The authors would like to thank Dr. Samuel H. Wilson at the National Institute of Environmental Health Sciences for the cell lines used in this work.

The authors have nothing to disclose.

Using the conditions described here, any confocal microscope with a UV-A or 405 nm laser, either integrated by the manufacturer or added by the end user, can induce DNA damage within the nucleus of a cell and allow the recruitment of DNA repair proteins to the site of induced DNA damage to be monitored. However, as noted in the protocol and representative results, wavelength selection, applied power, and damage characterization are all important issues that must be addressed first by the user in order to study a specific DNA repair pathway. Additionally, there are other considerations in experimental design that must also be considered by users.
In order to monitor a protein&#39;s behavior in live cells post micro-irradiation, a fluorescently labeled protein must be created. The availability of a wide range of fluorescent proteins that can be spectrally separated offers tools for creating protein fusions that can be used for characterizing cellular responses to the induction of DNA damage. Transient transfection of fluorescent proteins of interest allows for quick screening of damaging conditions, mutations, or even inhibitors, while stably transfected cells offer more control over expression levels and allow for other biochemical characterizations to be performed, validating micro-irradiation results. Spectrally distinct fluorescent proteins can also be utilized in the same cell to monitor interactions between proteins within the same pathway or in different pathways. Fluorescent proteins also eliminate the need for specific antibodies for each protein of interest and allow live cell monitoring of protein behavior for long periods of time after the induction of damage.
However, the use of fluorescent proteins also has drawbacks. Without genetic backgrounds deficient in the protein(s) of interest, endogenous proteins will compete with the fluorescently tagged proteins. Conjugation of the fluorescent tag to the N or C terminus of the protein may alter protein folding, hinder key protein-protein interactions, or block translocation signals; altering function and potentially impacting recruitment dynamics. These effects have been demonstrated in a number of reports where immunofluorescent staining demonstrated dramatically different recruitment and retention times for endogenous proteins at the damage site28. Use of transient transfection or stable clones can also alter the response observed, typically through variations in protein expression levels. Further, the use of multiple fluorescent proteins in a single experiment may also change the dynamics of recruitment, if protein levels are not equilibrated well or if recruitment of the larger fluorescently-tagged protein blocks the recruitment of other proteins. Finally, fluorescent proteins can act as weak sensitization agents, increasing the formation of reactive oxygen species, and potentially altering the DNA damage mixtures induced46,47. Despite these drawbacks, fluorescent proteins offer a number of distinct advantages in studying DNA repair with micro-irradiation, and if users incorporate appropriate controls, such as the damage characterization described here, they can provide new insight into damage response and protein-protein interactions3.
If live cell imaging is not required, immunofluorescence can be used to monitor the response to induced damage. By fixing cells at specific times post damage induction and staining with antibodies specific for proteins and lesions of interest, static snapshots of damage induction and the recruitment and retention of repair proteins can be built. Antibodies can be used to monitor the recruitment of multiple proteins of interest and/or post-translational modifications induced by the DNA damage response. Use of immunofluorescence eliminates the need for fluorescent proteins, and allows for endogenous protein behavior to be examined. However, this method too has its drawbacks. Highly specific antibodies are necessary, and permeabilization and blocking procedures need to be optimized to allow for detection of recruitment with sufficient signal intensity. Fixing procedures are not instantaneous, and this physical constraint limits the temporal resolution of this approach. Mapping the site of damage induction so cells can be re-located after staining can also present significant challenges. The confocal microscope used in this work allows for image-based registration as described above, so damaged cells can be relocated with high precision. If stage registration or etched coverglass is not available, the time investment involved in relocating cells without stage registration, coupled with the inherent delay between damage induction and the imaged recruitment, may make immunofluorescence unattractive to some users. However, the most accurate and complete micro-irradiation experimental designs will incorporate these types of approaches in parallel with the use of fluorescent proteins, as described in the presented protocol.
Here, both live cell imaging and immunofluorescence is used to demonstrate the utility of laser micro-irradiation. Immunofluorescent staining allows us to accurately examine the mixture of DNA damage created and the recruitment of proteins induced by each laser power, which allow us to better interpret the observed alterations in the recruitment and retention of XRCC1-GFP. Based on these results, the use of 405 nm lasers should be limited for examination of BER and SSBR proteins. Further, the optimal experimental design would include power measurements after the objective, a full damage mixture characterization for each cell line used, and validation of the recruitment and retention observed in fluorescently tagged proteins with immunofluorescence. Obviously, equipment, time, and cost considerations may make these optimal experimental designs impossible for some users. However, the importance of each of these elements is demonstrated here, and users should keep these considerations in mind when beginning micro-irradiation experiments.

