Name: Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts (Video) | JoVE Journal
Uploaded: 2022-04-13T13:00:00.000+00:00
Duration: 6 min 32 s
Description: Learn a rapid radiolabeled amino acid uptake assay for evaluating nutrient consumption in bone cells and isolated bone shafts. This sensitive method quantifies amino acid uptake using primary or transformed cells.

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5 minutes

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, 100 U/mL penicillin and 0.1 &#181;g/mL streptomycin (Pen/Strep). Plate extra wells of cells to quantify the cell number per condition for the normalizations in step 1.12. Incubate cells in a humidified cell culture incubator at 37 &#176;C with 5% CO2.</li>
	<li>Culture the cells for 2-3 days until confluent.</li>
	<li>On the day of the experiment, prepare the following solutions: 1x Phosphate Buffered Saline (PBS), pH 7.4 and Krebs Ringers HEPES (KRH) buffer, pH 8.0: (120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, NaHCO3, 5 mM HEPES, 1 mM D-Glucose). Prewarm to 37 &#176;C.</li>
	<li>Aspirate the medium and wash the cells twice with 1 mL of 1x PBS, pH 7.4. 
	NOTE: This protocol is also appropriate for rapidly dividing non-confluent cells. In this case, it is important to normalize the radioactivity to either absolute cell number or DNA content. In addition, consider increasing the size of the culture plate or flask to increase the overall cell number and cpm values. This is important to determine empirically on an individual basis.</li>
	<li>Aspirate 1x PBS and wash the cells once with 1 mL KRH.</li>
	<li>Make 4 &#181;Ci/mL L-[3,4-3H]-Glutamine working media by diluting 4 &#181;L of [1 &#181;Ci &#181;L-1] L-[3,4-3H]-Glutamine stock per 1 mL of KRH. 
	NOTE: Before using radioactive materials, please contact the Office of Radiation Safety at your home institution to obtain approvals. All the procedures related to radiation must be performed behind the plexiglass shield.</li>
	<li>Incubate cells with 0.5 mL of KRH containing 4 &#181;Ci/mL L-[3,4-3H]-Glutamine working media for 5 min.</li>
	<li>Collect the radioactive medium and dispense into the liquid waste container. Wash the cells three times briefly with ice-cold KRH to terminate the reaction. Collect and discard all the washes in the radioactive liquid waste container.</li>
	<li>Add 1 mL of 1% SDS to each well and triturate 10x to lyse and homogenize the cells. Transfer the cell lysates to 1.5 mL tubes. Discard cell culture plates, serological pipettes, and pipette tips in the solid radioactive waste container.</li>
	<li>Centrifuge at &#62;10,000 x g for 10 min. Transfer the supernatants to scintillation vials containing 8 mL of the scintillation solution. Mix by shaking the scintillation vials vigorously. Discard tubes and pipette tips in solid radioactive waste container.</li>
	<li>Read radioactivity in counts per minute (cpm) using a Scintillation counter. Discard scintillation vials in scintillation vial waste container.</li>
	<li>Trypsinize, resuspend, and count the cells from the remaining non-radioactive plates of cells (see step 1.1) to estimate the number of cells in the lysed, radioactive cultures. Using a hemocytometer, count the number of cells per non-radioactive well for each experimental condition. Normalize the cpm from step 1.11 to the estimated cell number from the non-radioactive plates.</li>
	<li>After completion of the experiment, decontaminate the cell culture hood, bench, and all instruments with a radioactivity decontaminant spray. Finally, perform wipe tests to ensure the working area is radiation-free.</li>
</ol>
2. Amino acid uptake in freshly isolated bone tissues (Protocol II)
<ol>
	<li>Prewarm KRH to 37 &#176;C.</li>
	<li>Euthanize a 3-day-old mouse and remove the skin on the arms. Disarticulate the humeri from the shoulder using scissors and dissect out both humeri. Remove all extemporaneous tissues using a scalpel and forceps. Remove the epiphyses from the bone. 
	NOTE: In addition to humerii, this protocol can be adapted to neonatal femora, tibiae and calvariae as well as humerii, femora and tibiae isolated from 2- and 4-month-old mice (unpublished data).</li>
	<li>Flush out marrow from the bone and weigh the bone shafts to normalize in step 2.14.</li>
	<li>Boil one humerus in 1x PBS at 100 &#176;C for 10 min to decellularize the bone. The decellularized boiled bone is used as a negative control. 
	NOTE: As a quality control, paraffin embed the boiled and non-boiled bones for histological stains to visually confirm that boiling was efficacious in decellularizing the bone.</li>
	<li>Equilibrate both humeri in 1 mL of KRH for 30 min in the cell culture incubator at 37 &#176;C.</li>
	<li>Make a working solution of 4 &#181;Ci/mL L-[2,3,4-3H]-Arginine in KRH by diluting 4 &#181;L of 1&#181;Ci/&#181;L L-[2,3,4-3H]- Arginine stock per 1 mL KRH. 
	NOTE: This protocol is appropriate to evaluate the uptake of whichever radiolabeled nutrient is chosen. L-[2,3,4-3H]-glutamine, L-[14CU]-alanine, and L-[2,3-3H]-proline have been tested with similar results. Arginine data is shown to highlight the utility of this protocol. 
	CAUTION: All the procedures using radiation must be performed behind the plexiglass shield while using appropriate personal protective equipment (PPE).</li>
	<li>Incubate both the experimental and boiled control bone separately in KRH containing 4 &#181;Ci/mL L-[2,3,4-3H]-Arginine for up to 90 min at 37 &#176;C. 
	NOTE: It is important to empirically determine the incubation times for different conditions including the age of the mice, different skeletal elements and the types of radiolabeled amino acids by performing a time course. Saturation mostly occurs after approximately 3-4 h. Uptake should be evaluated at a timepoint in the linear rage of uptake.</li>
	<li>Remove the radioactive medium and discard in the liquid radioactive waste container. Wash humeri three times using ice cold 1 mL of KRH to terminate the reaction. Discard all the washes in the liquid radioactive waste container.</li>
	<li>Transfer each bone into a 1.5 mL tube. Add 500 &#181;L of RIPA buffer [150 mM NaCl; 5 mM EDTA; 50 mM Tris (pH 8.0); 0.5% NP40 (v/v); 0.5% DOX (w/v); 0.1% SDS (w/v)]. Discard the culture plates, serological pipettes, and pipette tips in a radioactive solid waste container.</li>
	<li>Homogenize the humeri by chopping 100 times with scissors in a RIPA buffer.</li>
	<li>Sonicate (Amplitude: 35%, Pulse 1 s) the resulting homogenized bone for 10 s. 
	NOTE: Sonication is also known to produce aerosols. Sonication steps can be performed in a biological safety cabinet. To reduce aerosol formation, it is important to not overfill the tubes. Sonication of small sample volumes can result in incomplete sonication or sample loss due to foaming. If foaming occurs, centrifuge the sample for 5 min to settle.</li>
	<li>Clarify the lysate by centrifugation for 10 min at &#62;10,000 x g at room temperature. Transfer 200 &#181;L of the supernatant to scintillation vials containing 8 mL of the scintillation solution. Mix by shaking the scintillation vials vigorously.&#160;Discard all solid radioactive waste (e.g., used tubes, pipette tips, plates, etc.) in the solid radioactive waste container.</li>
	<li>Read radioactivity (cpm) using the Scintillation counter. Discard used scintillation vials in the radioactive waste container designated for scintillation vials.</li>
	<li>Subtract the cpm of boiled bone (basal value) from the cpm of experimental bone. Normalize the radioactivity with bone weight from step 2.3.</li>
	<li>Spray the cell culture hood, instruments, and bench with radioactivity decontaminant. Perform wipe tests to confirm the working area is radiation-free.</li>
</ol>

