Name: evaluation of amino acid consumption in cultured bone cells and isolated bone shafts
Uploaded: 2022-04-13T13:00:00
Description: Watch this Scientific Journal Video about Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts at JoVE.com

The Karner lab is supported by National Institute of Health R01 grants (AR076325 and AR071967) to C.M.K.

All mouse procedures described herein were approved by the Animal Studies Committees at the University of Texas Southwestern Medical Center at Dallas. The radiation protocol was approved by the Radiation Safety Advisory Committee at the University of Texas Southwestern Medical Center at Dallas.
1. Amino acid uptake in cells (Protocol I)
<ol>
	<li>Plate 5 x 104 ST2 cells in each well of a 12-well tissue culture plate. Plate cells in &#945;-MEM containing 10% FBS, 1...

<ol>
	<li>Xiao, M., 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=Inhibition+of+&#945;-KG-dependent+histone+and+DNA+demethylases+by+fumarate+and+succinate+that+are+accumulated+in+mutations+of+FH+and+SDH+tumor+suppressors.">Inhibition of &#945;-KG-dependent histone and DNA demethylases by fumarate and succinate that are accumulated in mutations of FH and SDH tumor suppressors.</a> Genes &amp; Development. 26 (12), 1326-1338 (2012).</li><li>Altman, B. J., Stine, Z. E., Dang, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Krebs+to+clinic:+glutamine+metabolism+to+cancer+therapy.">From Krebs to clinic: glutamine metabolism to cancer therapy.</a> Nature Reviews Cancer. 16 (10), 619-634 (2016).</li><li>Karner, C. M., Long, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signaling+and+cellular+metabolism+in+osteoblasts.">Wnt signaling and cellular metabolism in osteoblasts.</a> Cell and Molecular Life Sciences. 74 (9), 1649-1657 (2017).</li><li>Zarse, K., 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=Impaired+insulin/IGF1+signaling+extends+life+span+by+promoting+mitochondrial+L-proline+catabolism+to+induce+a+transient+ROS+signal.">Impaired insulin/IGF1 signaling extends life span by promoting mitochondrial L-proline catabolism to induce a transient ROS signal.</a> Cell Metabolism. 15 (4), 451-465 (2012).</li><li>Nagano, 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=Proline+dehydrogenase+promotes+senescence+through+the+generation+of+reactive+oxygen+species.">Proline dehydrogenase promotes senescence through the generation of reactive oxygen species.</a> Journal of Cell Science. 130 (8), 1413-1420 (2017).</li><li>Comes, 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=L-Proline+induces+a+mesenchymal-like+invasive+program+in+embryonic+stem+cells+by+remodeling+H3K9+and+H3K36+methylation.">L-Proline induces a mesenchymal-like invasive program in embryonic stem cells by remodeling H3K9 and H3K36 methylation.</a> Stem Cell Reports. 1 (4), 307-321 (2013).</li><li>Fan, 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=Glutamine-driven+oxidative+phosphorylation+is+a+major+ATP+source+in+transformed+mammalian+cells+in+both+normoxia+and+hypoxia.">Glutamine-driven oxidative phosphorylation is a major ATP source in transformed mammalian cells in both normoxia and hypoxia.</a> Molecular Systems Biology. 9, 712 (2013).</li><li>Hosios, A. M., 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=Amino+acids+rather+than+glucose+account+for+the+majority+of+cell+mass+in+proliferating+mammalian+cells.">Amino acids rather than glucose account for the majority of cell mass in proliferating mammalian cells.</a> Developmental Cell. 36 (5), 540-549 (2016).</li><li>Welbourne, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ammonia+production+and+glutamine+incorporation+into+glutathione+in+the+functioning+rat+kidney.">Ammonia production and glutamine incorporation into glutathione in the functioning rat kidney.</a> Canadian Journal of Biochemistry. 57 (3), 233-237 (1979).</li><li>Sullivan, L. B., 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=Supporting+aspartate+biosynthesis+is+an+essential+function+of+respiration+in+proliferating+cells.">Supporting aspartate biosynthesis is an essential function of respiration in proliferating cells.</a> Cell. 162 (3), 552-563 (2015).</li><li>Nelsen, C. 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=Amino+acids+regulate+hepatocyte+proliferation+through+modulation+of+cyclin+D1+expression.">Amino acids regulate hepatocyte proliferation through modulation of cyclin D1 expression.</a> The Journal of Biological Chemistry. 