This work was supported by the National Natural Science Foundation of China (81573992). We thank Emily K. Lo and Kathleen DiNapoli for their language editing assistance.

The protocol follows the animal care guidelines of Shanghai University of Traditional Chinese Medicine and is approved by the animal experiment Shanghai Animal Ethics Committee.
1. Protocol I: Preparation of EXD
<ol>
	<li>Calculation
	<ol>
		<li>Administer the EXD to a treatment group of 10 Six month-old female Imprinting Control Region (ICR)&#160;mice (0.02 kg/mouse), 5 days per week for 12 weeks. 
		NOTE: A total of 1.702 EXDs are needed. Because EXDs are measured in ...

<ol>
	<li>Zhang, L. G., Ouyang, X. W., Wu, T. T., Ni, L. J., Shi, W. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+in+vitro+effects+and+interactions+of+active+fractions+in+a+Chinese+medicinal+formula+(Yaotongning+Capsule)+on+rat+chondrocytes.">Quantitative evaluation of in vitro effects and interactions of active fractions in a Chinese medicinal formula (Yaotongning Capsule) on rat chondrocytes.</a> J Ethnopharmacol. 155 (3), 1424-1432 (2014).</li><li>Ogawa, Y., Fujii, Y., Sugiyama, R., Konishi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+seven+crude+drug+components+in+the+sleep-promoting+effect+of+Yokukansan.">The role of the seven crude drug components in the sleep-promoting effect of Yokukansan.</a> J Ethnopharmacol. 177, 19-27 (2016).</li><li>Kim, J. H., Doh, E. J., Lee, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Medicinal+Categorization+of+Atractylodes+japonica+Koidz.+by+Using+Internal+Transcribed+Spacer+Sequencing+Analysis+and+HPLC+Fingerprinting+Combined+with+Statistical+Tools.">Evaluation of Medicinal Categorization of Atractylodes japonica Koidz. by Using Internal Transcribed Spacer Sequencing Analysis and HPLC Fingerprinting Combined with Statistical Tools.</a> Evid Based Complement Alternat Med. 2016, 2926819 (2016).</li><li>Sheridan, H., 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+potential+of+metabolic+fingerprinting+as+a+tool+for+the+modernisation+of+TCM+preparations.">The potential of metabolic fingerprinting as a tool for the modernisation of TCM preparations.</a> J Ethnopharmacol. 140 (3), 482-491 (2012).</li><li>Planas, G. M., Kucacute, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contraceptive+Properties+of+Stevia+rebaudiana.">Contraceptive Properties of Stevia rebaudiana.</a> Science. 162 (3857), 1007 (1968).</li><li>Tashino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Serum+pharmacology"+and+"serum+pharmaceutical+chemistry":+from+pharmacology+of+Chinese+traditional+medicines+to+start+a+new+measurement+of+drug+concentration+in+blood.">"Serum pharmacology" and "serum pharmaceutical chemistry": from pharmacology of Chinese traditional medicines to start a new measurement of drug concentration in blood.</a> Ther Drug Monit Res. 5, 54-64 (1988).</li><li>Cao, Y., Liu, F., Huang, Z., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+Guanxin+Shutong+capsule+drug-containing+serum+on+tumor+necrosis+factor-alpha-induced+endothelial+dysfunction+through+nicotinamide+adenine+dinucleotide+phosphate+oxidase+and+the+nitric+oxide+pathway.">Protective effects of Guanxin Shutong capsule drug-containing serum on tumor necrosis factor-alpha-induced endothelial dysfunction through nicotinamide adenine dinucleotide phosphate oxidase and the nitric oxide pathway.</a> Exp Ther Med. 8 (3), 998-1004 (2014).</li><li>Fu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+Vivo+Stromal+Cell-Derived+Factor+1-Mediated+Differentiation+of+Mouse+Bone+Marrow+Mesenchymal+Stem+Cells+into+Hepatocytes+Is+Enhanced+by+Chinese+Medicine+Yiguanjian+Drug-Containing+Serum.">Ex Vivo Stromal Cell-Derived Factor 1-Mediated Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells into Hepatocytes Is Enhanced by Chinese Medicine Yiguanjian Drug-Containing Serum.</a> Evid Based Complement Alternat Med. 2016, 7380439 (2016).</li><li>Chen, X., 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=Application+of+serum+pharmacology+in+evaluating+the+antitumor+effect+of+Fuzheng+Yiliu+Decoction+from+Chinese+Medicine.">Application of serum pharmacology in evaluating the antitumor effect of Fuzheng Yiliu Decoction from Chinese Medicine.</a> Chin J Integr Med. 20 (6), 450-455 (2014).</li><li>Sze, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+mechanism:+Erxian+Decoction,+a+Chinese+medicine+formula,+for+relieving+menopausal+syndrome.">A novel mechanism: Erxian Decoction, a Chinese medicine formula, for relieving menopausal syndrome.</a> J Ethnopharmacol. 123 (1), 27-33 (2009).</li><li>Sze, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Erxian+decoction,+a+Chinese+medicinal+formulation,+on+serum+lipid+profile+in+a+rat+model+of+menopause.">Effects of Erxian decoction, a Chinese medicinal formulation, on serum lipid profile in a rat model of menopause.