Name: use of a linear accelerator for conducting in vitro radiobiology experiments
Uploaded: 2019-05-26T13:00:00
Description: Watch this Scientific Journal Video about Use of a Linear Accelerator for Conducting In Vitro Radiobiology Experiments at JoVE.com

We thank the Cleveland Clinic Department of Radiation Oncology for use of the linear accelerators. We thank Dr. Jeremy Rich for his generous gift of glioma stem-like cells. This research was supported by the Cleveland Clinic.

1. Cell preparation for suspension cell culture
<ol>
	<li>Culture glioma stem-like cells in stem cell culture media at approximately 5 x 106 cell/10 cm plates in a cell culture incubator with 5% CO2, 95% relative humidity at 37 &#176;C. 
	NOTE: The cell culture condition is the same throughout all the procedures. The media used in the protocol are complete media.</li>
	<li>Two days before scheduled irradiation, collect glioma stem-like cells from the culture plate with a sterile 5 mL pipette ...

<ol>
	<li>Stupp, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiotherapy+plus+concomitant+and+adjuvant+temozolomide+for+glioblastoma.">Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.</a> The New England Journal of Medicine. 352 (10), 987-996 (2005).</li><li>Stupp, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+radiotherapy+with+concomitant+and+adjuvant+temozolomide+versus+radiotherapy+alone+on+survival+in+glioblastoma+in+a+randomised+phase+III+study:+5-year+analysis+of+the+EORTC-NCIC+trial.">Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial.</a> The Lancet Oncology. 10 (5), 459-466 (2009).</li><li>Tao, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+imaging+in+upper+gastrointestinal+tumors+and+application+to+radiation+therapy.">Hypoxia imaging in upper gastrointestinal tumors and application to radiation therapy.</a> Journal of Gastrointestinal Oncology. 9 (6), 1044-1053 (2018).</li><li>Gajiwala, S., Torgeson, A., Garrido-Laguna, I., Kinsey, C., Lloyd, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+immunotherapy+and+radiation+therapy+strategies+for+pancreatic+cancer-targeting+multiple+steps+in+the+cancer+immunity+cycle.">Combination immunotherapy and radiation therapy strategies for pancreatic cancer-targeting multiple steps in the cancer immunity cycle.</a> Journal of Gastrointestinal Oncology. 9 (6), 1014-1026 (2018).</li><li>Liney, G. P., Whelan, B., Oborn, B., Barton, M., Keall, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI-Linear+accelerator+raiotherapy+systems.">MRI-Linear accelerator raiotherapy systems.</a> Clinical Oncology Journal | The Royal College of Radiologists. 30 (11), 686-691 (2018).</li><li>Hall, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+dose-rate:+a+factor+of+importance+in+radiobiology+and+radiotherapy.">Radiation dose-rate: a factor of importance in radiobiology and radiotherapy.</a> The British Journal of Radiology. 45 (530), 81-97 (1972).</li><li>Steel, G. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dose-rate+effect+in+human+tumour+cells.">The dose-rate effect in human tumour cells.</a> Radiotherapy &amp; Oncology. 9 (4), 299-310 (1987).</li><li>Ling, C. C., Gerweck, L. E., Zaider, M., Yorke, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose-rate+effects+in+external+beam+radiotherapy+redux.">Dose-rate effects in external beam radiotherapy redux.</a> Radiotherapy &amp; Oncology. 95 (3), 261-268 (2010).