Fluorescent microscopy has emerged as a powerful technique to visualize cellular architecture, examine protein localization, and monitor protein-protein and protein-DNA interactions. Using fluorescent microscopy to study DNA damage responses after the application of global DNA damaging agents, such as ultraviolet (UV) light, ionizing radiation, chemical oxidizing or alkylating agents, and/or chemotherapeutics has provided new insight into the initiation, signaling, and recruitment of DNA repair proteins to sites of DNA damage1,2. However, these global and asynchronous damaging events are limiting, if detailed information about the recruitment order, kinetics of association or dissociation, and the relationships between key DNA repair proteins are sought. Fortunately, advancements in laser scanning confocal microscopes, broader availability of damage-inducing laser wavelengths, and improvements in fluorescent proteins over the past 25 years have provided researchers with improved tools for examining these aspects of DNA repair, through targeted DNA damage induction.
Irradiation of cells with laser microbeams in order to study cellular and subcellular functions is a well-established tool in cell and radiation biology3. Application of this technique to the study of DNA repair emerged when Cremer and co-workers used a highly-focused UV (257 nm) laser microbeam system to induce DNA damage over a 0.5 &#181;m spot in Chinese hamster ovary (CHO) cells4 and established the induction of DNA photolesions by this system5. While offering significant improvements over UV microbeam methods at the time, adoption of this damaging system was limited due to its specialized set up, and its inability to generate double strand breaks (DSBs)6. Subsequent investigation of a variety of UV-B (290 - 320 nm) and UV-A (320 - 400 nm) wavelengths by a number of groups revealed that UV photoproducts, oxidative base lesions, single strand breaks (SSBs), and DSBs could be induced dependent on the laser wavelength and power applied4,7,8,9,10 (reviewed in 3). Further, combinations of these UV-B and UV-A wavelengths with sensitizing agents, like psoralen, bromodeoxyuridine (BrdU), and Hoechst dyes, were also found to induce DNA damage dependent on wavelength, power, and duration of exposure, though the power needed to induce damage is often lowered in the presence of these agents11,12,13,14,15. These advancements expanded the use of micro-irradiation, though there were still technical hurdles to be addressed for wider adoption of these methods.
Cremer and co-workers significantly advanced the field of micro-irradiation by precisely focusing the UV microbeam to apply significant damaging energy over a highly localized area in the cell. As confocal microscopes and laser microdissection systems advanced, tightly focused light was more broadly accessible; however, coupling UV sources to scopes and dealing with the chromatic aberrations they induced still presented significant challenges to most users3,6,16. As UV dyes increased in popularity throughout the 1990s, optics capable of focusing and capturing UV-excited fluorescence became more widely available16, and improvements in laser scanning offered users the ability to create highly focused UV excitation spots within cells6,17. However, it was not until the early 2000s that the true impact of this combination of tightly focused beams with higher intensity lasers was felt, when numerous reports emerged demonstrating that DNA strand breaks could be induced with and without sensitizers in the UV-A range6,10,18,19,20, 405 nm21,22,23,24,25,26, and even at longer visible wavelengths like 488 nm27. These advancements allowed for more widespread adoption of the micro-irradiation technique on a number of commercial systems. In parallel with these developments, two-photon techniques also emerged that allowed precise induction of DNA damage; though these advancements will not be discussed here, there are a number of review articles discussing these methodologies9,28,29,30.
With the current accessibility of confocal microscopes capable of delivering highly focused UV light and the wide-spread availability of fluorescent proteins to allow real-time tracking of DNA repair proteins, micro-irradiation techniques have evolved into powerful tools for examining DNA damage response and repair pathways. However, users need to be aware that the generation of DNA damage is highly dependent on the laser wavelength and the power applied to the sub-nuclear region. Use of UV-C (~260 nm) wavelengths allow direct DNA excitation and high selectivity for the induction of UV photoproducts7,8. UV-B and UV-A wavelengths produce mixtures of DNA damage (base lesion, SSBs, and DSBs), dependent on the applied power and the cellular background utilized7. Endogenous photosensitizers and anti-oxidant levels in the targeted cells can influence the DNA damage mixtures produced by these wavelengths. Additionally, the use of exogenous photosensitizers (BrdU, etc.) may assist in lowering the energy needed for the induction of DNA damage. However, these agents can induce DNA damage by themselves, and they may alter the cell cycle and chromatin structure, so their use may produce undesired effects that need to be considered by the user. Therefore, prior to using micro-irradiation to study DNA damage response and repair, careful consideration of the DNA repair pathway of interest, the wavelengths available for use, and the DNA damage mixture created is required.
Here, laser micro-irradiation is performed at two commonly used wavelengths, 355 nm and 405 nm, without sensitizers to demonstrate the mixtures of DNA damage induced by these wavelengths and the influences these damage mixtures have on examining the repair of SSBs and DSBs. Users should be aware that these wavelengths do not create a single species of strand breaks or base lesions. In order to discriminate between DNA repair pathways, users must carefully control the applied power over a specific region of the nucleus and characterize the induced damage using multiple strand break markers and DNA lesion antibodies. If properly applied and characterized, laser micro-irradiation can enrich some species of DNA damage, allowing users to assess repair of base lesions and SSBs or DSBs, with some specificity. Therefore, we have provided a method allowing users to reproducibly perform laser micro-irradiation, characterize the DNA damage mixtures induced by the applied laser dose, and perform data analysis.

