This study was supported by grants from the National Natural Science Foundation of China (81804116, 81673991, 81770107, 81603643, and 81330085), the program for Innovative Team, Ministry of Science and Technology of China (2015RA4002 to WYJ), &#160;the program for Innovative Team, Ministry of Education of China (IRT1270 to WYJ), Shanghai TCM Medical Center of Chronic Disease (2017ZZ01010 to WYJ), Three Years Action to Accelerate the Development of Traditional Chinese Medicine Plan (ZY(2018-2020)-CCCX-3003 to WYJ), and national key research development projects (2018YFC1704302).

All of the experimental procedures were performed with the approval of Institutional Animal Care and Use Committee of the Shanghai University of TCM (SZY201604005).
1. Preparation and administration of GSK working solution
<ol>
	<li>Calculate the equivalent doses of GSK granules for mouse.
	<ol>
		<li>Calculate body surface based on the Meeh-Rubner equation15: body surface = K x (body weight2/3)/1000, where the K values are 10.6 for human and 9.1 for mouse. Assuming a human body weight of 70 kg, then human body surface (m2) = 10.6 x (702/3)/1000 = 1.8 m2. Assuming a mouse body weight of 20 g (0.02 kg; e.g., 1 month old, female, C57/BL6), then mouse body surface (m2) = 9.1 x (0.022/3)/1000 = 0.0067 m2.</li>
		<li>Based on the calculated body surface, calculate the body transform ratio for human and mouse. Human: 70 kg/1.8 m2 = 39. Mouse: 0.02 kg/0.0067 m2 = 3. GSK granule = 20 g/70 kg x 39/3 = 3.72 g/kg &#8776; 4 g/kg.</li>
		<li>Based on a body weight of 20 g per mouse, calculate the equivalent dosage for mouse: 4 g/kg x 0.02 kg = 0.08 g.</li>
		<li>Calculate three equivalent doses of GSK granules based on 20 mice per group and an intervention lasting for 3 months (90 days): (1) GSKL (OVX + low-dose GSK granules [2 g/kg/day]): 0.04 g mouse/day x 20 mice x 90 days = 72 g. (2) GSKM (OVX + medium-dose GSK granules [4 g/kg/day]): 0.08 g mouse/day x 20 mice x 90 days = 144 g. (3) GSKH (OVX + high-dose GSK granules [8 g/kg/day]): 0.12 g mouse/day x 20 mice x 90 days = 216 g. 
		NOTE: Prepare an additional 20% of GSK granules in practice to offset the loss.</li>
	</ol>
	</li>
	<li>Calculate the volume of GSK granule per mouse based on the body weight15: e.g., volume (V) = 0.24 mL/mouse/day. 
	NOTE: The volume for intragastric administration for mouse is 0.12 mL/10 g.</li>
	<li>Weigh 10-days&#8217; worth of three doses of GSK granules. Weigh 8 g, 16 g, and 24 g of GSK granules and serve as GSKL, GSKM, and GSKH, respectively.</li>
	<li>Calculate the equivalent dose of calcium carbonate with vitamin D3 (CAL) for mouse based on the Meeh-Rubner equation15 as in steps 1.1.1 and 1.1.2: CAL dosage = 2 tablet/70 kg x 39/3 = 0.372 tablet/kg &#8776; 0.4 tablet/kg.</li>
	<li>Based on a body weight of 20 g per mouse (e.g., 1 month old, female, C57/BL6), calculate the equivalent dosage of CAL for mouse: 0.4 tablet/kg x 0.02 kg = 0.008 tablet. Then calculate the equivalent dose of CAL based on 20 mice per group and an intervention lasting for 3 months (90 days): 0.008 tablet x 20 x 90 = 14.4 tablets. Weigh 10-days&#8217; worth of CAL (1.6 tablets).</li>
	<li>Dissolution
	<ol>
		<li>Place 8 g of GSK granules into a 50 mL tube. Add 48 mL of saline and shake tube to completely dissolve. 
		NOTE: The standard for complete dissolution is the absence of sediment. Complete dissolution can be further confirmed if a gavage needle can draw up the working solution and then expel it smoothly.</li>
		<li>Repeat step 1.5.1 with 16 g and 24 g of GSK granules.</li>
		<li>Place 1.6 tablets (10-days&#8217; worth) of CAL into a 50 mL tube. Add 48 mL of saline and shake tube to completely dissolve. 
		NOTE: The working solutions can be stored at -4 &#176;C and prepared every 10 days.</li>
	</ol>
	</li>
	<li>Intragastric administration
	<ol>
		<li>Grasp the back of the mouse (1 month old, female, C57/BL6) with the mouse facing forward and ensure that it stays firmly in that position. Keep the mouse calm for 2&#8722;3 min before administration. 
		NOTE: Ensure that the researcher can clearly see the front of the mouse. Wear gloves to prevent mouse bites, particularly for new researchers.</li>
		<li>Place the gavage needle (size: #12, 40 mm) in the working solution of GSK granules and draw 0.24 mL of the working solution.</li>
		<li>Put the gavage needle into the mouse through one side of its mouth until the gavage needle reaches the stomach. 