</li><li>Castro, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amotosalen/UVA+treatment+inactivates+T+cells+more+effectively+than+the+recommended+gammadose+for+prevention+of+transfusion-associated+graft-versus-host+disease.">Amotosalen/UVA treatment inactivates T cells more effectively than the recommended gammadose for prevention of transfusion-associated graft-versus-host disease.</a> Transfusion. 58 (6), 1506-1515 (2018).</li><li>Gaddini, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposing+primary+rat+retina+cell+cultures+to+&#947;-rays:+An+in+vitro+model+for+evaluating+radiation+responses.">Exposing primary rat retina cell cultures to &#947;-rays: An in vitro model for evaluating radiation responses.</a> Experimental Eye Research. 166, 21-28 (2018).</li><li>Simara, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+double-strand+breaks+in+human+induced+pluripotent+stem+cell+reprogramming+and+long-term+in+vitro+culturing.">DNA double-strand breaks in human induced pluripotent stem cell reprogramming and long-term in vitro culturing.</a> Stem Cell Research &amp; Therapy. 8 (1), 73 (2017).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+the+biological+effects+of+125I+seeds+continuous+low-dose-rate+radiation+and+60Co+high-dose-rate+gamma+radiation+on+non-small+cell+lung+cancer+cells.">A comparison of the biological effects of 125I seeds continuous low-dose-rate radiation and 60Co high-dose-rate gamma radiation on non-small cell lung cancer cells.</a> PLoS One. 10 (8), 0133728 (2015).</li><li>Lasio, G., Guerrero, M., Goetz, W., Lima, F., Baulch, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+varying+dose-per-pulse+and+average+dose+rate+in+X-ray+beam+irradiation+on+cultured+cell+survival.">Effect of varying dose-per-pulse and average dose rate in X-ray beam irradiation on cultured cell survival.</a> Radiation and Environmental Biophysics. 53 (4), 671-676 (2014).</li><li>Karan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiobiological+effects+of+altering+dose+rate+in+filter-free+photon+beams.">Radiobiological effects of altering dose rate in filter-free photon beams.</a> Physics in Medicine and Biology. 58 (4), 1075-1082 (2013).</li><li>Sarojini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combination+of+high+dose+rate+(10X+FFF/2400+MU/min/10+MV+X-rays)+and+total+low+dose+(0.5+Gy)+induces+a+higher+rate+of+apoptosis+in+melanoma+cells+in+vitro+and+superior+preservation+of+normal+melanocytes.">A combination of high dose rate (10X FFF/2400 MU/min/10 MV X-rays) and total low dose (0.5 Gy) induces a higher rate of apoptosis in melanoma cells in vitro and superior preservation of normal melanocytes.</a> Melanoma Research. 25 (5), 376-389 (2015).</li><li>Hao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+extra+high+on+glioma+stem-like+cells.">The effects of extra high on glioma stem-like cells.</a> PLoS One. 13 (8), 0202533 (2018).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation-induced+G2/M+arrest+rarely+occurred+in+glioblastoma+stem-like+cells.">Radiation-induced G2/M arrest rarely occurred in glioblastoma stem-like cells.</a> International Journal of Radiation Biology. 94 (4), 394-402 (2018).</li><li>Mcdermott, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Physics and Technology of Radiation Therapy. , (2010).</li><li>Lohse, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+high+dose+per+pulse+flattening+filter-free+beams+on+cancer+cell+survival.">Effect of high dose per pulse flattening filter-free beams on cancer cell survival.</a> Radiotherapy &amp; Oncology. 101 (1), 226-232 (2011).</li></ol>