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<li>Nelsen, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amino+acids+regulate+hepatocyte+proliferation+through+modulation+of+cyclin+D1+expression%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Asparagine+promotes+cancer+cell+proliferation+through+use+as+an+amino+acid+exchange+factor%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((Branched-chain+amino+acid+catabolism+fuels+adipocyte+differentiation+and+lipogenesis%5BTitle%5D)+AND+%22Nature+Chemical+Biology%22%5BJournal%5D)">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/pubmed?term=((Methionine+metabolism+regulates+maintenance+and+differentiation+of+human+pluripotent+stem+cells%5BTitle%5D)+AND+%22Cell+Metabolism%22%5BJournal%5D)">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/pubmed?term=((Glutamine+metabolism+regulates+proliferation+and+lineage+allocation+in+skeletal+stem+cells%5BTitle%5D)+AND+%22Cell+Metabolism%22%5BJournal%5D)">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/pubmed?term=((Biphasic+regulation+of+glutamine+consumption+by+WNT+during+osteoblast+differentiation%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">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/pubmed?term=((Increased+glutamine+catabolism+mediates+bone+anabolism+in+response+to+WNT+signaling%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">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/pubmed?term=((The+amino+acid+sensor+Eif2ak4%2FGCN2+is+required+for+proliferation+of+osteoblast+progenitors+in+mice%5BTitle%5D)+AND+%22Journal+of+Bone+and+Mineral+Research%22%5BJournal%5D)">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/pubmed?term=((FoxO1+is+a+positive+regulator+of+bone+formation+by+favoring+protein+synthesis+and+resistance+to+oxidative+stress+in+osteoblasts%5BTitle%5D)+AND+%22Cell+Metabolism%22%5BJournal%5D)">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/pubmed?term=((ATF4+mediation+of+NF1+functions+in+osteoblast+reveals+a+nutritional+basis+for+congenital+skeletal+dysplasiae%5BTitle%5D)+AND+%22Cell+Metabolism%22%5BJournal%5D)">ATF4 mediation of NF1 functions in osteoblast reveals a nutritional basis for congenital skeletal dysplasiae.</a> Cell Metabolism. 4 (6), 441-451 (2006).</li>
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<li>Rennie, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+introduction+to+the+use+of+tracers+in+nutrition+and+metabolism%5BTitle%5D)+AND+%22The+Proceedings+of+the+Nutrition+Society%22%5BJournal%5D)">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/pubmed?term=((Amino+acid+transport+in+adult+diaphyseal+bone%3A+contrast+with+amino+acid+transport+mechanisms+in+fetal+membranous+bone%5BTitle%5D)+AND+%22Biochimica+Biophysica+Acta%22%5BJournal%5D)">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>
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</ol>