		NOTE: To confirm the gavage needle has reached the stomach: (1) The gavage needle encounters the feeling of resistance. Meanwhile, the mouse shows the action of swallowing before the gavage needle passes the physical narrowing of the esophagus. (2) Inject approximately 0.5 mL of the working solution into the mouse and wait for 1 min. If there is no solution coming out of the mouse, this means that the gavage needle has reached the stomach.</li>
		<li>Inject the working solution of GSK granule (0.24 mL/mouse) into the stomach and then draw out the gavage needle. Return the mouse into its cage.</li>
		<li>Repeat step 1.6.4 with the CAL solution and inject 0.24 mL of CAL solution per mouse. 
		NOTE: The volume of CAL solution is calculated as in step 1.2.</li>
	</ol>
	</li>
</ol>
2. Micro-CT scanning
<ol>
	<li>Tibia harvesting and preparation
	<ol>
		<li>Intraperitoneally anesthetize mouse with 300 mL/100 g of 80 mg/kg ketamine the day following the 90 day&#8217;s intervention. Use a needle pinch of the toes to confirm whether the mouse is completely anesthetized. No response indicates successful anesthesia. Then kill the mouse with cervical dislocation.</li>
		<li>Fix the mouse with both the arms and legs on foam with tacks.</li>
		<li>Cut off the skin with scissors (size: 8.5 cm) and tweezers (size: 10 cm) of the legs from the proximal to the distal end and then harvest tibias.</li>
		<li>Immediately put the tibias into 70% ethyl alcohol and wash for 3 times.</li>
	</ol>
	</li>
	<li>Wrap the left tibia of the mouse with sponge foam and put it into a sample tube (35 mm diameter, 140 mm length). 
	NOTE: The long axis of the specimen should be along with that of the sample tube. Ensure the proximal end of the tibia points upwards.</li>
	<li>Running the micro-CT 80 scan machine
	<ol>
		<li>Start the micro-CT 80 scan machine at room temperature.</li>
		<li>Set the sample tube into micro-CT 80 and start cross-section scanning with the following scanning parameters: pixel size 15.6 &#956;m, tube voltage 55 kV, tube current 72 &#956;A, integration time 200 ms, spatial resolution 15.6 &#956;m, pixel resolution 15.6 &#956;m, and image matrix 2048 x 2048. 
		NOTE: The cancellous bone is distinguished from the cortical bone by pre-scanning. The scan area of the tibia is defined as the cancellous bone area from 5 mm below the tibial plateau to the distal end.</li>
	</ol>
	</li>
	<li>Quantification of bone parameter
	<ol>
		<li>After completing cross-section scanning, obtain the images of the left tibias.</li>
		<li>Set the density threshold to 245&#8722;1000. Use the micro-CT evaluation program V6.6 to measure the following bone parameters: bone mineral densities (BMD), bone volume over total volume (BV/TV), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), as well as bone trabecular bone separation (Tb.Sp).</li>
	</ol>
	</li>
</ol>
3. Preparation of blood serum for in vitro experiments
<ol>
	<li>Calculation
	<ol>
		<li>Based on a rat body weight of 0.2 kg (1 month old, female, Sprague-Dawley), calculate the dosage of GSK granule: human dosage/day x body weight of human x K/body weight of rat = 20 g/70 kg/day x 70 kg x K (K = 0.018) /0.2 kg = 2 g/kg/day. 
		NOTE: K is the pharmacological transformation coefficient between human and mouse15 (K = 0.018).</li>
		<li>Repeat step 3.1.1 and calculate the following dosages.
		<ol>
			<li>Calculate dosage of GSKL: 10 g/70 kg/day x 70 kg x K/0.2 kg = 1 g/kg/day.</li>
			<li>Calculate dosage of GSKM: 20 g/70 kg/day x 70 kg x K/0.2 kg = 2 g/kg/day.</li>
			<li>Calculate dosage of GSKL: 40 g/70 kg/day x 70 kg x K/0.2 kg = 4 g/kg/day.</li>
			<li>Calculate dosage of CAL: 2 tablet /70 kg/day x 70 kg x K/0.2 kg = 0.2 tablet/kg/day.</li>
		</ol>
		</li>
		<li>Calculate the total dosage of GSK granule and CAL.
		<ol>
			<li>Calculate the total dosage for GSKL: 1 g/kg/day x 0.2 kg x 6 rats x 3 days = 3.6 g.</li>
			<li>Calculate the total dosage for GSKM: 2 g/kg/day x 0.2 kg x 6 rats x 3 days = 7.2 g.</li>
			<li>Calculate the total dosage for GSKH: 4 g/kg/day x 0.2 kg x 6 rats x 3 days = 14.4 g.</li>
			<li>Calculate the CAL dosage = 0.2 tablet/kg/day x 0.2 kg x 6 rats x 3 days = 0.72 tablet. 
			NOTE: A total of 10 mL of GSK granule-containing serum is needed to prepare 100 mL culture medium (20% GSK granule-containing serum). Each rat (6 rats/group) is expected to provide 1.5&#8722;2 mL of GSK granule-containing serum after centrifugation.</li>
		</ol>
		</li>
		<li>Calculate the volume of GSK granules applied per rat based on body weight15: e.g., volume (V) = 2 mL/rat/day. 
