Name: bioluminescent monitoring of graft survival in an adoptive transfer model of autoimmune diabetes in mice
Uploaded: 2022-11-18T15:00:00
Description: Watch this Scientific Journal Video about Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice at JoVE.com

我们感谢Erica P. Cai博士和Yuki Ishikawa博士开发本协议中描述的方法（见参考文献11）。S.K.和P.Y.实验室的研究得到了美国国立卫生研究院（NIH）（R01DK120445，P30DK036836），JDRF，哈佛干细胞研究所和Beatson基金会的资助。T.S.得到了国家糖尿病，消化和肾脏疾病研究所（NIDDK）（T32 DK007260-45）的博士后奖学金的支持，K.B.得到了Mary K. Iacocca基金会的部分奖学金支持。

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64836/64836fig01.jpg"> 图 1：小鼠糖尿病过继转移模型中移植和成像移植物的工作流程。 表达萤火虫转基因荧光素酶（luc2）的NIT-1细胞皮下移植到NOD-scid小鼠中。同时向小鼠注射从自发糖尿病NOD小鼠分离的自身反应性脾细胞。通过非侵入性生物发光成像定期对移植物进行成像。BioRend...

<ol>
	<li>Katsarou, A., 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=Type+1+diabetes+mellitus.">Type 1 diabetes mellitus.</a> Nature Reviews Disease Primers. 3, 17016 (2017).</li><li>Shapiro, A. M., Pokrywczynska, M., Ricordi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+pancreatic+islet+transplantation.">Clinical pancreatic islet transplantation.</a> Nature Reviews Endocrinology. 13 (5), 268-277 (2017).</li><li>Pagliuca, F. 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=Generation+of+functional+human+pancreatic+beta+cells+in+vitro.">Generation of functional human pancreatic beta cells in vitro.</a> Cell. 159 (2), 428-439 (2014).</li><li>Russ, H. A., 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=Controlled+induction+of+human+pancreatic+progenitors+produces+functional+beta-like+cells+in+vitro.">Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro.</a> EMBO Journal. 34 (13), 1759-1772 (2015).</li><li>Rezania, A., 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=Reversal+of+diabetes+with+insulin-producing+cells+derived+in+vitro+from+human+pluripotent+stem+cells.">Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells.</a> Nature Biotechnology. 32 (11), 1121-1133 (2014).</li><li>Vertex Announces Positive Day 90 Data for the First Patient in the Phase 1_2 Clinical Trial Dosed With VX-880, a Novel Investigational Stem Cell-Derived Therapy for the Treatment of Type 1 Diabetes. Businesswire Available from: <a target="_blank" href="https://www.businesswire.com/news/home/20211018005226/en/Vertex-Announces-Positive-Day-90-Data-for-the-First-Patient-in-the-Phase-12-Clinical-Trial-Dosed-With-VX-880-a-Novel-Investigational-Stem-Cell-Derived-Therapy-for-the-Treatment-of-Type-1-Diabetes">https://www.businesswire.com/news/home/20211018005226/en/Vertex-Announces-Positive-Day-90-Data-for-the-First-Patient-in-the-Phase-12-Clinical-Trial-Dosed-With-VX-880-a-Novel-Investigational-Stem-Cell-Derived-Therapy-for-the-Treatment-of-Type-1-Diabetes</a> (2021)</li><li>Gamble, A., Pepper, A. R., Bruni, A., Shapiro, A. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+journey+of+islet+cell+transplantation+and+future+development.">The journey of islet cell transplantation and future development.</a> Islets. 10 (2), 80-94 (2018).</li><li>Pearson, J. A., Wong, F. S., Wen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+the+Non+Obese+Diabetic+(NOD)+mouse+model+in+autoimmune+diabetes.">The importance of the Non Obese Diabetic (NOD) mouse model in autoimmune diabetes.</a> Journal of Autoimmunity. 66, 76-88 (2016).</li><li>Hamaguchi, K., Gaskins, R. H., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIT-1,+a+pancreatic+&#946;-cell+line+established+from+a+transgenic+NOD/Lt+mouse.">NIT-1, a pancreatic &#946;-cell line established from a transgenic NOD/Lt mouse.</a> Diabetes. 40 (7), 842-849 (1991).</li><li>Christianson, S. W., Shultz, L. D., Leiter, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+transfer+of+diabetes+into+immunodeficient+NOD-scid/scid+mice:+Relative+contribution+of+CD4++and+CD8++T-cells+from+diabetogenic+versus+prediabetic+NOD.NON-Thy1a+donors.">Adoptive transfer of diabetes into immunodeficient NOD-scid/scid mice: Relative contribution of CD4+ and CD8+ T-cells from diabetogenic versus prediabetic NOD.NON-Thy1a donors.</a> Diabetes. 42 (1), 44-55 (1993).</li><li>Cai, E. 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=Genome-scale+in+vivo+CRISPR+screen+identifies+RNLS+as+a+target+for+beta+cell+protection+in+type+1+diabetes.">Genome-scale in vivo CRISPR screen identifies RNLS as a target for beta cell protection in type 1 diabetes.</a> Nature Metabolism. 2 (9), 934-945 (2020).</li><li>Parent, A. V., 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=Selective+deletion+of+human+leukocyte+antigens+protects+stem+cell-derived+islets+from+immune+rejection.">Selective deletion of human leukocyte antigens protects stem cell-derived islets from immune rejection.</a> Cell Reports. 36 (7), 109538 (2021).</li><li>Brehm, M. A., 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=Lack+of+acute+xenogeneic+graft-versus-host+disease,+but+retention+of+T-cell+function+following+engraftment+of+human+peripheral+blood+mononuclear+cells+in+NSG+mice+deficient+in+MHC+class+I+and+II+expression.">Lack of acute xenogeneic graft-versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression.</a> FASEB Journal. 33 (3), 3137-3151 (2019).</li><li>Abdulreda, M. 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=In+vivo+imaging+of+type+1+diabetes+immunopathology+using+eye-transplanted+islets+in+NOD+mice.">In vivo imaging of type 1 diabetes immunopathology using eye-transplanted islets in NOD mice.</a> Diabetologia. 62 (7), 1237-1250 (2019).</li></ol>