Radiotherapy is an integral part of cancer management. Ongoing efforts seek to improve the efficacy and efficiency of radiation treatment. Advancements in linear accelerator technology have provided the opportunity to treat patients with unprecedented accuracy and safety. Because most patients are treated with high energy X-rays from linear accelerators, studies examining the biologic effects of a large range of dose rates performed on linear accelerators may be readily applied to patients. There have been several report...

Radiotherapy is an effective treatment for many types of cancer1,2,3,4. Extra high dose rate irradiation is relatively new in radiation therapy and is made possible by recent technological advances in linear accelerators5. Clinical advantages of extra high dose rate over standard dose rate irradiation include shortened treatment time and improved patient experience. Linear accelerators also provide a clinical setting for cell culture based radiation biology studies. The biological and therapeutic implications of radiation dose and dose rates have been a focus of interest of radiation oncologists and biologists for decades6,7,8. But, the radiobiology of extra high dose rate irradiation and flash irradiation - an extremely high dose rate of radiation - has yet to be thoroughly investigated.
Gamma ray irradiation is widely used in cell culture based radiation biology9,10,11. Radiation is achieved by gamma-rays emitted from decaying radioactive isotope sources, typically Cesium-137. Use of radioactive sources is highly regulated and often restricted. With source-based irradiation, it is challenging to test a wide range of dose rates, limiting its utility in the analysis of the biologic effects of clinical achievable dose rates12.
There have been several studies that illustrate both dose and dose rate effects12,13,14,15,16,17. In these studies, both gamma-irradiation generated from radioactive isotopes or X-rays generated from linear accelerators were used. A variety of cell lines representing lung cancer, cervical cancer, glioblastoma, and melanoma were used. Radiation effects on cell survival, cell cycle arrest, apoptosis and DNA damage were evaluated as readouts12,13,14,15,16,17. Here, we describe a method to define the biological effects of clinically relevant radiation dose and dose rates by delivering X-ray based radiation using a linear accelerator. These studies should be performed with close collaboration between the biologist, radiation oncologist and medical physicist.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Material</td>
			<td style="width:269px;"></td>
			<td style="width:183px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">glioma stem-like cell 4121</td>
			<td style="width:269px;"></td>
			<td style="width:183px;"></td>
			<td>gift from Dr. Jeremy Rich</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">293 cells</td>
			<td style="width:269px;">ATCC</td>
			<td style="width:183px;">CRL-1573</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:307px;">neuron stem cell culture media</td>
			<td style="width:269px;">Thermo Fisher Scientific</td>
			<td style="width:183px;">21103049</td>
			<td>NeurobasalTM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">DMEM</td>
			<td style="width:269px;">Thermo Fisher Scientific</td>
			<td style="width:183px;">10569044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Fetal Bovine Serum</td>
			<td style="width:269px;">Thermo Fisher Scientific</td>
			<td style="width:183px;">16000044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Penicillin/Streptomycin</td>
			<td style="width:269px;">Thermo Fisher Scientific</td>
			<td style="width:183px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Recombinant Human EGF Protein</td>
			<td style="width:269px;">R&#38;D Systems</td>
			<td>236-EG-01M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Recombinant Human FGF basic</td>
			<td style="width:269px;">R&#38;D Systems</td>
			<td>4114-TC-01M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">B-27&#8482; Supplement</td>
			<td style="width:269px;">Thermo Fisher Scientific</td>
			<td style="width:183px;">17504044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Sodium Pyruvate</td>
			<td style="width:269px;">Thermo Fisher Scientific</td>
			<td style="width:183px;">11360070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">L-Glutamine</td>
			<td style="width:269px;">Thermo Fisher Scientific</td>
			<td style="width:183px;">25030164</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Tripsin-EDTA</td>
			<td style="width:269px;">Thermo Fisher</td>
			<td style="width:183px;">25200056</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:307px;">extracellular proten matrix</td>
			<td style="width:269px;">Corning</td>
			<td style="width:183px;">354277</td>
			<td style="width:167px;">MatrigelTM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Ethanol</td>
			<td style="width:269px;">Fisher chemical</td>
			<td style="width:183px;">A4094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Equipment</td>
			<td style="width:269px;"></td>
			<td style="width:183px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">10 cm cell culture dish</td>
			<td style="width:269px;">Denville</td>
			<td style="width:183px;">T1110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">3.5 cm cell culture dish</td>
			<td style="width:269px;">USA Scientific Inc.</td>
			<td style="width:183px;">CC7682-3340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">22x22mm glass cover slip</td>
			<td style="width:269px;">electron microscopy sciences</td>
			<td style="width:183px;">72210-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">15 ml centrifuge tube</td>
			<td style="width:269px;">Thomas Scientific</td>
			<td style="width:183px;">1159M36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">50 ml centrifuge tube</td>
			<td style="width:269px;">Thomas Scientific</td>
			<td style="width:183px;">1158R10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">5 ml Pipette</td>
			<td style="width:269px;">Fisher Scientific</td>
			<td style="width:183px;">14-955-233</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">pipet aid</td>
			<td style="width:269px;">Fisher Scientific</td>
			<td style="width:183px;">13-681-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Vortex mixer</td>
			<td style="width:269px;">Fisher Scientific</td>
			<td style="width:183px;">02-215-414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Centrifuge</td>
			<td style="width:269px;">Eppendorf</td>
			<td style="width:183px;">5810R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Linear Accelerator</td>
			<td style="width:269px;">Varian</td>
			<td style="width:183px;">n/a</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:307px;">water equivalent material</td>
			<td style="width:269px;">Sun Nuclear corporation</td>
			<td style="width:183px;">557</td>
			<td>Solid waterTM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Reagent preparation</td>
			<td style="width:269px;"></td>
			<td style="width:183px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:307px;">DMEM media</td>
			<td style="width:269px;">10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/mL penicillin G, 100 &#181;g/mL streptomycin in 500 ml DMEM media</td>
			<td style="width:183px;"></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:307px;">stem cell culture media</td>
			<td style="width:269px;">10 ml B27 supplement, 20 &#181;g hFGF, 20 &#181;g hEGF, 2 mM L-glutamine, 100 units/mL penicillin G, 100 &#181;g/mL streptomycin in 500 ml Neurobasal media</td>
			<td style="width:183px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