		NOTE: The volume for intragastric administration for rat is 0.1 mL/10 g.</li>
	</ol>
	</li>
	<li>Weigh 3-days&#8217; worth of three doses of GSK granules. Weigh 3.6 g, 7.2 g, and 14.4 g of GSK granules and serve as GSKL, GSKM, and GSKH, respectively. Weigh 0.72 tablet for the CAL group.</li>
	<li>Place 7.2 g of GSK granules into a 50 mL tube. Add 36 mL of saline and shake tube to completely dissolve. Repeat this with 3.6 g and 14.4 g of GSK granules.</li>
	<li>Repeat section 1.6 for intragastric administration with 2 mL of GSK working solution. 
	NOTE: Administer the same volume of saline (2 mL per rat) to prepare serum and serves as a blank control group for in vitro assays.</li>
	<li>Preparation of the GSK-containing serum 
	<ol>
		<li>Intraperitoneally anesthetize the rats with 300 mL/100 g of 80 mg/kg ketamine 1 h after the last administration of GSK granules. Use a needle pinch of the toes to confirm whether the rat is completely anesthetized. No response indicates successful anesthesia.</li>
		<li>Expose the abdomen to the bottom of the thorax of rats using straight operating scissors after incising the skin and peritoneum. 
		NOTE: The surgical instrument must be sterilized at high temperatures and high pressures prior to use. The surgical area must be sterilized with 70% ethanol during blood collection.</li>
		<li>Remove the connective tissue of the abdominal aorta with tissue paper to expose the vessel clearly.</li>
		<li>Draw blood from the abdominal aorta using a 10 mL, 22 G syringe. Then remove the needle and transfer the blood to a 15 mL sterile tube. Usually, 6&#8722;8 mL of blood can be obtained from one rat. 
		NOTE: Each rat must be kept living when drawing blood. One indicator is that the abdominal aorta pulsates when the rat is alive. The rat is dead after blood draw.</li>
		<li>Keep the tube upright at room temperature for 30&#8722;60 min until the blood is clotted in the tube. Then centrifuge the tube at 500&#8722;600 x g for 20 min. Transfer all the supernatant (serum) from one group (6 rats) to one 50 mL sterile tube and shake to mix.</li>
		<li>Inactivate the serum by incubating in a 56 &#176;C water bath for 30 min. Filter the serum using a 0.22-&#956;m-pore-size hydrophilic polyethersulfone syringe filter. Store at -80 &#176;C for long-term usage (less than 1 year). 
		NOTE: The filtered serum can be used for in vitro osteoclastogenesis and osteoblastogenesis.</li>
	</ol>
	</li>
	<li>Application
	<ol>
		<li>In vitro osteoclastogenesis
		<ol>
			<li>Dilute the three dosages of the GSK-containing serum (GSKL, GSKM, GSKH) at the ratio of 1:4 with minimum Eagle&#8217;s medium (&#945;-MEM) containing L-glutamine, ribonucleosides, and deoxyribonucleosides. 
			NOTE: Ensure that the final concentration of GSK-containing serum for in vitro osteoclastogenesis and osteoblastogenesis is 20%.</li>
			<li>Add the diluted GSK-containing serum (200 &#956;L/well) from step 3.6.1.1 to bone marrow macrophages (BMMs) from 4&#8722;6 week old C57BL/6 mice for osteoclastogenesis and stimulate BMMs with macrophage colony-stimulating factor (M-CSF, 10 ng/mL) and receptor activator for nuclear factor-&#954;B ligand (RANKL, 100 ng/mL) as previously described2.</li>
		</ol>
		</li>
		<li>In vitro osteoblastogenesis
		<ol>
			<li>Repeat step 3.6.1.1.</li>
			<li>Add the diluted GSK-containing serum (2 mL/well) to bone mesenchymal stem cells (BMSCs) from 4&#8722;6 week old C57BL/6 mice to generate osteoblast as previously described16.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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Bone Mineral Research. 11, 1043-1051 (1996).</li><li>Ammann, 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=Transgenic+mice+expressing+soluble+tumor+necrosis+factor-receptor+are+protected+against+bone+loss+caused+by+estrogen+deficiency.">Transgenic mice expressing soluble tumor necrosis factor-receptor are protected against bone loss caused by estrogen deficiency.</a> Journal Clinical Investigation. 99, 1699-1703 (1997).</li><li>Kimble, R. 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=Simultaneous+block+of+interleukin-1+and+tumor+necrosis+factor+is+required+to+completely+prevent+bone+loss+in+the+early+postovariectomy+period.">Simultaneous block of interleukin-1 and tumor necrosis factor is required to completely prevent bone loss in the early postovariectomy period.</a> Endocrinology. 136, 3054-3061 (1995).</li></ol>