</a> Chin Med. 6, 40 (2011).</li><li>Zhong, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+randomized,+double-blind,+controlled+trial+of+a+Chinese+herbal+formula+(Er-Xian+decoction)+for+menopausal+symptoms+in+Hong+Kong+perimenopausal+women.">A randomized, double-blind, controlled trial of a Chinese herbal formula (Er-Xian decoction) for menopausal symptoms in Hong Kong perimenopausal women.</a> Menopause. 20 (7), 767-776 (2013).</li><li>Wang, S. W., 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=Steroidogenic+effect+of+Erxian+decoction+for+relieving+menopause+via+the+p-Akt/PKB+pathway+in+vitro+and+in+vivo.">Steroidogenic effect of Erxian decoction for relieving menopause via the p-Akt/PKB pathway in vitro and in vivo.</a> J Ethnopharmacol. , (2016).</li><li>Lin, L., Wu, S., Tang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Clinical+observation+and+experimental+study+of+the+treatment+of+aplastic+anemia+by+warming+and+tonifying+the+spleen+and+kidney].">[Clinical observation and experimental study of the treatment of aplastic anemia by warming and tonifying the spleen and kidney].</a> Zhong Xi Yi Jie He Za Zhi. 10 (5), 272-274 (1990).</li><li>Nian, H., 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=Antiosteoporotic+activity+of+Er-Xian+Decoction,+a+traditional+Chinese+herbal+formula,+in+ovariectomized+rats.">Antiosteoporotic activity of Er-Xian Decoction, a traditional Chinese herbal formula, in ovariectomized rats.</a> J Ethnopharmacol. 108 (1), 96-102 (2006).</li><li>Qin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiosteoporotic+chemical+constituents+from+Er-Xian+Decoction,+a+traditional+Chinese+herbal+formula.">Antiosteoporotic chemical constituents from Er-Xian Decoction, a traditional Chinese herbal formula.</a> J Ethnopharmacol. 118 (2), 271-279 (2008).</li><li>Xue, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+HNMR-based+metabonomics+study+of+postmenopausal+osteoporosis+and+intervention+effects+of+Er-Xian+Decoction+in+ovariectomized+rats.">A HNMR-based metabonomics study of postmenopausal osteoporosis and intervention effects of Er-Xian Decoction in ovariectomized rats.</a> Int J Mol Sci. 12 (11), 7635-7651 (2011).</li><li>Lu, X. N., Xu, X. R., Lin, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Clinical+observation+of+bushen+er'xian+decoction+in+treating+premature+ovarian+failure].">[Clinical observation of bushen er'xian decoction in treating premature ovarian failure].</a> Zhongguo Zhong Xi Yi Jie He Za Zhi. 28 (7), 594-596 (2008).</li><li>Yu, X., 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=Anti-angiogenic+activity+of+Erxian+Decoction,+a+traditional+Chinese+herbal+formula,+in+zebrafish.">Anti-angiogenic activity of Erxian Decoction, a traditional Chinese herbal formula, in zebrafish.</a> Biol Pharm Bull. 35 (12), 2119-2127 (2012).</li><li>Chu, E. 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=An+in+vitro+and+in+vivo+investigation+of+the+antimetastatic+effects+of+a+Chinese+medicinal+decoction,+erxian+decoction,+on+human+ovarian+cancer+models.">An in vitro and in vivo investigation of the antimetastatic effects of a Chinese medicinal decoction, erxian decoction, on human ovarian cancer models.</a> Integr Cancer Ther. 12 (4), 336-346 (2013).</li><li>Zhu, Z., Li, L., Jin, X., Fang, J., Zhang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Er-Xian+Decoction,+a+traditional+Chinese+herbal+formula,+intervening+early+in+hypothalamic-pituitary+axis+of+male+rats+with+delayed+puberty.">Er-Xian Decoction, a traditional Chinese herbal formula, intervening early in hypothalamic-pituitary axis of male rats with delayed puberty.</a> Pharmacogn Mag. 10 (40), 517-521 (2014).</li><li>Miao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Experimental animals and technology. 1997, 145 (1997).</li><li>Soleimani, M., Nadri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+isolation+and+culture+of+mesenchymal+stem+cells+from+mouse+bone+marrow.">A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow.</a> Nat Protoc. 4 (1), 102-106 (2009).</li><li>Lu, S., Huang, J., Wang, 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=Er-Xian+Decoction+Stimulates+Osteoblastic+Differentiation+of+Bone+Mesenchymal+Stem+Cells+in+Ovariectomized+Mice+and+Its+Gene+Profile+Analysis.">Er-Xian Decoction Stimulates Osteoblastic Differentiation of Bone Mesenchymal Stem Cells in Ovariectomized Mice and Its Gene Profile Analysis.</a> Stem Cells International. 2016, 4079210 (2016).</li><li>Turner, P. V., Brabb, T., Pekow, C., Vasbinder, 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=Administration+of+substances+to+laboratory+animals:+routes+of+administration+and+factors+to+consider.">Administration of substances to laboratory animals: routes of administration and factors to consider.