278 (28), 25853-25858 (2003).</li><li>Krall, A. S., Xu, S., Graeber, T. G., Braas, D., Christofk, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asparagine+promotes+cancer+cell+proliferation+through+use+as+an+amino+acid+exchange+factor.">Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor.</a> Nature Communications. 7, 11457 (2016).</li><li>Green, C. 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=Branched-chain+amino+acid+catabolism+fuels+adipocyte+differentiation+and+lipogenesis.">Branched-chain amino acid catabolism fuels adipocyte differentiation and lipogenesis.</a> Nature Chemical Biology. 12 (1), 15-21 (2016).</li><li>Shiraki, N., 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=Methionine+metabolism+regulates+maintenance+and+differentiation+of+human+pluripotent+stem+cells.">Methionine metabolism regulates maintenance and differentiation of human pluripotent stem cells.</a> Cell Metabolism. 19 (5), 780-794 (2014).</li><li>Yu, Y., 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=Glutamine+metabolism+regulates+proliferation+and+lineage+allocation+in+skeletal+stem+cells.">Glutamine metabolism regulates proliferation and lineage allocation in skeletal stem cells.</a> Cell Metabolism. 29 (4), 966-978 (2019).</li><li>Shen, L., Sharma, D., Yu, Y., Long, F., Karner, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biphasic+regulation+of+glutamine+consumption+by+WNT+during+osteoblast+differentiation.">Biphasic regulation of glutamine consumption by WNT during osteoblast differentiation.</a> Journal of Cell Science. 134 (1), (2021).</li><li>Karner, C. M., Esen, E., Okunade, A. L., Patterson, B. W., Long, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+glutamine+catabolism+mediates+bone+anabolism+in+response+to+WNT+signaling.">Increased glutamine catabolism mediates bone anabolism in response to WNT signaling.</a> Journal of Clinical Investigation. 125 (2), 551-562 (2015).</li><li>Hu, 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+amino+acid+sensor+Eif2ak4/GCN2+is+required+for+proliferation+of+osteoblast+progenitors+in+mice.">The amino acid sensor Eif2ak4/GCN2 is required for proliferation of osteoblast progenitors in mice.</a> Journal of Bone and Mineral Research. 35 (10), 2004-2014 (2020).</li><li>Rached, M. 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=FoxO1+is+a+positive+regulator+of+bone+formation+by+favoring+protein+synthesis+and+resistance+to+oxidative+stress+in+osteoblasts.">FoxO1 is a positive regulator of bone formation by favoring protein synthesis and resistance to oxidative stress in osteoblasts.</a> Cell Metabolism. 11 (2), 147-160 (2010).</li><li>Elefteriou, F., 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=ATF4+mediation+of+NF1+functions+in+osteoblast+reveals+a+nutritional+basis+for+congenital+skeletal+dysplasiae.">ATF4 mediation of NF1 functions in osteoblast reveals a nutritional basis for congenital skeletal dysplasiae.</a> Cell Metabolism. 4 (6), 441-451 (2006).</li><li>Maleknia, S. D., Johnson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+of+amino+acids+and+proteins.">Mass spectrometry of amino acids and proteins.</a> Amino Acids, Peptides and Proteins in Organic Chemistry. , 1-50 (2011).</li><li>Rennie, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+the+use+of+tracers+in+nutrition+and+metabolism.">An introduction to the use of tracers in nutrition and metabolism.</a> The Proceedings of the Nutrition Society. 58 (4), 935-944 (1999).</li><li>Hahn, T. J., Downing, S. J., Phang, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acid+transport+in+adult+diaphyseal+bone:+contrast+with+amino+acid+transport+mechanisms+in+fetal+membranous+bone.">Amino acid transport in adult diaphyseal bone: contrast with amino acid transport mechanisms in fetal membranous bone.</a> Biochimica Biophysica Acta. 183 (1), 194-203 (1969).</li><li>Rosenbusch, J. P., Flanagan, B., Nichols, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+transport+of+amino+acids+into+bone+cells.">Active transport of amino acids into bone cells.</a> Biochimica Biophysica Acta. 135 (4), 732-740 (1967).</li><li>Kandasamy, P., Gyimesi, G., Kanai, Y., Hediger, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acid+transporters+revisited:+New+views+in+health+and+disease.">Amino acid transporters revisited: New views in health and disease.</a> Trends in Biochemical Sciences. 43 (10), 752-789 (2018).</li></ol>