<table style="border-collapse:collapse;width:548pt" width="730">
	<colgroup>
		<col style="width:220pt" width="293" />
		<col style="width:119pt" width="158" />
		<col style="width:84pt" width="112" />
		<col style="width:125pt" width="167" />
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	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl23" height="21" style="width:220pt;height:15.75pt" width="293">Nikon A1rsi laser scanning confocal microscope</td>
			<td class="xl21" style="width:119pt" width="158">Nikon</td>
			<td class="xl21" style="width:84pt" width="112"></td>
			<td class="xl23" style="width:125pt" width="167"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl23" height="21" style="height:15.75pt">NIS Elements software</td>
			<td class="xl21" style="width:119pt" width="158">Nikon</td>
			<td class="xl21" style="width:84pt" width="112"></td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">355 nm laser</td>
			<td class="xl23">PicoQuant VisUV</td>
			<td class="xl21" style="width:84pt" width="112"></td>
			<td class="xl23">Radiation source</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Galvanometer photoactivation miniscanner</td>
			<td class="xl21" style="width:119pt" width="158">Bruker</td>
			<td class="xl21" style="width:84pt" width="112"></td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Microscope slide photodiode power sensor</td>
			<td class="xl21" style="width:119pt" width="158">THORLabs</td>
			<td class="xl21" style="width:84pt" width="112">S170C</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Fiber photodiode power sensor</td>
			<td class="xl21" style="width:119pt" width="158">THORLabs</td>
			<td class="xl21" style="width:84pt" width="112">S150C</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Digital handheld optical power and energy meter</td>
			<td class="xl21" style="width:119pt" width="158">THORLabs</td>
			<td class="xl21" style="width:84pt" width="112">PM100D</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">CHO-K1</td>
			<td class="xl21" style="width:119pt" width="158"></td>
			<td class="xl21" style="width:84pt" width="112"></td>
			<td class="xl23">From Dr. Samuel H. Wilson, NIEHS</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Minimal essential media</td>
			<td class="xl28">Hyclone</td>
			<td class="xl21" style="width:84pt" width="112">SH3026501</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Fetal bovine serum</td>
			<td class="xl21" style="width:119pt" width="158">Atlanta biologicals</td>
			<td class="xl21" style="width:84pt" width="112">S11550</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">XRCC1-GFP</td>
			<td class="xl21" style="width:119pt" width="158">Origene</td>
			<td class="xl27">RG204952</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Jetprime</td>
			<td class="xl21" style="width:119pt" width="158">Polyplus transfection</td>
			<td class="xl27">11407</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Geneticin</td>
			<td class="xl21" style="width:119pt" width="158">ThermoFisher</td>
			<td class="xl27">10131035</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">4 chambered coverglass</td>
			<td class="xl21" style="width:119pt" width="158">ThermoFisher</td>
			<td class="xl21" style="width:84pt" width="112">155382</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">8 chambered coverglass</td>
			<td class="xl21" style="width:119pt" width="158">ThermoFisher</td>
			<td class="xl21" style="width:84pt" width="112">155409</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Anti 53BP-1</td>
			<td class="xl21" style="width:119pt" width="158">Novus</td>
			<td class="xl21" style="width:84pt" width="112">NB100304</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Anti-phospho-histone H2AX&#160;</td>
			<td class="xl21" style="width:119pt" width="158">Millipore</td>
			<td class="xl21" style="width:84pt" width="112">05-636-I</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Anti cyclobutane pyrimidine dimer</td>
			<td class="xl21" style="width:119pt" width="158">Cosmo Bio clone</td>
			<td class="xl21" style="width:84pt" width="112">CAC-NM-DND-001</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Anti 8-oxo-2&#180;-deoxyguanosine&#160;</td>
			<td class="xl21" style="width:119pt" width="158">Trevigen</td>
			<td class="xl21" style="width:84pt" width="112">4354-MC-050</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Alexa 488 goat anti-mouse</td>
			<td class="xl21" style="width:119pt" width="158">ThermoFisher</td>
			<td class="xl21" style="width:84pt" width="112">A11029</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Alexa 546 goat anti-rabbit</td>
			<td class="xl21" style="width:119pt" width="158">ThermoFisher</td>
			<td class="xl21" style="width:84pt" width="112">A11010</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">4&#8217;,6-Diamidino-2-Phenylindole (DAPI)</td>
			<td class="xl21" style="width:119pt" width="158">ThermoFisher</td>
			<td class="xl21" style="width:84pt" width="112">R37606</td>
			<td class="xl23">Caution toxic!</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Phosphate buffered saline</td>
			<td class="xl21" style="width:119pt" width="158">ThermoFisher</td>
			<td class="xl21" style="width:84pt" width="112">0780</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Normal goat serum</td>
			<td class="xl21" style="width:119pt" width="158">ThermoFisher</td>
			<td class="xl21" style="width:84pt" width="112">31873</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Bovine serum albumin (BSA)</td>
			<td class="xl21" style="width:119pt" width="158">Jackson Immuno Research</td>
			<td class="xl21" style="width:84pt" width="112">001-000-162</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">37% Formaldehyde</td>
			<td class="xl21" style="width:119pt" width="158">ThermoFisher</td>
			<td class="xl21" style="width:84pt" width="112">9311</td>
			<td class="xl23">Caution toxic!</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Ethanol</td>
			<td class="xl20">Decon Labs</td>
			<td class="xl20">2716</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Methanol</td>
			<td class="xl21" style="width:119pt" width="158">VWR</td>
			<td class="xl21" style="width:84pt" width="112">BDH1135</td>
			<td class="xl23">Caution toxic!</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">HCl</td>
			<td class="xl20">Fisher</td>
			<td class="xl20">SA49</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Sodium azide</td>
			<td class="xl21" style="width:119pt" width="158">Sigma-Aldrich</td>
			<td class="xl18" style="width:84pt" width="112">S2002</td>
			<td class="xl23">Caution toxic!</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:220pt;height:15.75pt" width="293">Tris Hydrochloride</td>
			<td class="xl20">Amresco</td>
			<td class="xl20">O234</td>
			<td class="xl23"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:220pt;height:15.75pt" width="293">Triton X-100</td>
			<td class="xl21" style="width:119pt" width="158">Sigma-Aldrich</td>
			<td class="xl21" style="width:84pt" width="112">T8787</td>
			<td class="xl29"></td>
		</tr>
	</tbody>
</table>