The authors have nothing to disclose.

Granules of TCM agents have become one of the common choices for formulations or prescriptions. GSK granules are composed of several herbal medicines based on clinical experiences or the TCM theory, and they exert better curative effects with fewer side effects4. Compared with water decoction, the granules have these advantages: good taste, convenience of delivery, long-term storage, standard protocol and consistent curative effects, as well as higher productivity. Currently, granules are one of t...

Traditional Chinese medicine (TCM) is one of the important complementary and alternative approaches to treat osteoporosis1,2. Water decoction is the basic and most commonly used form of the formula3. However, drawbacks also exist: bad taste, inconvenience for carriage, short shelf life and inconsistent protocols, limiting the uses as well as the curative effects. To avoid the above disadvantages as well as to pursue better effects, granules were developed and have been widely used4. Although many studies have explored the pharmacological mechanisms of one or more effective components from the granules5,6,7, the exact mechanisms and underlying pharmacological processes are still difficult to identify. This is because too many effective components from one granule may simultaneously exert similar or opposed effects4. Therefore, the development of one standard protocol to prepare the granules before delivering to the body not only would have a great impact on the therapeutic outcomes but is also required for both in vivo and in vitro assays.
Moreover, the curative effects of granules in the clinic are difficult to confirm and exactly identify using in vitro or ex vivo studies, which creates a challenge because the pharmacological mechanisms are too complex. To resolve this, the preparation of drug-containing serum was first proposed by Tashino in 1980s8. From then on, numerous researchers applied drug-containing serum to herbal medicine, including granules9,10,11. Currently, the choice of drug-containing serum for in vitro investigations is regarded as one strategy that closely mimics physiological conditions.
Gushukang (GSK) granules were developed to treat postmenopausal osteoporosis (POP) based on clinical practice in light of the theory of TCM. GSK granules prevent bone loss in ovariectomized (OVX) mice in vivo, inhibit osteoclastic bone resorption, and stimulate osteoblastic bone formation4. Consequently, Li et al.12 found that GSK granules have bone protective effects in OVX mice by enhancing the activities of calcium receptor to stimulate bone formation. To confirm the bone-protective effects as well as the pharmacological effects of GSK granules, the authors here provide a detailed procedure for preparation of working solutions and drug (GSK granule)-containing serum. Moreover, this article describes the application of GSK granules in an OVX-induced osteoporotic mouse model and GSK granule-containing serum for in vitro osteoclastogenesis/osteoblastogenesis.
GSK granules are composed of several herbs13,14 and can be completely dissolved in saline easily. Therefore, saline serves as the vehicle. Sham-operated mice (Sham) and OVX mice were administered the same volume of saline as the granule-administered mice. The equivalent doses of GSK granules for the mouse were calculated based on the Meeh-Rubner equation15. This equation not only has the advantage of obtaining safe dosages but also guarantees pharmacological effects15. The three dosages of GSK granules were generated as the follows: (1) GSKL: OVX + low-dose GSK granules, 2 g/kg/day. (2) GSKM: OVX + medium-dose GSK granules, 4 g/kg/day. (3) GSKH: OVX + high-dose GSK granules, 8 g/kg/day. Mice in the GSKL, GSKM and GSKH groups were intragastrically administered GSK granules. Calcium carbonate (600 mg/tablet) with vitamin D3 (125 international unit/tablet), for example, in a mature and marketed product (e.g., Caltrate [CAL]) for treating and preventing osteoporosis, was used as a positive control.