The protocol described herein provides a fast and sensitive approach to evaluate amino acid uptake in response to various experimental permutations either in vitro or ex vivo. When compared to commercially available kits (e.g., Glutamine and Glutamate Determination Kit), this method is much more sensitive, quicker, and less labor intensive16,17,25. In our protocol, we evaluate uptake in the Krebs Ringers HEPES ...

Amino acids are organic compounds that contain an amino (-NH2) and carboxyl (-COOH) functional groups with a variable side chain that is specific to each amino acid. In general, amino acids are well known as the basic constituent of protein. More recently, novel uses, and functions of amino acids have been elucidated. For example, individual amino acids can be metabolized to generate intermediate metabolites that contribute to bioenergetics, function as enzymatic cofactors, regulate reactive oxygen species or are used to synthesize other amino acids1,2,3,4,5,6,7,8,9,10. Many studies demonstrate that amino acid metabolism is critical for cell pluripotency, proliferation, and differentiation in various contexts3,6,11,12,13,14,15,16,17.
Osteoblasts are secretory cells that produce and secrete the Collagen Type 1 rich extracellular bone matrix. To sustain high rates of protein synthesis during bone formation, osteoblasts demand a constant supply of amino acids. To meet this demand, osteoblasts must actively acquire amino acids. Consistent with this, recent studies reveal the importance of amino acid uptake and metabolism in osteoblast activity and bone formation15,16,17,18,19,20.
Osteoblasts acquire cellular amino acids from three major sources: extracellular milieu, intracellular protein degradation and de novo amino acid biosynthesis. This protocol will focus on the evaluation of amino acid uptake from extracellular environment. The most common methods to measure amino acid uptake rely on either radiolabeled (e.g., 3H or 14C) or heavy isotope labeled (e.g., 13C) amino acids. Heavy isotopomer assays can analyze amino acid uptake and metabolism more thoroughly and safely but are more time consuming taking multiple days to complete as it takes a day to prepare and derivatize samples and multiple days to analyze on the mass spectrometer depending on the number of samples21,22. By comparison, radiolabeled amino acid uptake assays are not informative about downstream metabolism but are cheap and relatively fast, being able to be completed within 2-3 h from the start of the experiment23,24. Here, we describe an easily modifiable basic protocol designed to evaluate radiolabeled amino acid uptake in cultured primary cells or cell lines in vitro or individual bone shafts ex vivo. The application of these two protocols can be extended to other radiolabeled amino acids and other bone associated cell types and tissues.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% trypsin</td>
			<td>Gibco</td>
			<td>25200</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Corning</td>
			<td>3513</td>
			<td></td>
		</tr>
		<tr>
			<td>1mL syringe</td>
			<td>BD precision</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>30G Needle</td>
			<td>BD precision</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>Arginine Monohydrochloride L-[2,3,4-3H]-, 1mCi</td>
			<td>PerkinElmer</td>
			<td>NET1123001MC</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman LS6500 scintillation counter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr>
			<td>choline chloride</td>
			<td>Sigma</td>
			<td>C7077</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose solution</td>
			<td>Sigma</td>
			<td>G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Tool</td>
			<td></td>
			<td></td>
			<td>Forceps, scissors, scapels</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Sigma</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES(1M)</td>
			<td>Gibco</td>
			<td>15630</td>
			<td></td>
		</tr>
		<tr>
			<td>L-[3,4-3H(N)]-Glutamine</td>
			<td>PerkinElmer</td>
			<td>NET551250UC</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid scintilation vials</td>
			<td>Sigma</td>
			<td>Z190535</td>
			<td></td>
		</tr>
		<tr>
			<td>lithium chloride solution, 8M</td>
			<td>Sigma</td>
			<td>L7026</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM&#945;</td>
			<td>Gibco</td>
			<td>12561</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube, 15mL</td>
			<td>Biotix</td>
			<td>89511-256</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Sigma</td>
			<td>492016</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium chloride</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Deoxycholate</td>
			<td>Sigma</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Sigma</td>
			<td>436143</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Sonic&#38;Materials</td>
			<td>VCX130</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Sigma</td>
			<td>648311</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultima Gold (Scintillation solution)</td>
			<td>PerkinElmer</td>
			<td>6013329</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-(Methylamino)isobutyric acid</td>
			<td>Sigma</td>
			<td>M2383</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of amino acid consumption in cultured bone cells and isolated bone shafts