</a> J Am Assoc Lab Anim Sci. 50 (5), 600-613 (2011).</li><li>Wu, C., 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+determination+of+seven+flavonoids+in+dog+plasma+by+ultra-performance+liquid+chromatography-tandem+mass+spectrometry+and+its+application+to+a+bioequivalence+study+of+bioactive+components+in+Herba+Epimedii+and+Er-Xian+Decoction.">Simultaneous determination of seven flavonoids in dog plasma by ultra-performance liquid chromatography-tandem mass spectrometry and its application to a bioequivalence study of bioactive components in Herba Epimedii and Er-Xian Decoction.</a> J Pharm Biomed Anal. 54 (1), 186-191 (2011).</li><li>Hu, Y. 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=Identification+of+the+major+chemical+constituents+and+their+metabolites+in+rat+plasma+and+various+organs+after+oral+administration+of+effective+Erxian+Decoction+(EXD)+fraction+by+liquid+chromatography-mass+spectrometry.">Identification of the major chemical constituents and their metabolites in rat plasma and various organs after oral administration of effective Erxian Decoction (EXD) fraction by liquid chromatography-mass spectrometry.</a> Biomed Chromatogr. 24 (5), 479-489 (2010).</li><li>Xue, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+and+interaction+of+icariin,+curculigoside,+and+berberine+in+er-xian+decoction,+a+traditional+chinese+medicinal+formula,+on+osteoclastic+bone+resorption.">Effects and interaction of icariin, curculigoside, and berberine in er-xian decoction, a traditional chinese medicinal formula, on osteoclastic bone resorption.</a> Evid Based Complement Alternat Med. , (2012).</li><li>Nair, A. B., Jacob, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+practice+guide+for+dose+conversion+between+animals+and+human.">A simple practice guide for dose conversion between animals and human.</a> J Basic Clin Pharm. 7 (2), 27-31 (2016).</li><li>Xu, X., 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=Protective+effect+of+the+traditional+Chinese+medicine+xuesaitong+on+intestinal+ischemia-reperfusion+injury+in+rats.">Protective effect of the traditional Chinese medicine xuesaitong on intestinal ischemia-reperfusion injury in rats.</a> Int J Clin Exp Med. 8 (2), 1768-1779 (2015).</li><li>Li, Z., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+methods+for+Chinese+herbs+serum+pharmcology.">On the methods for Chinese herbs serum pharmcology.</a> Zhong Guo Zhong Yi Yao Xing Xi Za Zhi. 9 (2), 5-6 (2002).</li><li>Jiang, Y. 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=Effect+of+chinese+herbal+drug-containing+serum+for+activating-blood+and+dispelling-toxin+on+ox-LDL-induced+inflammatory+factors'+expression+in+endothelial+cells.">Effect of chinese herbal drug-containing serum for activating-blood and dispelling-toxin on ox-LDL-induced inflammatory factors' expression in endothelial cells.</a> Chin J Integr Med. 18 (1), 30-33 (2012).</li><li>Guo, C. Y., Ma, X. J., Liu, Q., Yin, H. J., Shi, D. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Effect+of+Chinese+herbal+drug-containing+serum+for+activating+blood,+activating+blood+and+dispelling+toxin+on+TNF-alpha-induced+adherence+between+endothelial+cells+and+neutrophils+and+the+expression+of+MAPK+pathway].">[Effect of Chinese herbal drug-containing serum for activating blood, activating blood and dispelling toxin on TNF-alpha-induced adherence between endothelial cells and neutrophils and the expression of MAPK pathway].</a> Zhongguo Zhong Xi Yi Jie He Za Zhi. 35 (2), 204-209 (2015).</li><li>Li, Y., Xia, J. Y., Chen, W., Deng, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Ling+Qi+Juan+Gan+capsule+drug-containing+serum+on+PDGF-induced+proliferation+and+JAK/STAT+signaling+of+HSC-T6+cells].">Effects of Ling Qi Juan Gan capsule drug-containing serum on PDGF-induced proliferation and JAK/STAT signaling of HSC-T6 cells].</a> Zhonghua Gan Zang Bing Za Zhi. 21 (9), 663-667 (2013).</li><li>Li, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Some+issues+in+methology+of+Chinese+herbs+serum+pharmcology].">[Some issues in methology of Chinese herbs serum pharmcology].</a> Zhong Yao Xin Yao Yu Lin Chuang Yao Li. 10 (5), 263 (1999).</li><li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[A+review+of+Chinese+herbs+serum+pharmcology+methodological+study].">[A review of Chinese herbs serum pharmcology methodological study].</a> Nan Jing Zhong Yi Yao Da Xue Xue Bao. 18 (4), 254 (2002).</li><li>Zhang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Issues+and+strategies+for+study+of+serum+pharmcology+in+oncology].">[Issues and strategies for study of serum pharmcology in oncology].</a> Zhong Yi Yan Jiu. 17 (5), 13-14 (2004).</li></ol>