application of laser micro-irradiation for examination of single and double strand break repair in mammalian cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Characterization of induced DNA damage 
Induction of base lesions and strand breaks is dependent on the laser dose applied to the selected nuclear area and the cellular microenvironment of the cell model used7. Fluorescent proteins fused to repair proteins, like XRCC1, 53BP1, Ku70, or Rad51, provide useful single and double strand break markers for establishing the minimal energy required to see accumulation of a fluorescent protein within a damage ROI above the background fluorescence9,19,31. Once conditions that induce a response are found, it is critical to characterize the damage mixture induced by that specific wavelength and dose. Attenuation of the dose and duration at the wavelength used can allow the user to minimize the formation of complex damage mixtures. Low laser doses in the UV-A range have been demonstrated to produce predominantly SSBs and a small amount of base lesions, appropriate for studying SSBR and BER pathways10,28. Increasing the dose creates more complex base lesions, oxidative and UV induced, and induces more significant numbers of DSBs7,10. While the induction of a single species of DNA damage is desirable for examination of specific DNA repair pathways, it is more likely that users are inducing a mixture of DNA lesions, with a specific lesion like SSBs being much more frequent than base lesions or DSBs. This is similar to DNA damage mixtures induced by chemical agents like hydrogen peroxide (H2O2) or methyl methanesulfonate (MMS)32. Users need to be aware when they report results that damage mixtures can occur, and careful characterization of the dose and lesions at the induced damage site are necessary to ensure the reproducibility and comparability of their results.
In micro-irradiation studies, XRCC1-GFP is often used as a marker for the induction of base lesions and SSBs9,28. XRCC1 is a scaffold protein that plays an important role in SSB repair (SSBR) and base excision repair (BER), and also participates in other repair pathways, like nucleotide excision repair (NER)33,34,35. It plays an important coordinating role in DNA repair, interacting with a number of key proteins, including poly(ADP-ribose) polymerase 1 (PARP-1), DNA polymerase &#946; (Pol &#946;), and DNA ligase III. We utilized XRCC1-GFP stably expressed in CHO-K1 cells to determine the laser doses required to generate SSBs and DSBs. We first identified the minimum dose required to induce an observable recruitment of XRCC1-GFP for each wavelength (Figure 1). For the 355 nm wavelength, a 2 s dwell time over the defined damage ROI generated an increased fluorescent signal within that ROI, indicating the induction of DNA damage that was detectable over the background (Figure 1A). For 405 nm, an 8 fps scan rate was needed to generate an observable recruitment to the damage ROI (Figure 1A). The dose was then increased (10 s for 355 nm and 0.5 fps for 405 nm) to create a more intense damage ROI (Figure 1A).
Recruitment and retention of XRCC1-GFP at the site of induced damage was then monitored by timelapse imaging. Retention of the protein at the site of DNA damage may indicate on-going DNA repair, while dissociation of the protein from the site of induced damage is often considered a marker for completion of BER or SSBR. However, there has been no clear evidence linking the dissociation of XRCC1 from laser-induced DNA damage sites with the completion of repair. Recruitment of the protein to the site of damage is measured by reporting the mean intensity of the fluorescent signal within the damaged ROI over the mean fluorescent signal measured for the entire nucleus (Figure 1B). This type of normalization helps address intensity fluctuations in the nuclear signal, though other normalization techniques can be employed depending on the cellular distribution of the protein of interest. Here, XRCC1 is localized in the nuclear compartment, so normalization to the nuclear area measures the redistribution of the signal to the damage ROI. The ROI mean fluorescent intensity is then recorded for each image in the timelapse, including the pre-damage image, and graphed as a function of time (Figure 1C).
