The authors would like to thank the Microscope and Animal Models Imaging Facilites at WVU for the use of their equipment supported by grant number P20GM103434. Additionally, this work&#160;was supported by grant&#160;number P20GM121322 from the&#160;National Institue of General&#160;Medical Sciences, by National&#160;Cancer Institute grant number&#160;F99CA25376801,&#160;and the Mylan Chair Endowment Fund.

<ol>
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A., Rodermond, H. M., Stap, J., Haveman, J., van Bree, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonogenic+assay+of+cells+in+vitro.">Clonogenic assay of cells in vitro.</a> Nature Protocols. 1 (5), 2315-2319 (2006).</li><li>Ford, E. 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The authors have no disclosures to make.

The above protocol describes a user-friendly approach for radiation dosimetry, determination of &#945;/&#946; values in cancer cell lines, and a brief example of an approach for irradiation in a preclinical model of breast cancer brain metastasis. These methods can be used to study any model of cancer and are not just limited to brain metastasis of breast cancer. In this section we will discuss the relevant intricacies underlying preclinical radiotherapy experiments.
Dosimetry involves two parts: 1) calibrate the output with a farmer chamber, so that the dose rate of the x-ray unit is established, and 2) prepare a practical dosimetry measurement system using radiochromic film. With regards to output calibration, TG-61 provides a reproducible method in water. The protocol here uses Gammex RMI 457 solid water, as recommended by XStrahl, the manufacturer of the irradiator. Although relative dosimetry (profiles or depth dose curves normalized to maximum dose) analysis with solid water, agrees to better than 1% with that of water, there is a difference of about 3 to 4% in absolute dose due to a higher mass energy absorption coefficient for solid water compared to water. However, as all installations of the XStrahl system use the solid water protocol for output calibration, we did not correct for these differences. Knowing the output allows the calculation of the exposure time required to deliver a desired dose. Placing film in the same setup as the farmer chamber allows us to deliver known doses to the film. Scanning the film then provides optical densities. The dose to the film can then be graphed against the corresponding net optical density (difference in optical density after and before exposure). This produces a film calibration curve. When we change experimental setups, the dose rate in that situation could change, since dose rate depends on field size, depth and the material being irradiated. Exposing film with the experimental setup provides us with a net optical density, and using the film calibration curve, we can then determine the corresponding dose. Dividing this dose by the time the film was irradiated, we get the dose rate. This dose rate can then be used to calculate the exposure time to deliver a desired dose for the given experimental setup. The protocol described above handles several nuances associated with film dosimetry. For example, after exposure, the film requires approximately 24 hours for the chemical reactions in the film&#8217;s active layer to be virtually complete. Not waiting for this amount of time will lead to a lower optical density.
For any study to have reproducible dosimetry it is important to know and understand several of the key elements of a given irradiator. In particular, it is crucial to know and detail to other researchers the make and model of the irradiator used, the source type (x-ray, radioactive, etc.), energy, half-value layer, field size, source to surface and source to isocenter distances, size of material irradiated, attenuation before and backscatter after the irradiated material, experiment-specific dose rate, fractionation schema, exact dosimetry equipment utilized, and the dosimetry protocol used. All of these points of information are what cohesively describe the beam quality of a given irradiator prior to delivering a dose to any animal or cell19. Another pertinent point of information from this protocol and others is that the dose rate achieved in Protocol 1 is simply the output of the irradiator being used. For any given experiment it is important to define the dose rate for that particular setup (Protocol 4) by comparison with a generated radiochromic film calibration curve (Protocol 2).
In vitro experimentation provides important details about the radiobiologic behavior of cancer cell lines. In vitro clonogenic cell survival assays accurately estimate and quantify the inherent radio-sensitivity of a cell line20, aiding in the design of fractionation schedules in subsequent cellular or small animal experiments21. Specifically, these assays approximate values for the parameters &#945; and &#946; that are used in the linear-quadratic model to predict cell death in response to radiotherapy according to the equation:
<img alt="Equation 9" src="/files/ftp_upload/61692/61692equ09.jpg" /> (Equation 9)
where SF is the surviving fraction of clonogenically viable cells, D is radiation dose in Gy, and &#945; and &#946; are fitted parameters22. The ratio &#945;/&#946; provides an inherent measure of cellular radio-sensitivity, with higher values correlating with increased sensitivity of a cell line22. Because this functional relationship is non-linear with respect to dose, the biologic effects of a radiotherapy fractionation scheme are not only related to the total delivered dose but also the number and size of fractions23. The biologic effective dose (BED) is a measure of the true biological dose delivered to a tissue and permits direct comparison of different fractionations schemes24,25. The BED equation only requires an estimate of &#945;/&#946;, and is displayed below:
<img alt="Equation 14" src="/files/ftp_upload/61692/61692equ14.jpg" /> (Equation 14)
where n is the number of fractions of dose D. Clonogenic cell survival assays estimate &#945;/&#946; and facilitate the direct comparison of radiotherapy fractionation schemes via the BED equation. Incorrect conclusions may be drawn regarding a tissue or organ response to radiotherapy (or combinations of radiotherapy with other modalities) if the BED in the treatment groups is not equitable within or between experiments. For example, 2 fractions of 10 Gy compared with 4 fractions of 5 Gy do not yield the same BED, and thus these dosing schemes cannot be directly compared in terms of biologic response. The BED equation, while imperfect due to inherent limitations in the linear-quadratic model, reliably estimates equitable effects for a wide range of experimental treatment conditions24,25.
Clonogenic cell survival assays clearly play an important role in studying radiotherapy effects in cancer models, but in vitro experimentation offers a number of additional options to further explore mechanistic details of cancer cell radiobiology. Simple modifications of the clonogenic cell survival assay were used to determine the modes of action for some radio-sensitizing chemotherapies, such as paclitaxel or etoposide26,27. Further in vitro experimental options include immunocytochemistry studies to examine specific cellular repair pathways, such as &#947;-H2AX foci and/or 53BP1 staining for double-stranded DNA break repair28. These experiments may be of particular interest when comparing radiotherapy as a single modality with combination therapies, especially when probing mechanistic details for a given cell line. Other experimental options include cytokine measurements to examine the innate role of a cell&#8217;s inflammatory response to irradiation or analyses of the mode of cell death (i.e., apoptosis, necrosis, mitotic catastrophe, etc.) under different therapeutic conditions29,30,31. This type of experimentation can complement or replace animal experimentation and provide a more complete understanding of a cancer cell line&#8217;s radiobiology. Regardless of the choice of additional experiments to conduct, a standard clonogenic cell survival assay as described in protocol 3 is an important initial radiobiologic assessment of a cell line.
Clonogenic assays and radiation dosimetry provide the researcher with a means to precisely plan experiments to more directly resemble clinical scenarios. With the addition of preclinical cancer small rodent models, it is possible to study the response to radiation alone or in the context of a treatment plan in vivo. Prior to using animals, it is important to determine the relative dose output of the specific setup if it differs from the setup used for determination of dose output32,33. When it comes to determining a dose rate for field sizes of &#60;10 mm, use of an ionization chamber becomes less accurate due to alignment within a small field and partial volume averaging effects33. The use of radiochromic film to determine output in combination with in vivo immunohistochemical experiments has been used to determine output and dose deposition in the past16,34,35,36,37,38.