In recent years, more attention has been paid to herbal medicines, one type of alternative medicine that has been applied in the clinical setting in the Eastern world for thousands of years. Different from the &#34;bench to bed&#34; pattern of modern medicine, traditional herbal medicine first require the &#34;bed to bench&#34; pattern to explain their mechanisms. This can be followed by validation performed at the bench stage and processing for the development of newly optimized drugs. So far, there are several extracti...

Interest in the study and application of traditional herbal medicine is currently growing. As opposed to modern drugs, in which chemical ingredients are definite, herbal formulas have some unknown ingredients and require extraction processes to enable the delivery of their active compounds. Although many studies try to select one small compound with a well-known structure as a representative of the whole herb or herbal formula, neither the pharmacological efficacies nor mechanisms can be considered equivalent1,2. While fingerprinting enables the analysis of the constituents of complex herbal formulas, some constituents are still not clearly analyzed, leading to a challenge when combining all extracts for study3. The interactions of numerous constituents/extracts mediate the therapeutic effects of herbal medicines. To retain this advantage, the traditional extract form&#8212;decoction&#8212;is still widely used in the clinic. Since the extract procedure has a great impact on the therapeutic efficacy, a standard protocol of traditional water decoction is necessary, especially for in vivo studies4,5.
On the other hand, when exploring pharmacological mechanisms, the administration of herbal decoctions during in vitro or ex vivo studies is also a challenge. The concept of a drug-containing serum was first proposed by Tashino in 19886. Since then, increasing numbers of researchers have applied it to herbal medicine7,8,9. Although the method of drug-containing serum has some limitations, such as the influence of certain components of the serum, it is still considered to be a method that closely mimics physiological conditions.
The er-xian decoction was developed in the early 1950s to relieve menopausal symptoms10,11,12,13. It has also been applied to the treatment of aplastic anemia14, menopausal osteoporosis15,16,17, premature ovarian failure18, breast cancer19, ovarian cancer20, and delayed puberty21. Here, we present detailed protocols for the preparation of both the er-xian decoction (EXD) and its drug-containing serum. In addition, we describe the application of EXD and EXD-containing serum to murine menopausal osteoporotic models.
An EXD is composed of 9 g each of Curculigo orchioides Gaertn, Herbaa Epimedii, Radix Morindae Officinalis, and Radix Angelicae Sinensis and 6 g each of Cortex Phellodendri and Rhizoma Anemarrhenae per adult patient per day. The equivalent dose for a mouse is 0.1418 EXD/kg/day based on the following equation22: dB = dA * RB / RA * (WA / WB) 1/3. dA and dB refer to the dose per bodyweight (mg/kg) of the human and mouse, respectively. The dose, in mg/kg, is replaced by the number of EXD/kg. RA and RB represent the human body factor and mouse body factor, respectively, which are proportional to the (body surface area (m2) / body weight (kg))2/3 (See Table 1). WA and WB indicate the bodyweight (kg) of the human and mouse, respectively.