We then further characterized the damage induced by the two selected laser doses to examine the formation of DNA base lesions. First, formation of CPD, a bulky UV-induced lesion, was probed by immunofluorescence as a marker for NER type lesions (Figure 2A). Then, the oxidatively induced base lesion8-oxodG was probed as a marker for BER type lesions (Figure 2B). No significant increase in CPD lesions were observed at the low dose exposure for both wavelengths (2 s for 355 nm and 8 fps for 405 nm), while the high dose treatment at both wavelengths (10 s for 355 nm and 0.5 fps for 405 nm) did show a significant increase in fluorescent signal observed within the damage ROI (Figure 2A). A scatter plot of the CPD ROI mean intensity for each damaged cell shows heterogeneity in damage formation and detection at both wavelengths and doses, indicating that a low level of CPD lesions may be present at the lower doses, but the load may not be significantly detected until a higher dose is applied. The scatterplot also suggests that inefficiency in antibody detection that may limit accurate quantification of the damage mixtures.
This is further highlighted in the detection of oxidatively induced DNA lesions by the marker 8-oxodG. No clear increase in fluorescent signal within the damage ROI was observed for 8-oxodG at either laser wavelength or dose used (Figure 2B). The antibody used for this work is consistent with previous publications9,10,36; however, it should be noted that there can be limitations in observing the formation of 8-oxodG with antibodies37,38. Confirmation of the lack of oxidatively induced lesions is also recommended by a second marker, like recruitment of 8-Oxoguanine DNA Glycosylase (OGG1), the enzyme responsible for removal of 8-oxodG from the DNA10. We did not observe OGG1 recruitment to our DNA damage sites; however, the formation of low levels of oxidatively induced DNA damage cannot be ruled out completely.
Finally, we examined the formation of DSBs using two markers, &#947;H2AX and 53BP-1, at the selected laser doses by immunofluorescence (Figure 3 &#38; 4). &#947;H2AX is commonly used as a strand break marker, but its specificity for DSBs has been questioned in a number of reports39,40. Additionally, it is a phosphorylation event that propagates from the strand break site, so localization of the signal to a strand break can be limited due to this signal propagation. Therefore, combining &#947;H2AX with 53BP-1 allows for more accurate assessment of DSB formation within the damage ROI.
The response of &#947;H2AX and 53BP-1 to micro-irradiation is both wavelength and dose dependent. The low dose (2 s) stimulation at 355 nm elicits no response at 5 and 20 min, and a weak and variable response 10 min post irradiation (Figure 3A) for both markers. The high dose (10 s) of 355 nm micro-irradiation induces an increased fluorescent signal within the damage ROI at 5, 10, and 20 min post irradiation that is reduced at 40 min (Figure 3B). These results indicate that careful titration of the 355 nm dose is required to minimize cross-stimulation of repair pathways, as demonstrated by the reduced detection of the double strand break marker &#947;H2AX at the early time points (&#60; 20 min) at the low dose applied.
Similar experiments were performed using both low (8 fps) and high dose (0.5 fps) 405 nm laser stimulation (Figure 4). At this wavelength significant accumulation of fluorescent intensity within the damage ROI was observed for both 53BP-1 and &#947;H2AX regardless of applied dose, indicating that these doses generate a complex mixture of single and double strand breaks almost immediate after the induction of DNA damage (Figure 4). Additionally, the high doses of 405 nm show an increase in pan-nuclear &#947;H2AX staining within 10 min of damage induction (Figure 4B, bottom) that makes detection of the damage ROI difficult to report, while the 53BP-1 accumulation is more contained within the damage ROI.
These results clearly demonstrate that 405 nm micro-irradiation is not appropriate for monitoring SSBR or BER, and that multiple markers for DNA adducts and strand breaks should be employed to fully characterize the induced lesions and DNA repair responses.
Dose-dependent alteration in recruitment and retention of XRCC1-GFP 
Once the induced DNA damage has been characterized, laser micro-irradiation can be an ideal platform for studying the dynamics of DNA repair proteins. The retention and dissociation kinetics of the XRCC1-GFP shows a dose dependency (Figure 1), which is not unexpected given the induction of different damage mixtures by each wavelength. The highest irradiation doses (10 s and 0.5 fps) show a higher intensity recruitment of XRCC1-GFP relative to the lower doses and longer retention of the XRCC1-GFP at the site of damage over the 20 min time course (Figure 1C). This indicates that the DNA damage created at higher doses for both 355 and 405 nm is likely not resolved during the course of the experiment, which is consistent with the appearance and retention of DSB markers, &#947;H2AX and 53BP-1 (Figures 3 &#38; 4).