Bone development and homeostasis is dependent upon the differentiation and activity of bone forming osteoblasts. Osteoblast differentiation is sequentially characterized by proliferation followed by protein synthesis and ultimately bone matrix secretion. Proliferation and protein synthesis require a constant supply of amino acids. Despite this, very little is known about amino acid consumption in osteoblasts. Here we describe a very sensitive protocol that is designed to measure amino acid consumption using radiolabeled amino acids. This method is optimized to quantify changes in amino acid uptake that are associated with osteoblast proliferation or differentiation, drug or growth factor treatments, or various genetic manipulations. Importantly, this method can be used interchangeably to quantify amino acid consumption in cultured cell lines or primary cells in vitro or in isolated bone shafts ex vivo. Finally, our method can be easily adapted to measure the transport of any of the amino acids as well as glucose and other radiolabeled nutrients.

Amino acid transport is regulated by many membrane-bound amino acid transporters that have been categorized into distinct transport systems based on numerous characteristics, including substrate specificity, kinetics, as well as ion and pH dependence25. For example, glutamine uptake can be mediated by the Na+-dependent transport systems A, ASC, &#947;+L and N or the Na+-independent System L. The Na+-dependent systems are distinguished by the ability to substitute L...

Watch this Scientific Journal Video about Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts at JoVE.com

Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts

This protocol presents a radiolabeled amino acid uptake assay, which is useful for evaluating amino acid consumption either in primary cells or in isolated bones.

Bone development and homeostasis is dependent upon the differentiation and activity of bone forming osteoblasts. Osteoblast differentiation is ...