Cancers collectively represent the second-leading cause of death in the U.S. and in many countries around the globe1. Radiation therapy is a cornerstone of treatment for many tumor subtypes and is administered to about half of all cancer patients2,3. Patient outcomes for nearly all cancers have improved over time as equipment used to deliver radiation doses has steadily advanced and some effective multimodal therapy approaches were developed4,5,6, but recurrence and mortality rates for patients with certain types of tumors remain high7,8,9. Thus, radiotherapy for cancer continues to be an active area of basic and clinical research. Many pre-clinical radiotherapy studies employ the use of small-scale irradiators to deliver radiation doses to in vitro or animal models of cancers. With a multitude of potential experiments to conduct exploring mechanistic radiobiology details or novel treatments, common pitfalls may be encountered that lead to incorrect conclusions, poor reproducibility, and wasted resources. These pitfalls fall within three important areas: irradiator dosimetry, in vitro characterization of model cell lines, and in vivo irradiation dosing schedule and setup. Accurate and reproducible results from more advanced experiments are difficult to achieve without prior attention to these fundamental aspects of radiotherapy research.
The protocol detailed herein describes a generalized strategy for avoiding or mitigating these issues and draws upon several previously developed methodologies intended for independent use. These distinct methods have been merged so that a researcher interested in beginning or improving preclinical radiotherapy experiments could use this as a robust experimental layout. The suggested framework includes methodology for the commissioning of small-scale animal irradiators, for determining basic radiobiologic properties of model cancer cell lines, and for appropriately designing and administering a dosing and fractionation schedule for in vivo tumor models.