<table>
	<tbody>
		<tr>
			<td>&#945;-MEM</td>
			<td>Hyclone 
			laboratories</td>
			<td>SH30265.018</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>&#946;-Glycerophosphate</td>
			<td>Sigma</td>
			<td>G5422</td>
			<td>Osteoblastogenesis</td>
		</tr>
		<tr>
			<td>Caltrate (CAL)</td>
			<td>Wyeth</td>
			<td>L96625</td>
			<td>Animal interventation</td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>SLAC Laboratory 
			Animal Co. Ltd.</td>
			<td>Random</td>
			<td>Ainimal preparation</td>
		</tr>
		<tr>
			<td>Dexamethsome</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D2438</td>
			<td>Cell frozen</td>
		</tr>
		<tr>
			<td>Ethylene Diamine Tetraacetic Acid (EDTA)</td>
			<td>Sangon Biotech</td>
			<td>60-00-4</td>
			<td>Samples treatmnet</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>FL-24562</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Gushukang granules</td>
			<td>kangcheng companyin china</td>
			<td>Z20003255</td>
			<td>Herbal prescription</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Olympus BX50</td>
			<td>Olympus BX50</td>
			<td>Images for osteoclastogenesis</td>
		</tr>
		<tr>
			<td>L-Ascorbic acid 2-phosphate sequinagneium slat hyclrate</td>
			<td>Sigma</td>
			<td>A8960-5G</td>
			<td>Osteoblastogenesis</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>DMI300B</td>
			<td>Osteocast and osteoblast imagine</td>
		</tr>
		<tr>
			<td>M-CSF</td>
			<td>Peprotech</td>
			<td>AF-300-25-10</td>
			<td>Osteoclastogenesis</td>
		</tr>
		<tr>
			<td>&#924;icro-CT</td>
			<td>Scanco 
			Medical AG</td>
			<td>&#956;CT80 radiograph microtomograph</td>
			<td>Bone Structural analsysis</td>
		</tr>
		<tr>
			<td>RANKL</td>
			<td>Peprotech</td>
			<td>11682-HNCHF</td>
			<td>Osteoclastogenesis</td>
		</tr>
		<tr>
			<td>Sprague Dawley</td>
			<td>SLAC Laboratory 
			Animal Co. Ltd.</td>
			<td>Random</td>
			<td>Blood serum collection</td>
		</tr>
		<tr>
			<td>Tartrate-Resistant Acid Phosphate (TRAP) Kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A-1KT</td>
			<td>TRAP staining</td>
		</tr>
	</tbody>
</table>

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.

Micro-CT scanning results indicated that the OVX mice showed significant bone loss compared to saline control mice (Figure 1A). The intervention (90 days) of GSK granules greatly increased the BMD, particularly in the GSKM group (Figure 1B). The bone structure parameters, such as BMD, BV/TV, Tb.N, and Tb.Th, were quantified. GSK granule treatments led to increased BMD, BV/TV, Tb.N and Tb.Th but decreased Tb.Sp (<strong class="xfi...

Watch this Scientific Journal Video about Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments at JoVE.com

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.

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 ...

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7.5K Views.  Shanghai University of Traditional Chinese Medicine. 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.

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

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

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 ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

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.

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. ...