use of a linear accelerator for conducting in vitro radiobiology experiments

Radiation therapy remains one of the cornerstones of cancer management. For most cancers, it is the most effective, nonsurgical therapy to debulk tumors. Here, we describe a method to irradiate cancer cells with a linear accelerator. The advancement of linear accelerator technology has improved the precision and efficiency of radiation therapy. The biological effects of a wide range of radiation doses and dose rates continue to be an intense area of investigation. Use of linear accelerators can facilitate these studies using clinically relevant doses and dose rates.

To investigate the cell cycle effect of standard dose rate and extra high dose rate irradiation by a linear accelerator, three samples of glioma stem-like cells were prepared using this protocol and collected 24 h after irradiation17: one control sample that was not irradiated (Figure 2A), one sample irradiated with 400 MU/min (monitor unit, 4.2 Gy/min standard dose rate, Figure 2B) to 4 Gy, and another s...

Watch this Scientific Journal Video about Use of a Linear Accelerator for Conducting In Vitro Radiobiology Experiments at JoVE.com

Use of a Linear Accelerator for Conducting In Vitro Radiobiology Experiments

Clinical linear accelerators can be used to determine biologic effects of a wide range of dose rates on cancer cells. We discuss how to set up a linear accelerator for cell-based assays and assays for cancer stem-like cells grown as tumorspheres in suspension and cell lines grown as adherent cultures.

Radiation therapy remains one of the cornerstones of cancer management. For most cancers, it is the most effective, nonsurgical therapy to debulk tumors. ...