<table>
	<tbody>
		<tr>
			<td>Acetic acid, glacial</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td>This or comparable glacial acetic acid products are acceptable.</td>
		</tr>
		<tr>
			<td>Crystal Violet</td>
			<td>Sigma-Aldrich</td>
			<td>C6158</td>
			<td>This or comparable crystal violet products are acceptable.</td>
		</tr>
		<tr>
			<td>Digital Baraometer</td>
			<td>Fisher Scientific</td>
			<td>14-650-118</td>
			<td>For pressure and temperature measurements.</td>
		</tr>
		<tr>
			<td>Electrometer</td>
			<td>Standard Imaging</td>
			<td>CDX 2000B</td>
			<td>Calibrated by an ADCL; Need correction factor, Pelec</td>
		</tr>
		<tr>
			<td>Film</td>
			<td>Gafchromic</td>
			<td>EBT3 Film</td>
			<td>Comes in sheets of 25; calibration films and experimental films must come from same set</td>
		</tr>
		<tr>
			<td>Ionization Chamber</td>
			<td>Farmer</td>
			<td>PTW TN30013</td>
			<td>Calibrated by an ADCL @ two calibration points</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td>This or comparable methanol products are acceptable.</td>
		</tr>
		<tr>
			<td>Photo Scanner</td>
			<td>Epson</td>
			<td>Perfection V700</td>
			<td>Equivalent scanners are V800, V10000, V11000, V12000</td>
		</tr>
		<tr>
			<td>XenX</td>
			<td>Xstrahl</td>
			<td>NA</td>
			<td>Irradiator used.</td>
		</tr>
	</tbody>
</table>

irradiator commissioning and dosimetry for assessment of lq &alpha; and &beta; parameters, radiation dosing schema, and in vivo dose deposition

Radiation dosimetry is critical in the accurate delivery and reproducibility of radiation schemes in preclinical models for high translational relevance. Prior to performing any in vitro or in vivo experiments, the specific dose output for the irradiator and individual experimental designs must be assessed. Using an ionization chamber, electrometer, and solid water setup, the dose output of wide fields at isocenter can be determined. Using a similar setup with radiochromic films in the place of the ionization chamber, dose rates for smaller fields at different depths can also be determined. In vitro clonogenic survival assays of cancer cells in response to radiation treatment are inexpensive experiments that provide a measure of inherent radio-sensitivity of cell lines by fitting these data with the traditional linear-quadratic model. Model parameters estimated from these assays, combined with the principles of biologic effective doses, allows one to develop varying fractionation schedules for radiation treatment that provide equivalent effective doses in tumor-bearing animal experiments. This is an important factor to consider and correct for in comparing in vivo radiation therapy schedules to eliminate potential confounding of results due to variance in the delivered effective doses. Taken together, this article provides a general method for dose output verification preclinical animal and cabinet irradiators, in vitro assessment of radio-sensitivity, and verification of radiation delivery in small living organisms.