Amino acid uptake and metabolism are essential for osteoblast specification, differentiation, and bone formation. This protocol provides a rapid and sensitive method to evaluate amino acid uptake. Our protocol uses radiolabeled amino acids to quantify amino acid uptake.It is a cheap, sensitive and a fast method which can be easily modified for different conditions. This protocol can easily be modified to evaluate the uptake of other radiolabeled nutrients like glucose and fatty acids in a variety of tissues as well as primary or transform cells. To begin amino acid uptake assay plate five times 10 to the fourth ST2 cells in each well of a 12 well tissue culture plate in alpha MEM medium supplemented with FBS, penicillin and streptomycin.Plate extra wells of cells to quantify the cell number per condition for the normalizations. Next, incubate cells in a humidified cell culture incubator at 37 degrees Celsius with 5%carbon dioxide for two to three days until confluent. After incubation aspirate the medium and wash the cells twice with one milliliter of PBS at pH 7.4.Then aspirate the PBS before washing the cells once with one milliliter Krebs-Ringer's HEPES or KRH. Incubate cells with 0.5 milliliters of freshly prepared KRH containing four microcuries per milliliter L34 tritiated glutamine working media for five minutes. Post incubation, collect the radioactive medium to dispense into the liquid waste container.Then wash the cells three times with ice cold KRH to terminate the reaction. Next, add one milliliter of 1%sodium dodecyl sulfate to each well and triturate the mixture 10 times to lyse and homogenize the cells. Then transfer the cell lysates to a 1.5 milliliter tube and clarify the lysate by centrifuging at a minimum speed of 10, 000 G for 10 minutes.Transfer the supernatant to a scintillation vial containing eight milliliters of the scintillation solution, and read radio activity and counts per minute using the scintillation counter. From the remaining non-radioactive plates of cells, trypsinize, resuspend, and count the cells to estimate the number of cells in the lysed radioactive cultures. Using a hemacytometer, count the number of cells per non-radioactive well for each experimental condition.To determine amino acid uptake, flush out the marrow from the bone before weighing the bone shaft. To decellularize the bone, boil one humerus in PBS at 100 degrees Celsius for 10 minutes. Equilibrate both live and boiled humeri in one milliliter of KRH for 30 minutes in the cell culture incubator at 37 degrees Celsius.Next, incubate the experimental and boiled control bones separately in freshly prepared KRH containing four microcurries per milliliter L234 triciated arginine for up to 90 minutes at 37 degrees Celsius. After removing the radioactive medium, terminate the reaction by washing humerus three times with one milliliter of ice cold KRH. Transfer each bone into a 1.5 milliliter tube with 500 microliters of RIPA Buffer.Then homogenize the humerus and RIPA Buffer by chopping 100 times with scissors, followed by sonication at 35%amplitude and one second pulse for 10 seconds. Clarified the lysate by centrifugation for 10 minutes at 10, 000 times G at room temperature. Post centrifugation, transfer 200 microliters of the supernatant to scintillation vials containing eight milliliters of the scintillation solution.Read radio activity using the scintillation counter. Kinetic properties of in vitro L34 tritiated glutamine uptake and confluent ST2 cells were evaluated in the presence or absence of sodium methyl amino isobutyric acid or lithium. The removal of sodium led to a 90%reduction in glutamine uptake.The presence of lithium increased glutamine uptake by 2%compared to the sodium free condition. Whereas methyl amino isobutyric acid did not affect glutamine uptake. The estimation percentage contributions of system A, N, L or ASC and gamma plus L indicated that most glutamine uptake is mediated by systems ASC and gamma plus L.System N is responsible for roughly 2%sodium dependent glutamine uptake.And system A showed 0%contribution and uptake. X-VIVO L34 tritiated arginine uptake in boiled and live neonatal humeri was analyzed over a time course of two hours. Arginine uptake increased linearly throughout the experiment and live humeri.The boiled humeri did not exhibit a dynamic increase of arginine uptake in the experiment. The representative analysis shows the normalized and corrected arginine uptake. It is important to boil the contralateral bone as a active control.This step is essential as the bone matrix may absorb radioactive amino acids, resulting in inaccurate results. The protocol has allowed us to confirm amino acid uptake kinetics both in vitro and in vivo.

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Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection may result in sustained inflammation, osteomyelitis, and eventual bone non-union. Establishment of a feasible, reproducible animal model is important to bone infection research and antibiotic treatment. As an in vivo model, the rabbit model is widely used in bone infection research. However, previous studies on rabbit bone infection models showed that the infection status was inconsistent, as the amount of bacteria was variable. This study presents an improved surgical method for inducing bone infection on a rabbit, by blocking the bacteria in the bone marrow. Then, multi-level evaluations can be carried out to verify the modelling method.
In general, debriding necrotic tissue and implantation of vancomycin-loaded calcium sulphate (VCS) are predominant in antibiotic treatment. Although calcium sulphate in VCS benefits osteocyte crawling and new bone growth, massive bone defects occur after debriding. Autogenous bone (AB) is an appealing strategy to overcome bone defects for the treatment of massive bone defects after debriding necrotic bone.
In this study, we used the tail bone as an autogenous bone implanted in the bone defect. Bone repair was measured using micro-computed-tomography (micro-CT) and histological analysis after animal sacrifice. As a result, in the VCS group, bone non-union was consistently obtained. In contrast, the bone defect areas in the VCS-AB group were decreased significantly. The present modeling method described a reproducible, feasible, stable method to prepare a bone infection model. The VCS-AB treatment resulted in lower bone non-union rates after antibiotic treatment. The improved bone infection model and the combination treatment of VCS and autogenous bone could be helpful in studying the underlying mechanisms in bone infection and bone regeneration pertinent to traumatology orthopedic applications.

This study presents an improved rabbit model infected with Staphylococcus aureus by blocking the same amount of bacteria in bone marrow. Vancomycin loaded calcium sulphate and autogenous bone are used for antibiotic and bone repair treatment. The protocol could be helpful for studying bone infection and regeneration.