Radiation therapy is most frequently delivered using linear accelerators. This protocol allows researchers to assess the biological effects of radiation on cells using a linear accelerator. The advantage of this technique is that by using a linear accelerator, researchers can study a wide range of clinically relevant doses and dose rates.Furthermore, this protocol can be used for all types of cancer cells. This method can be performed using similar equipment to that shown here. For example, linear accelerators manufactured by other vendors.Individuals not experienced in performing dosimetric calculations using a linear accelerator may struggle to calculate the correct number of monitor units required to deliver the desired dose to the sample. They would benefit by seeking advice from an experience medical physicist or dosimetrist. Delivering dose to the sample accurately using a linear accelerator requires careful placement.Two days before scheduled irradiation, working in a culture hood, use a sterile five milliliter pipette to collect glioma stem-like cells from a culture plate into a 15 milliliter centrifuge tube. Centrifuge the cells at 200 times g for three minutes in a countertop centrifuge. Discard the supernatant and resuspend the cell pellet with one milliliter of trypsin-EDTA.Incubate at room temperature for five minutes to digest the pellet and make a single cell suspension in trypsin-EDTA. Every two minutes during digestion, gently shake the bottom of the centrifuge tube to make sure the cells are thoroughly digested. Then add three milliliters of stem cell culture media to quench trypsin, gently mix, and centrifuge at 200 times g for three minutes in a countertop centrifuge.After discarding the supernatant, resuspend the cell pellet with five milliliters of cell culture media and count the cells with a hemocytometer. Plate five million cells divided in two 10 centimeter plates containing 10 milliliters of cell culture media and incubate 48 hours at 37 degrees Celsius. Right before the scheduled irradiation, collect the cells and centrifuge as done before.Discard the supernatant and resuspend the cell pellet with five milliliters of cell culture media. Transfer one milliliter of cell suspension into a 35 millimeter plate containing two milliliters of cell culture medium, making sure that the medium reaches a height of one centimeter. Transfer the plated cells to a secondary container to reduce the risk of contamination and bring the cells in the container on a utility cart to the irradiation facility to irradiate the cells.One day before irradiation, working in the cell culture hood, use a Pasteur pipette connected to a vacuum to remove DMEM medium from the cell culture plate to the attached cell line. Then wash the cells with five milliliters of sterile PBS to rinse off residual medium. Add one milliliter of trypsin-EDTA into the culture dish and gently tilt the culture plate to make sure that the entire dish is covered.After incubating at room temperature for five minutes, quench the trypsin-EDTA reaction with three milliliters of DMEM media. Use a five milliliter pipette to collect the cells into 15 milliliter centrifuge tube, centrifuge, and then culture the cells as described in the manuscript. To irradiate these previously prepared cells using the LINAC's console software, set the accelerator gantry and collimator to zero degrees, open the x and y jaws to a symmetrical five by five square centimeters field size, and retract the multileaf collimators.Add at least five centimeters of water-equivalent material on the treatment couch and place the cell dish to be irradiated onto it at the center of the LINAC crosshairs. Place the cells at a depth of maximum dose in a six megavolt X-ray beam around 1.5 centimeters and add an additional one centimeter of water-equivalent material on top of the dish. Affix the front pointer to the head of the LINAC and extend it until it touches the surface of the buildup material.Adjust the table height until the distance from the source to the buildup surface is 100 centimeters. Leave the treatment vault, making sure that all other individuals have left. Verify that there are no other cells in the room, and then close the vault door.Confirm the field size, monitor units, and monitor units per minute at the console, and then enable the beam for a cell of radiation. Three samples of glioma stem-like cells were prepared using this protocol and collected 24 hours after irradiation. There cell cycle profiles were calculated and shown with the percentages of cells in different phases of the cell cycle.G2 cell cycle arrest was observed after irradiation of glioma stem cells with either a standard dose rate or an extra high dose rate, compared with non-irradiated control cells. In order to have the right dose delivered, it is most important to measure the height of the media in the culture dish to make sure it reaches one centimeter. Following this procedure, other biological assays can be performed, such as immunostaining, western blot, and cell cycle analysis.Since this procedure provides the researchers clinically relevant dose and dose rate settings, it can be used to evaluate the radiation effect on many cell culture based models.