Interestingly, the lower damage doses (2 s and 8 fps) show rapid recruitment of XRCC1-GFP to the damage sites and dissociation of XRCC1-GFP over the experimental time course to pre-irradiation levels (Figure 1C). Without the complete characterization of the damage mixture, this may lead to the conclusion that SSBs and base lesions are completely resolved using these damaging conditions. However, the presence of &#947;H2AX and 53BP-1 at 40 min for 355 nm and at 5 min for the 405 nm may lead to different interpretations. For the 355 nm 2 s dose, the damage mixture may be predominantly SSBs, so the appearance of DSB markers at 40 min may indicate that some unrepaired lesions may be leading to DSBs or that DSBs generated by this energy are repaired on a longer time scale. Time scale differences between SSBR and DSB repair have been previously reported28,41,42. Similarly, the 405 nm low dose (8 fps) dissociation may indicate a low level of SSBs or a clustering of SSBs that are rapidly converted to DSBs, which has been noted for high damage micro-irradiation and other DNA damaging agents previously43,44,45.
Together these results highlight the importance of characterizing the induced damage mixtures and utilizing multiple DNA repair proteins and markers for interpreting the recruitment and retention of DNA repair proteins at sites of induced damage.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56265/56265fig1.jpg" /> 
Figure 1. Laser micro-irradiation induces recruitment of XRCC1-GFP.&#160; 
(A) CHO-K1 cells stably expressing XRCC1-GFP were irradiated and imaged before and immediately after damage induction. Arrows indicate location of micro-irradiation dose and scale bar is 10 &#181;m. (B) Recruitment is measured by determining the mean fluorescent intensity within the damage ROI and normalizing to the mean fluorescent intensity of the entire nucleus. (C) Dynamics of recruitment can be measured for each timelapse image.Graphs are representative of two independent experiments with error bars representing the SEM (n=24).&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56265/56265fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56265/56265fig2.jpg" /> 
Figure 2. Laser micro-irradiation induces nucleotide damage.&#160; 
CHO-K1 cells were subjected to laser micro-irradiation and fixed immediately after damage induction. Immunofluorescence was performed to detect CPD and 8-oxodG adducts. (A) Top, scatter plot of the ROI mean fluorescence intensities observed in damaged cells after CPD staining. Bottom, representative images of CPD staining. Arrows indicate location of micro-irradiation and the scale bar is 10 &#181;m (n=12). (B) Representative images for 8-oxodG staining. Arrows indicate location of micro-irradiation and the scale bar is 10 &#181;m.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56265/56265fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56265/56265fig3.jpg" /> 
Figure 3. DNA double strand break markers respond to 355 nm micro-irradiation in a dose dependent manner.&#160; 
CHO-K1 cells were subjected to micro-irradiation and fixed at the time points indicated post-stimulation. Immunofluorescence for the DSB markers &#947;H2AX and 53BP-1 was performed. (A) Scatter plot of normalized damage ROI mean fluorescence intensities measured for undamaged and damaged cells. Error bars are representative of the SEM (n=12). (B) Representative images for &#947;H2AX and 53BP-1 staining. Arrows indicate location of micro-irradiation and the scale bar is 10 &#181;m.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56265/56265fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56265/56265fig4.jpg" /> 
Figure 4. DNA double strand break markers robustly respond to 405 nm micro-irradiation.&#160; 
CHO-K1 cells were subjected to micro-irradiation and fixed at the time points indicated post stimulation. Immunofluorescence for the DSB markers &#947;H2AX and 53BP-1. (A) Scatter plot of normalized damage ROI mean fluorescence intensities measured for undamaged and damaged cells. Error bars are representative of the SEM (n=12). (B) Representative images for &#947;H2AX and 53BP-1 staining. Arrows indicate location of micro-irradiation and the scale bar is 10 &#181;m.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56265/56265fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