<table border="0" cellpadding="0" cellspacing="0" width="651">
	
	<tbody>
		<tr height="165">
			<td height="165" style="height:165px;width:216px;">Curculigo orchioides Gaertn (9&#8201;g), Herbaa Epimedii (9&#8201;g), Radix Morindae Officinalis (9&#8201;g), Radix Angelicae Sinensis (9&#8201;g), Cortex Phellodendri (6&#8201;g), and Rhizoma Anemarrhenae (6&#8201;g)</td>
			<td style="width:149px;">Kang-qiao Chinese Medicine Yinpian Co. Ltd (Shanghai, CN)</td>
			<td style="width:119px;">160922</td>
			<td style="width:167px;">EXD components</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Filter paper</td>
			<td style="width:149px;">GElifesciences</td>
			<td style="width:119px;">99-103-952</td>
			<td style="width:167px;">Filter EXD decoction before concentration</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Imprinting Control Region (ICR) mice</td>
			<td style="width:149px;">Shanghai Laboratory Animal Center</td>
			<td style="width:119px;">SCXK 2007-0005</td>
			<td style="width:167px;">In vivo study</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Sprague Dawley rats</td>
			<td style="width:149px;">Shanghai Laboratory Animal Center</td>
			<td style="width:119px;">SCXK 2007-0005</td>
			<td style="width:167px;">EXD-containing serum preparation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Syringe filter</td>
			<td style="width:149px;">Millipore</td>
			<td style="width:119px;">SLGP033RB</td>
			<td style="width:167px;">0.22 &#181;m</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">gavage needles (10 ml)</td>
			<td style="width:149px;">Shanghai BO Ge trade sales department</td>
			<td align="right" style="width:119px;">59104274</td>
			<td style="width:167px;">Adminstration of EXD</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Ketamine (80 mg/kg)&#160;</td>
			<td style="width:149px;">Fujian Gutian Pharma Co. Ltd</td>
			<td style="width:119px;">H35020148</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Xylazine (10 mg/kg)</td>
			<td style="width:149px;">Sunway Pharma Co. Ltd</td>
			<td style="width:119px;">CB07591</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">Dulbecco&#8217;s modification of Eagle&#8217;s medium Dulbecco (DMEM) culture medium</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:119px;">12800-116</td>
			<td style="width:167px;">DMEM with 2 mM L-glutamine and without ribonucleosides and ribonucleotides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Streptomycin</td>
			<td style="width:149px;">Sigma</td>
			<td align="right" style="width:119px;">1277</td>
			<td style="width:167px;">100 &#181;g / ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin</td>
			<td style="width:149px;">Sigma</td>
			<td align="right" style="width:119px;">4687</td>
			<td style="width:167px;">100 &#181;g / ml</td>
		</tr>
	</tbody>
</table>