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Medicine

Use of a Hanging Weight System for Coronary Artery Occlusion in Mice

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of knockout mouse studies is particularly important 7, 14, 30, 39. Despite the fact that previous studies have already successfully performed cardiac ischemia and reperfusion in mice, this model is technically very challenging. Particularly, visual identification of the coronary artery, placement of the suture around the vessel and coronary occlusion by tying off the vessel with a supported knot is technically difficult. In addition, re-opening the knot for intermittent reperfusion of the coronary artery during IP without causing surgical trauma adds additional challenge. Moreover, if the knot is not tied down strong enough, inadvertent reperfusion due to imperfect occlusion of the coronary may affect the results. In fact, this can easily occur due to the movement of the beating heart.
Based on potential problems associated with using a knotted coronary occlusion system, we adopted a previously published model of chronic cardiomyopathy based on a hanging weight system for intermittent coronary artery occlusion during IP 39. In fact, coronary artery occlusion can thus be achieved without having to occlude the coronary by a knot. Moreover, reperfusion of the vessel can be easily achieved by supporting the hanging weights which are in a remote localization from cardiac tissues.
We tested this system systematically, including variation of ischemia and reperfusion times, preconditioning regiments, body temperature and genetic backgrounds39. In addition to infarct staining, we tested cardiac troponin I (cTnI)
as a marker of myocardial infarction in this model. In fact, plasma levels of cTnI correlated with infarct sizes (R2=0.8). Finally, we could show in several studies that this technique yields highly reproducible infarct sizes during murine IP and myocardial infarction6, 8, 30, 40, 41. Therefore, this technique may be helpful for researchers who pursue molecular mechanisms involved in cardioprotection by IP using a genetic approach in mice with targeted gene deletion. Further studies on cardiac IP using transgenic mice may consider this technique.

A murine model for myocardial ischemia and ischemic preconditioning is an important tool study cardioprotective mechanisms in vivo. Here, we report an easy applicable in situ model for cardiac IP using a hanging-weight system for coronary artery occlusion.

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. ...

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

Use of a Hanging-weight System for Liver Ischemia in Mice

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and can result in liver failure which has a high mortality rate1-2. Therefore murine studies of hepatic ischemia have become an important field of research by providing the opportunity to utilize pharmacological and genetic studies3-9. Specifically, conditional mice with tissue specific deletion of a gene (cre, flox system) provide insights into the role of proteins in particular tissues10-13 . Because of the technical difficulty associated with manually clamping the portal triad in mice, we performed a systematic evaluation using a hanging-weight system for portal triad occlusion which has been previously described3. By using a hanging-weight system we place a suture around the left branch of the portal triad without causing any damage to the hepatic lobes, since also the finest clamps available can cause hepatic tissue damage because of the close location of liver tissue to the vessels. Furthermore, the right branch of the hepatic triad is still perfused thus no intestinal congestion occurs with this technique as blood flow to the right hepatic lobes is preserved. Furthermore, the portal triad is only manipulated once throughout the entire surgical procedure. As a result, procedures like pre-conditioning, with short times of ischemia and reperfusion, can be easily performed. Systematic evaluation of this model by performing different ischemia and reperfusion times revealed a close correlation of hepatic ischemia time with liver damage as measured by alanine (ALT) and aspartate (AST) aminotransferase serum levels3,9. Taken together, these studies confirm highly reproducible liver injury when using the hanging-weight system for hepatic ischemia and intermittent reperfusion. Thus, this technique might be useful for other investigators interested in liver ischemia studies in mice. Therefore the video clip provides a detailed step-by-step description of this technique.

We established a novel murine model of a hanging weight system for portal triad occlusion. This technique may be useful for future investigations of ischemia in murine hepatic models.

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and ...

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Setting Up a Stroke Team Algorithm and Conducting Simulation-based Training in the Emergency Department - A Practical Guide

Time is of the essence when caring for an acute stroke patient. The ultimate goal is to restore blood flow to the ischemic brain. This can be&#160;achieved by either&#160;thrombolysis with recombinant tissue-plasminogen activator (rt-PA), the standard therapy for stroke patients who present within the first hours of symptom onset without contraindications, or by an endovascular approach, if a proximal brain vessel occlusion is detected. As the efficacy of both therapies declines over time, every minute saved along the way will improve the patient&#39;s outcome.
This critical situation requires thorough work and precise communication with the patient, the family and colleagues from different professions to acquire all relevant information and reach the right decision while carefully monitoring the patient. This is a high fidelity situation. In nonmedical high-fidelity environments such as aviation, Crew Resource Management (CRM) is used to enhance safety and team efficiency.
This guide shows how a Stroke Team algorithm, which is transferable to other hospital settings, was established and how regular simulation-based trainings were performed. It requires determination and endurance to maintain these time-consuming simulation trainings on a regular basis over the course of time. However, the resulting improvement of team spirit and excellent door-to-needle times will benefit both the patients and the work environment in any hospital.
A dedicated Stroke Team of 7 persons who are notified 24/7 by a collective call via speed dial and run a binding algorithm that takes approximately 20 min, was established. To train everybody involved in this algorithm, a simulation-based team training for all new Stroke Team members was conceived and conducted at monthly intervals. This led to a relevant and sustained reduction of the mean door-to-needle time to 25 min, and enhanced the feeling of stroke readiness especially in junior doctors and nurses.