Following protocol 1 will provide a dose rate in Gy/min, which is specific to the irradiator being used. However, regardless of the type of irradiator, with a known dose rate a calibration curve can be generated using protocol 2 yielding similar films and a similar calibration curve to that in Figure 2A-B. A successful assay from protocol 3 will yield distinct, well-demarcated colonies of cells that stain homogenously violet. The estimate of &#945;/&#946; can be compared to literature values or other treatment groups to interpret the radio-sensitivity of the given cell line. Utilizing the calibration curve developed following protocol 2 and displayed in Figure 2B, protocol 4 will yield two film samples resembling Figure 2A that can be used to estimate required experimental irradiation times. If an on-board portal imaging camera is available for the irradiator being used, radiograms of small animals can be obtained with and without collimation. Overlaying these images will demonstrate the exact positioning of the collimated radiation beam relative to the small animal being treated as depicted in Figure 4A. Successful dose-deposition in protocol 5 can be confirmed following protocol 6. One indication that radiation is being deposited in an in vivo or in vitro systems is through the detection of double stranded DNA breaks. Illustrated in Figure 4B, the same mouse treated solely through the right hemisphere in Figure 4A, demonstrates positive &#947;H2AX staining only in the treated hemisphere. In this figure, the nuclei are stained with DAPI to show two things; 1) the whole are of the brain which the anti &#947;H2AX antibody was applied to during histological analysis, and 2) the untreated hemisphere of the brain remains unstained.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61692/61692fig01.jpg" /> 
Figure 1: Rough set up of ionization chamber and water phantom set up for determination of dose output.&#160;The pictogram illustrates a basic setup utilizing the various components required for dosimetry using an ionization chamber and solid water phantoms within the cabinet of the irradiator. <a href="https://www.jove.com/files/ftp_upload/61692/61692fig01large.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/61692/61692fig02.jpg" /> 
Figure 2: Generation of a calibration curve using radiochromic film.&#160;(A) Representative color change of radiochromic film with increasing dose. Top left (0 cGy); bottom right (2000 cGy). (B) Potential radiochromic film calibration curve comparing net optical density and dose. <a href="https://www.jove.com/files/ftp_upload/61692/61692fig02large.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/61692/61692fig03.jpg" /> 
Figure 3: Clonogenic Assay of Cancer Cells.&#160;Radiation treatment of cells can be done prior to plating in six well plates/petri dishes (A), or after (B). In panel (C), a representative image is displayed of a successful clonogenic assay with MDA-MB-231 breast cancer cells after following Protocol Section 3. <a href="https://www.jove.com/files/ftp_upload/61692/61692fig03large.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/61692/61692fig04.jpg" /> 
Figure 4: Use of dual overlayed radiograms for positioning (if available) and positive &#947;H2AX immunohistochemical staining for confirmation of dose deposition.&#160;(A) Representative overlayed radiograms depicting placement of radiation beam. (B) Representative results indicating dose deposition to the right hemisphere as demonstrated by increased &#947;H2AX intensity. <a href="https://www.jove.com/files/ftp_upload/61692/61692fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Correction Factor</td>
			<td colspan="2">Explanation</td>
		</tr>
		<tr>
			<td>N&#954;</td>
			<td colspan="2">Air kerma calibration factor</td>
		</tr>
		<tr>
			<td>[(&#181;en/&#961;)Wair]water</td>
			<td colspan="2">Ration of mass energy absorption coefficients of water to air; approximately 1.05</td>
		</tr>
		<tr>
			<td>Pq,Cham</td>
			<td colspan="2">Correction accounting for chamber stem affecting photon fluence perterbation by chamber; approximately 1.022</td>
		</tr>
		<tr>
			<td>Psheath</td>
			<td colspan="2">Correction accounting for sheath protecting ionization chamber; value of 1, as chamber is waterproof</td>
		</tr>
		<tr>
			<td>Ppol</td>
			<td colspan="2">Correction factor accounting for polarity; determined in Protocol 1</td>
		</tr>
		<tr>
			<td>Pion</td>
			<td colspan="2">Correction factor accounting for ion recombination; determined in Protocol 1</td>
		</tr>
		<tr>
			<td>PTp</td>
			<td colspan="2">Correction factor acocunting for temerpature and pressure on day of experiment; determined in Protocol 1</td>
		</tr>
	</tbody>
</table>
Table 1: Correction factors needed for determination of dose rate in Protocol 1.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dose</td>
			<td>N</td>
		</tr>
		<tr>
			<td>0.5</td>
			<td>3</td>
		</tr>
		<tr>
			<td>1</td>
			<td>3</td>
		</tr>
		<tr>
			<td>2</td>
			<td>3</td>
		</tr>
		<tr>
			<td>3</td>
			<td>3</td>
		</tr>
		<tr>
			<td>4</td>
			<td>3</td>
		</tr>
		<tr>
			<td>6</td>
			<td>3</td>
		</tr>
		<tr>
			<td>8</td>
			<td>3</td>
		</tr>
		<tr>
			<td>10</td>
			<td>3</td>
		</tr>
		<tr>
			<td>12*</td>
			<td>3</td>
		</tr>
		<tr>
			<td>15*</td>
			<td>3</td>
		</tr>
		<tr>
			<td>20*</td>
			<td>3</td>
		</tr>
		<tr>
			<td colspan="2">*Only necessary for doses exceeding 10 for individual experiments.</td>
		</tr>
	</tbody>
</table>
Table 2: Doses to be used in generation of radiochromic film calibration curve.