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9.6K Views.  University of South Alabama Mitchell Cancer Institute. Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

Video: Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of &#947;H2AX and 53BP1, the combined analysis of &#947;H2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on &#947;H2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the &#947;H2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of &#947;H2AX and 53BP1.

This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of &#947;H2AX and 53BP1.

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas ...

The Application of 1% Methylene Blue Dye As a Single Technique in Breast Cancer Sentinel Node Biopsy

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery (BCS), a separate incision in the lower axilla hairline was made to find the sentinel nodes (SNs). In mastectomy, the SNs were identified through the same mastectomy incision. The SNs were described as blue nodes or nodes with lymphatic blue channels. An anatomical landmark in the axilla was used to facilitate SNs identification. The SNs metastases were evaluated by intraoperative frozen section analysis and histopathology examination as it is a gold standard. Here, we described the MBD as the lone technique in breast cancer sentinel node biopsy (SNB) which could be useful when radioisotope tracer or patent or isosulfan blue dye (PBD) cannot be provided.

In Indonesia, sentinel node biopsy (SNB) is not routinely performed for breast cancer surgery because of the limitation to provide radioisotope tracer and isosulfan or patent blue dye (PBD). To overcome this obstacle, we applied 1% methylene blue dye (MBD) as a single agent to map the sentinel nodes (SNs).

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery ...

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% with current standard of care. Tumors often recur within 9 months following initial surgery, radiation and chemotherapy, at which point treatment options become limited. This highlights the pressing need for the development of better therapeutics to prolong survival and increase the quality of life for these patients.
Tumor Treating Fields (TTFields) therapy was developed to take advantage of the effect of low frequency alternating electrical fields on cells for cancer therapy. TTFields have been demonstrated to disrupt cells during mitosis and slow tumor growth. There is also growing evidence that they act through stimulating immune responses within exposed tumors. The advantages of TTFields therapy include its noninvasive approach and increased quality of life compared to other treatment modalities such as cytotoxic chemotherapies. The Food and Drug Administration approved TTFields therapy for the treatment of recurrent glioblastoma in 2011 and for newly diagnosed glioblastoma in 2015. We report on the effects of TTFields during mitosis, the results of electric fields modeling, and proper transducer array placement. Our protocol outlines the clinical application of TTFields on a patient post-surgery, using the second-generation device.