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection ...

Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow cytometry (MFC) for myeloid disease diagnosis in France. The protocol presented here was agreed-upon and applied between September 2013 and November 2015 in six French diagnostic laboratories (University Hospitals of Saint-Etienne, Grenoble, Clermont-Ferrand, Nice, and Lille and Institut Paoli-Calmettes in Marseille) and allowed the standardization of bone marrow sample preparation and data acquisition. Three maturation databases were developed for neutrophil, monocytic, and erythroid lineages with bone marrow from "healthy" donor individuals (individuals without any evidence of a hematopoietic disease). A robust method of analysis for each myeloid lineage should be applicable for routine diagnostic use. New cases can be analyzed in the same manner and compared against the usual databases. Thus, quantitative and qualitative phenotypic abnormalities can be identified and those above 2SD compared with data of normal bone marrow samples should be considered indicative of pathology. The major limitation is the higher variability between the data achieved using the monoclonal antibodies obtained with the methods based on hybridoma technologies and currently used in clinical diagnosis. Setting criteria for technical validation of the data acquired may help improve the utility of MFC for MDS diagnostics. The establishment of these criteria requires analysis against a database. The reduction of investigator subjectivity in data analysis is an important advantage of this method.

The MDS diagnosis is difficult in the absence of morphological criteria or non-informative cytogenetics. MFC could help refine the MDS diagnostic process. To become useful for clinical practice, the MFC analysis must be based on parameters with sufficient specificity and sensitivity, and data should be reproducible between different operators.

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow ...

Using a Knee Arthrometer to Evaluate Tissue-specific Contributions to Knee Flexion Contracture in the Rat

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ROM is called a joint contracture and results in increased morbidity. Due to the difficulty of reversing established knee contractures, early detection is important, and hence, knowing risk factors for their development is essential. The rat represents a good model with which the effect of an intervention can be studied due to the similarity of rat knee anatomy to that of humans, the rat's ability to tolerate long durations of knee immobilization in flexion, and because mechanical data can be correlated with histologic and biochemical analysis of knee tissue.
Using an automated arthrometer, we demonstrate a validated, precise, reproducible, user-independent method of measuring the extension ROM of the rat knee joint at specific torques. This arthrometer can be used to determine the effects of interventions on knee joint ROM in the rat.

The goal of the protocol is to measure the extension range of motion of the rat knee. The effects of various diseases that increase the stiffness of the knee joint and the effectiveness of treatments can be quantified.

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing continuous remodeling with the competing activity of osteoblasts, which produce the new bone matrix, and osteoclasts, whose function is to eliminate mineralized bone. [18F]-NaF is a positron emission tomography (PET) radiotracer that enables visualization of bone metabolism. [18F]-NaF is chemically absorbed into hydroxyapatite in the bone matrix by osteoblasts and can thus noninvasively detect osteoblastic activity, which is occult to conventional imaging techniques. Kinetic modeling of dynamic [18F]-NaF-PET data provides detailed quantitative measures of bone metabolism. Conventional semi-quantitative PET data, which utilizes standardized uptake values (SUVs) as a measure of radiotracer activity, is referred to as a static technique due to its snapshot of tracer uptake in time.&#160; Kinetic modeling, however, utilizes dynamic image data where tracer levels are continuously acquired providing tracer uptake temporal resolution. From the kinetic modeling of dynamic data, quantitative values like blood flow and metabolic rate (i.e., potentially informative metrics of tracer dynamics) can be extracted, all with respect to the measured activity in the image data. When combined with dual modality PET-MRI, region-specific kinetic data can be correlated with anatomically registered high-resolution structural and pathologic information afforded by MRI. The goal of this methodological manuscript is to outline detailed techniques for performing and analyzing dynamic [18F]-NaF-PET-MRI data. The lumbar facet joint is a common site of degenerative arthritis disease and a common cause for axial low back pain.&#160; Recent studies suggest [18F]-NaF-PET may serve as a useful biomarker of painful facetogenic disease.&#160; The human lumbar facet joint will, therefore, be used as a prototypical region of interest for dynamic [18F]-NaF-PET-MRI analysis in this manuscript.

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.