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.

The effect of EXD on the bone density of OVX mice
Hematoxylin and eosin (H&#38;E) staining of the lumbar vertebra section shows increased bone trabeculae after EXD treatment in vivo (Figure 1A, right panel) compared with those in the OVX group (Figure 1A, left panel). Figure 1B shows the representative &#181;CT images of the 4th lumbar in OVX mice (Figur...

Watch this Scientific Journal Video about Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments at JoVE.com

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.

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 ...

The overall goal of this procedure is to prepare an er-xian decoction, a traditional Chinese herbal medicine for its in vivo and in vitro application. This method can help answer key questions in herbal medicine field about preparation and application of a traditional Chinese medicine. The main advantage of this technique is that the water extraction steps are similar to those used in a clinical setting.Place the raw materials for a single er-xian decoction into a container with a lid, and add enough distilled water that the herbs are covered by about an inch. Put the lid on the container, and macerate the raw herbs for one hour. Then use a gas cooker on high power to heat the herbs until the water boils.Turn the power down to a low simmer, and decoct the herbs for two hours. At the end of the first water decoction, cover a 500-milliliter glass beaker with gauze and cotton, and carefully pour the decoction liquid only into the beaker through the filter. Return any herbal residue on the filter paper to the container, and add enough fresh distilled water to cover the herbs with another inch of water.After a second water decoction as just demonstrated, filter the decoction into the 1, 000-milliliter beaker, and place the beaker onto a gas cooker covered with asbestos-free wire gauze gas. Heat the decoction with a low simmer using a glass rod to slowly and continuously stir the solution. When the decoction reduces to 200 milliliters, transfer the solution to a 500-milliliter beaker, and reduce the volume to 100 milliliters as just demonstrated.After confirming the appropriate level of sedation by toe pinch, use straight operating scissors to make a five-centimeter long, 0.5-centimeter deep incision in the skin and peritoneum of an er-xian decoction-treated animal one hour after the last decoction treatment from the abdomen to the bottom of the thorax. Use tissue paper to move the abdominal viscera, and remove the connective tissue of the abdominal aorta to expose the vessel. Next, use a 10-milliliter syringe equipped with a 22-gauge needle to slowly draw eight to 10 milliliters of blood from the abdominal aorta, and transfer the blood to a 15-milliliter sterile tube.Clot the blood for 30 to 60 minutes at room temperature, then collect the cells by centrifugation. Carefully pool the serum from each animal into a single sterile 50-milliliter tube, and heat and activate the serum in a 56 degrees Celsius water bath for 30 minutes. Finally, use a syringe filter with a 0.22-micrometer pore size, and hydrophilic polyethersulfone membrane to filter the serum into a 50-milliliter conical tube for immediate use.Hematoxylin and eosin staining of the lumbar vertebral section of an ovariectomized or OVX mouse reveals an increase in bone trabeculae after er-xian decoction treatment in vivo. Compare to control OVX animals. Representative micro-CT images of the fourth lumbar of a control OVX mouse and an OVX mouse treated with the er-xian decoction for two weeks reveals that more trabecular bones are observed on the inside of the lumbar vertebra of the er-xian decoction-treated animals than those of the control OVX mice.Indeed, quantitative analysis of these micro-CT data indicates an increase in bone to tissue volume, trabecular number and thickness, and a decrease in the trabecular spacing of the fourth lumbar of the er-xian decoction-treated animals. Fat droplets automatically form when bone mesenchymal stem cells from OVX mice are cultured for seven days. In cultures from er-xian decoction-treated animals, bone nodules form instead.Further, more alkaline phosphatase-positive cells can be detected in er-xian decoction-treated bone mesenchymal stem cells than in stem cells from control OVX animals. In addition, hierarchical clustering demonstrates that the expression of 389 genes in bone mesenchymal cells as reveal from microarray is full change greater than 1.5 times between OVX and er-xian decoction-treated mice in vivo. Once mastered, the er-xian decoction can be prepared in eight hours, and the contained herb serum can be prepared in two hours.For internal gastric demonstration, first confirm that the IG tube is properly positioned within the esophagus, then gently delivers the decoction to the animal. According to tradition, preparation method of a Chinese herbal medicine, the medicine or materials should be extracted with water rather than external. Rotary evaporators are commonly used for concentrating the decoction but gas can be used as necessary.Traditional Chinese medicine requires the standardized preparation procedure and general medicinal materials for in this procedure, the consistent quality will be controlled. After watching this video, you should have a good understanding of how to standardize medicinal materials extraction, and to prepare contained herbal serum. Please keep in mind that the boiling decoction is very hot.Wear waterproof heat-resistant gloves as necessary.