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Application of Hemostatic Devices in Laparoscopic Hepatectomy

Laparoscopic hepatectomy is considered a conventional method for treating benign and malignant liver diseases because it is a minimally invasive method. Despite its non-invasive aspect, bleeding and bile leakage occur in liver parenchyma tissue resection during the operation or in the post-operation period, indicating the requirement for high-grade hemostatic devices, such as ultrasonic surgical aspiration, bipolar electrocoagulation, etc. The lack of availability of these high-grade hemostatic devices prevents laparoscopic hepatectomy from becoming a generalized procedure in basic medical organizations. In view of the situation mentioned above, a suite of simple and easy hemostatic devices is developed in this protocol, which includes a harmonic scalpel, monopole electrocoagulation, and a single lumen catheter, to innovatively perform liver parenchyma tissue resection. First of all, the porta hepatis or hepatic pedicle is occluded intermittently by a single lumen catheter, followed by clamping for 15 min and releasing for 5 min. Subsequently, using the harmonic scalpel, clamping and crushing of the liver are done to cut off the hepatic parenchyma tissue and to reveal the intrahepatic arteries, veins, and bile ducts. Lastly, the bleeding spots are coagulated by using monopole electrocoagulation at each spot. Intrahepatic pipeline structures are then visible by using these methods, which could stop bleeding easily, reduce the incidence rate of bile leakage, and improve the safety and feasibility of laparoscopic hepatectomy. Therefore, the simple and easy hemostatic devices shown here are suitable for conducting procedures in primary medical institutions.

High-grade hemostatic devices are essential for laparoscopic hepatectomy. However, these devices are not generalized in basic medical organizations. Therefore, a suite of simple and easy hemostatic devices is shown in this article, which can make the laparoscopic hepatectomy easier to perform.

Laparoscopic hepatectomy is considered a conventional method for treating benign and malignant liver diseases because it is a minimally invasive method. ...

A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves different RNA-binding proteins in the nucleus. Mature miRNAs frequently mediate gene silencing, and this has become an important tool for comprehending post-transcriptional events. Besides that, it can be explored as a promising methodology for gene therapies. However, there is currently a lack of direct methods for assessing miRNA expression in mammalian cell cultures. Here, we describe an efficient and simple method that aids in determining miRNA biogenesis and maturation through confirmation of its interaction with target sequences. Also, this system allows the separation of exogenous miRNA maturation from its endogenous activity using a doxycycline-inducible promoter capable of controlling primary miRNA (pri-miRNA) transcription with high efficiency and low cost. This tool also allows modulation with RNA-binding proteins in a separate plasmid. In addition to its use with a variety of different miRNAs and their respective targets, it can be adapted to different cell lines, provided these are amenable to transfection.

We developed an intronic microRNA biogenesis reporter assay to be used in cells in vitro with four plasmids: one with intronic miRNA, one with the target, one to overexpress a regulatory protein, and one for Renilla luciferase. The miRNA was processed and could control luciferase expression by binding to the target sequence.

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves ...

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural microenvironment and stromal cell interaction, cannot be entirely replicated with in vitro assays; thus, animal models are critical for investigating and understanding the effects of therapeutic intervention. However, most brain tumor xenografting methods do not produce brain metastases consistently in terms of the time frame and tumor burden. Brain metastasis models generated by intracardiac injection of cancer cells can result in unintended extracranial tumor burden and lead to non-brain metastatic morbidity and mortality. Although intracranial injection of cancer cells can limit extracranial tumor formation, it has several caveats, such as the injected cells frequently form a singular tumor mass at the injection site, high leptomeningeal involvement, and damage to brain vasculature during needle penetration. This protocol describes a mouse model of brain metastasis generated by internal carotid artery injection. This method produces intracranial tumors consistently without the involvement of other organs, enabling the evaluation of therapeutic agents for brain metastasis.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Most brain metastasis mouse models are complicated by systemic metastases confounding analysis of mortality and therapeutic intervention outcomes. Presented here is a protocol for internal carotid injection of cancer cells that produces consistent intracranial tumors with minimal systemic tumors.

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Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D tumoroids. Since patient derived tumor organoids can retain the characteristics of the original tumor, these reliable preclinical models enable cancer drug screening and the study of drug resistance mechanisms. However, CRC related death in patients is mostly associated with the presence of metastatic disease. It is therefore essential to evaluate the efficacy of anti-cancer therapies in relevant in vivo models that truly recapitulate the key molecular features of human cancer metastasis. We have established an orthotopic model based on the injection of CRC patient-derived cancer cells directly into the cecum wall of mice. These tumor cells develop primary tumors in the cecum that metastasize to the liver and lungs, which is frequently observed in patients with advanced CRC. This CRC mouse model can be used to evaluate drug responses monitored by microcomputed tomography (&#181;CT), a clinically relevant small-scale imaging method that can easily identify primary tumors or metastases in patients. Here, we describe the surgical procedure and the required methodology to implant patient-derived cancer cells in the cecum wall of immunodeficient mice.

This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. The model recapitulates advanced colorectal cancer metastatic disease and allows for the evaluation of new therapeutic drugs in a clinically relevant scenario of lung and liver metastases.

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...