The authors have no disclosures.

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><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>1 mL 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&amp;alpha;</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&amp;amp;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>&amp;alpha;-(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

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, γ+L and N or the Na+-independent System L. The Na+-dependent systems are distinguished by the ability to substitute Li<sup...

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 ...

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Medicine

A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

In this video, we describe a method for the detection of osteoblast-secreted calcium deposits using an anionic dye, Alizarin Red S. The cultured osteoblasts are first stained and then the dye is recovered for colorimetric quantification of calcium deposits or the degree of osteoblast mineralization.

In Vitro Mineralization Assay: A Colorimetric Method to Quantify Osteoblast Calcium Deposits on Bone Graft Substitute by Alizarin Red S Staining and Extraction

This video demonstrates an in vitro technique for estimating amino acid uptake by bone marrow-derived stromal cells. Na+-dependent amino acid transporters mediate the uptake of radiolabeled amino acids inside the cells, and the cellular uptake is estimated by measuring the radioactivity.

Radiolabeled Amino Acid Uptake Assay to Quantify Cellular Uptake of Amino Acids

We describe here how to prepare bone-conditioned medium (BCM) and test its activity in vitro.

Bone Conditioned Medium: Preparation and Bioassay

Uranium is known to affect bone metabolism. Here, we present a protocol aimed at investigating the effect of natural uranium exposure on the viability, the differentiation, and the function of osteoclasts, the cells in charge of bone resorption.

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

We present a protocol to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. Their mineralization profiles were analyzed by Alizarin Red-S (AR-S) staining, ultraviolet (UV) light visualization, transmission electron microscopy (TEM) imaging and energy dispersive X-ray microanalysis (EDX).

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

Here, we present a method for ex vivo culture of long murine bones at both fetal and newborn stages, suitable for analyzing bone and cartilage development and homeostasis in controlled conditions while recapitulating the in vivo process.

Culturing and Measuring Fetal and Newborn Murine Long Bones

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

Here, we present a simple and effective assay procedure for resorption pit assays using calcium phosphate coated cell culture plates.

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

This protocol describes the fabrication of 3D bone constructs using human osteocytes embedded in methacrylated gelatin (GelMA) hydrogels supplemented with decellularized extracellular matrix. The method is low-cost, scalable, and compatible with basic laboratory equipment, providing a translational platform for studying musculoskeletal disease processes.

A 3D Bone Culture Platform Using Human Osteocytes and Decellularized Extracellular Matrix for Modeling Musculoskeletal Diseases

Learn a rapid radiolabeled amino acid uptake assay for evaluating nutrient consumption in bone cells and isolated bone shafts. This sensitive method quantifies amino acid uptake using primary or transformed cells.

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

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