preparation-herbal-medicine-er-xian-decoction-er-xian-containing

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JoVE Journal

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10.8K ビュー.  Shanghai University of Traditional Chinese Medicine. The overall goal of this procedure is to prepare an er-xian decoction, a traditional Chinese herbal medicine for its in vivo and in vitro application. This method can help answer key questions in herbal medicine field about preparation and application of a traditional Chinese medicine. The main advantage of this technique is that the water extraction steps are similar to those used in a clinical setting.Place the raw materials for a single er-xian decoction into a container with a lid,...

ビデオ: 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...

preparation of gushukang (gsk) granules for in vivo and in vitro experiments

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...

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

gavage strategy for decoction formula of traditional chinese medicine in osteosarcoma model mice

Author Spotlight: Standardized Herbal Decoction Protocol for Enhanced Animal Studies

The protocol provides a detailed standardization process of decoction formula and gavage technique with Yiqi Jiedu decoction in the osteosarcoma mouse model as an example. It describes animal protection and improves reliability of research data, providing effective strategies for investigating therapeutic efficacy and molecular mechanisms of decoction formulas in...

Herb Decoction Formulation and Gavage Standardization in Osteosarcoma Mice

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effects of desmodium caudatum on gastrointestinal hormones and intestinal flora in rats with gastritis

Author Spotlight: Exploring the Mysteries of Sichuan&#039;s Herbal Medicine in Chinese Medicine Research

The present protocol describes the method for analyzing intestinal flora using Illumina-based 16S rRNA gene sequencing and provides a framework for evaluating the effectiveness of herbal...

&lt;i&gt;Desmodium caudatum &lt;/i&gt; Decoction&#039;s Impact on Intestinal Microbiota of Rats

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Gavage Strategy for Decoction Formula of Traditional Chinese Medicine in Osteosarcoma Model Mice

split-luciferase reassembly assay to measure endoplasmic reticulum-mitochondria contacts in live cells

Author Spotlight: Regulation and Dysregulation of ER-Mitochondria Contacts &#8212; Implications for Neurodegenerative Disease Pathogenesis

We have established a split-luciferase reassembly assay to monitor the endoplasmic reticulum-mitochondria contacts in live cells. Using this assay, we describe a protocol to quantitatively measure the level of these inter-organelle couplings in HEK293T cells, under the condition of chemical...