Watch this Scientific Journal Video about Irradiator Commissioning and Dosimetry for Assessment of LQ ÃŽÂ± and ÃŽÂ² Parameters, Radiation Dosing Schema, and in vivo Dose Deposition at JoVE.com

Irradiator Commissioning and Dosimetry for Assessment of LQ &#945; and &#946; Parameters, Radiation Dosing Schema, and in vivo Dose Deposition

Radiation dosimetry provides a technique for enhancing the accuracy of preclinical experiments and ensuring that the radiation doses delivered are closely related to clinical parameters. This protocol describes steps to be taken at each phase during preclinical radiation experiments to ensure proper experimental design.

Irradiator Commissioning and Dosimetry for Assessment of LQ &alpha; and &beta; Parameters, Radiation Dosing Schema, and in vivo Dose Deposition

Radiation dosimetry is critical in the accurate delivery and reproducibility of radiation schemes in preclinical models for high translational relevance. ...

irradiator-commissioning-dosimetry-for-assessment-lq-parameters

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JoVE Journal

Biology

7.1K Views.  West Virginia University. Radiation dosimetry provides a technique for enhancing the accuracy of preclinical experiments and ensuring that the radiation doses delivered are closely related to clinical parameters. This protocol describes steps to be taken at each phase during preclinical radiation experiments to ensure proper experimental design.

Video: Irradiator Commissioning and Dosimetry for Assessment of LQ α and β Parameters, Radiation Dosing Schema, and in vivo Dose Deposition

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &amp;#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Dosimetry for Cell Irradiation using Orthovoltage (40-300 kV) X-Ray Facilities

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose determination using low energy X-ray facilities, but depending on the irradiation configurations, samples, materials or beam quality, it is sometimes difficult to know which protocol is the most appropriate to employ. We, therefore, propose a dosimetry protocol for cell irradiations using low energy X-ray facility. The aim of this method is to perform the dose estimation at the level of the cell monolayer to make it as close as possible to real cell irradiation conditions. The different steps of the protocol are as follows: determination of the irradiation parameters (high voltage, intensity, cell container etc.), determination of the beam quality index (high voltage-half value layer couple), dose rate measurement with ionization chamber calibrated in air kerma conditions, quantification of the attenuation and scattering of the cell culture medium with EBT3 radiochromic films, and determination of the dose rate at the cellular level. This methodology must be performed for each new cell irradiation configuration as the modification of only one parameter can strongly impact the real dose deposition at the level of the cell monolayer, particularly involving low energy X-rays.

Dosimetry for Cell Irradiation using Orthovoltage 40-300 kV X-Ray Facilities

This document describes a new dosimetry protocol for cell irradiations using low energy X-ray equipment. Measurements are performed in conditions simulating real cell irradiation conditions as much as possible.

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose ...

Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) human corneal epithelial cell cultures. Cells were exposed to UV radiation and toxic doses of benzalkonium chloride (BAK), hydrogen peroxide (H2O2), and sodium dodecyl sulfate (SDS). Metabolic activity was measured using a metabolic assay. The release of inflammatory cytokines was measured using a multi-plex interleukin (IL)-1&#946;, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-&#945;) assay, and cells were evaluated for viability using fluorescent dyes. 
The damaging effects of UV on cell metabolic activity and cytokine release occurred at 5 min of UV exposure for iHCEC and 20 min for pHCEC. Similar percent drops in metabolic activity of the iHCEC and pHCEC occurred after exposure to BAK, H2O2, or SDS, and the most significant changes in cytokine release occurred for IL-6 and IL-8. Microscopy of fluorescently stained iHCEC and pHCEC BAK-exposed cells showed cell death at 0.005% BAK exposure, although the degree of ethidium staining was greater in the iHCECs than pHCECs. Utilizing multiple methods of assessing toxic effects using microscopy, assessments of metabolic activity, and cytokine production, the toxicity of UV radiation and chemical toxins could be determined for both primary and immortalized cell lines.

This article describes the procedures used to evaluate the toxicity of UV radiation and chemical toxins on a primary and immortalized cell line.

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) ...