Every min counts in acute stroke care. This guide shows how to establish a Stroke Team algorithm and enhance its performance with regular simulation training. The principles of Crew Resource Management (CRM) facilitate a straight workflow, reduce door-to-needle times and increase staff satisfaction.

Time is of the essence when caring for an acute stroke patient. The ultimate goal is to restore blood flow to the ischemic brain. This can ...

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

Traditional herbal medicine, an alternative medicine in the clinical setting, has received increased attention in recent years. Before delivery to the body, an additional extraction procedure is commonly required to release the active constituents from raw herbs. Water decoction is a classical extraction procedure that is still broadly used in the clinical settings. Here, we propose a detailed protocol for er-xian decoction (EXD) in order to apply herbal decoctions to experimental studies. The calculation of an animal-appropriate dose is described, as well as the four main steps of EXD: soaking, water decoction, filtration, and concentration. In addition, serum-containing EXD is introduced to rats as a means of in vitro validation. Here, rats were orally administered EXD for three days. Blood samples were then collected, inactivated, centrifuged, and filtered. The serum, diluted with the culture medium, can be utilized to treat cells or tissues in vitro. For example, EXD was applied to both in vivo and in vitro studies and demonstrated that EXD enhances osteogenesis. This protocol can be used as a reference for the preparation and application of herbal medicines.

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

Here, we present the preparation of an er-xian decoction (EXD) in four steps&#8212;soaking, decoction, filtration, and concentration&#8212;and demonstrate the administration of a prepared EXD-containing serum to rats. These methods are applicable to the in vivo and in vitro study of herbal decoctions such as traditional Chinese medicines.

Traditional herbal medicine, an alternative medicine in the clinical setting, has received increased attention in recent years. Before delivery to the ...

Conducting Maximal and Submaximal Endurance Exercise Testing to Measure Physiological and Biological Responses to Acute Exercise in Humans

Regular physical activity has a positive effect on human health, but the mechanisms controlling these effects remain unclear. The physiologic and biologic responses to acute exercise are predominantly influenced by the duration and intensity of the exercise regimen. As exercise is increasingly thought of as a therapeutic treatment and/or diagnostic tool, it is important that standardizable methodologies be utilized to understand the variability and to increase the reproducibility of exercise outputs and measurements of responses to such regimens. To that end, we describe two different cycling exercise regimens that yield different physiologic outputs. In a maximal exercise test, exercise intensity is continually increased with a greater workload resulting in an increasing cardiopulmonary and metabolic response (heart rate, stroke volume, ventilation, oxygen consumption and carbon dioxide production). In contrast, during endurance exercise tests, the demand is increased from that at rest, but is raised to a fixed submaximal exercise intensity resulting in a cardiopulmonary and metabolic response that typically plateaus. Along with the protocols, we provide suggestions on measuring physiologic outputs that include, but are not limited to, heart rate, slow and forced vital capacity, gas exchange metrics, and blood pressure to enable the comparison of exercise outputs between studies. Biospecimens can then be sampled to assess cellular, protein, and/or gene expression responses. Overall, this approach can be easily adapted into both short- and long-term effects of two distinct exercise regimens.

To assess the influence of exercise intensity on physiologic and biologic responses, two different exercise testing protocols were utilized. Methods outlining exercise testing on a cycle ergometer as an incremental maximal oxygen consumption test and endurance, steady state submaximal endurance test are described.

Regular physical activity has a positive effect on human health, but the mechanisms controlling these effects remain unclear. The physiologic and biologic ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...