A Simple Assay to Measure ER-Mitochondria Contacts for High-Throughput Screens

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site-directed mutagenesis for in vitro and in vivo experiments exemplified with rna interactions in escherichia coli

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

ultra high-performance liquid chromatography-mass spectrometry and self-established database analysis of chinese herbal medicine components

Ultra High-Performance Liquid Chromatography-Mass Spectrometry and Self-established Database Analysis of Chinese Herbal Medicine Components

We describe a general protocol and systematic design that could be applied to separate and recognize complex components in alpine yarrow herb, Achillea millefolium L., a Chinese herbal...

chemical treatment and luciferase assay to measure the level of er-mitochondria contacts in live cells

Chemical Treatment and Luciferase Assay to Measure the Level of ER-Mitochondria Contacts in Live Cells

direct imaging of er calcium with targeted-esterase induced dye loading (ted)

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

mitochondria-associated er membranes (mams) and glycosphingolipid enriched microdomains (gems): isolation from mouse brain

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial...

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

monitoring er/sr calcium release with the targeted ca2+ sensor catcher+

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

We present protocols for the application of our targeted genetically-encoded calcium indicator (GECI) CatchER+ for monitoring rapid calcium transients in the endoplasmic/sarcoplasmic reticulum (ER/SR) of HEK293 and skeletal muscle C2C12 cells using real-time fluorescence microscopy. A protocol for the in situ Kd measurement and calibration is also...

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

medicated thread moxibustion therapy of zhuang medicine and its application in a rat model of cold-congealing syndrome of primary dysmenorrhea

Author Spotlight: Demonstrating Therapeutic Effect of Automated Zhuang Medicine Line Moxibustion Simulator

This article demonstrates the standardized operation process of Zhuang medicine line moxibustion in experimental animals. To achieve repeatability and accuracy of Zhuang medicine line moxibustion, we developed a simulator to maintain strength, heat, and frequency. We constructed a dysmenorrhea model for experimental verification in rats with cold coagulation...

Standardized Operation Process of Zhuang Medicine Line Moxibustion in Rats

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in vitro wedge slice preparation for mimicking in vivo neuronal circuit connectivity

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation...

preparation and application of zhuang medicine threads for moxibustion in dysmenorrhea treatment

Preparation and Application of Zhuang Medicine Threads for Moxibustion in Dysmenorrhea Treatment

live cell calcium imaging combined with sirna mediated gene silencing identifies ca2+ leak channels in the er membrane and their regulatory mechanisms

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

visualization and quantification of endogenous intra-organelle protein interactions at er-mitochondria contact sites by proximity ligation assays

Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays

The need for new approaches to study membrane contact sites (MCSs) has grown due to increasing interest in studying these cellular structures and their components. Here, we present a protocol that integrates previously available microscopy technologies to identify and quantify intra-organelle and inter-organelle protein complexes that reside at...

preparation, characteristics, toxicity, and efficacy evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Here, we present detailed methods for the preparation and evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in...

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

an in vitro dissolution determination of multi-index components in tibetan medicine rhodiola granules

An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

Here, we test the dissolution of Rhodiola granules (RG) in vitro, draw dissolution curves of salidroside, gallic acid, and ethyl gallate in ultrapure water, and fit the curves to different mathematical models. This protocol provides information and guidance for in vivo bioequivalence and in vivo-in vitro correlation studies of...

An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

use of a linear accelerator for conducting in vitro radiobiology experiments

Use of a Linear Accelerator for Conducting In Vitro Radiobiology Experiments

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

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. ...

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 ...

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 ...

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.

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

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

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. ...

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...