue-fluorescent Hoechst 33342 was used as a nuclear counterstain, and Alexa Fluor 647 phalloidin was used to visualize F-actin (pseudocolored magenta). The image on the left shows untreated cells with intact F-actin cytoskeletons and no evidence of cytotoxicity or genotoxicity. The image on the right shows cells treated with 120 $\mu$M valinomycin, which completely disrupted the actin cytoskeletons, increased levels of DNA damage and compromised plasma membrane integrity.}
\label{figure_15_5_10}
\end{figure}