T1D是一种毁灭性的疾病，目前尚无治愈方法。β细胞替代疗法为患有这种疾病的患者提供了一种有希望的治疗方法，但这种策略的关键障碍是针对移植的β细胞的复发性自身免疫攻击的可能性。SC-β细胞的基因工程以降低其免疫可见性或易感性是这个问题的一个潜在解决方案。这里描述的是用于对移植的β细胞进行非侵入性成像的方案，以测量它们在小鼠自身免疫性糖尿病的过继转移模型中的存活?...

1型糖尿病（T1D）是由胰腺产生胰岛素的β细胞的自身免疫性破坏引起的。β细胞质量的损失导致胰岛素缺乏和高血糖。T1D 患者每天依赖多次注射外源性胰岛素，并终生经历严重高血糖和低血糖发作。与这些发作相关的并发症包括糖尿病视网膜病变、肾功能下降和神经病变1。
胰岛素注射是T1D的治疗方法，但不能治愈。然而，替换丢失的β细胞团有可能通过使患者产生自己的胰岛素来逆转疾病。然而，尸体供体胰岛的供应有限2。干细胞来源的胰岛（SC-胰岛）可以为移植提供几乎无限的β细胞供应。几个小组已经证明，人类胚胎干细胞（ESC）和诱导多能干细胞（iPSCs）可以分化以产生功能性β样细胞3，4，5。有希望的早期临床试验数据表明，这些细胞在移植后仍能维持其功能，并可能使患者成为胰岛素非依赖性6。然而，需要慢性免疫抑制，从而增加他们对癌症和感染的易感性。此外，免疫抑制剂长期可能对移植物具有细胞毒性7。为了消除免疫抑制的需要，可以对SC胰岛进行基因改造，以保护它们免受复发性自身免疫以及移植后的同种异体免疫。
干细胞研究对成本和劳动力的要求很高。小鼠细胞系和动物模型是用于初始鉴定和实验验证保护移植细胞免受自身免疫的策略的有用工具。NOD小鼠发展为自发性自身免疫性糖尿病，与人类T1D8有许多相似之处，NIT-1胰岛素瘤细胞系与该小鼠品系9共享遗传背景。糖尿病可以通过从NOD小鼠注射糖尿病脾细胞来过继转移到相关的免疫缺陷NOD-scid小鼠品系，以便在复制实验小鼠10中暂时同步糖尿病的发作。该模型可用于相对快速且廉价地识别遗传靶标，以便在SC胰岛中进行进一步验证。最近，该方法被用于鉴定和验证RNLS，RNLS是一种靶标，被发现可以保护原代人胰岛免受体内自身免疫和iPSC衍生胰岛免受体外β细胞应激的影响11。这里描述的是移植基因工程NIT-1细胞并在小鼠自身免疫性糖尿病的过继转移模型中非侵入性监测其存活率的简单方案。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin, 0.53 mM EDTA</td>
			<td>Corning</td>
			<td>25-052-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>293FT</td>
			<td>Invitrogen</td>
			<td>R70007</td>
			<td>Fast-growing, highly transfectable clonal isolate derived from human embryonal kidney cells transformed with the SV40 large T antigen</td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>Gibco</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol prep pads, 70% Isopropyl alcohol</td>
			<td>Amazon/Ever Ready First Aid</td>
			<td>B08NWF31DX</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 5ml Syringe Luer-Lok Tip</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Needle 26G x 5/8 (0.45 mm x 16 mm) Sub-Q</td>
			<td>BD</td>
			<td>305115</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 1 mL TB Syringe Slip Tip</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin S HCl</td>
			<td>Corning</td>
			<td>&#160;30-100-RB</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer premium SureStrain, 70 &#181;m, sterile</td>
			<td>Southern Labware</td>
			<td>C4070</td>
			<td>Or use similar sterile strainer with 40-70um pore size</td>
		</tr>
		<tr>
			<td>CellDrop automated cell counter</td>
			<td>Denovix</td>
			<td>CellDrop BF-PAYG</td>
			<td>Or use similar cell counter device</td>
		</tr>
		<tr>
			<td>Corning 100 mL Penicillin-Streptomycin Solution, 100x</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Aspirating Pipets, Polystyrene, Sterile, Capacity=2 mL</td>
			<td>VWR</td>
			<td>414004-265</td>
			<td>Or use similar aspirating pipette</td>
		</tr>
		<tr>
			<td>D-Luciferin, Potassium Salt , Molecular Biology Grade, Powder, &#62;99%</td>
			<td>Goldbio</td>
			<td>LUCK-100</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, pyruvate, no glutamine</td>
			<td>Gibco</td>
			<td>10313039</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon BD tubes, 50 mL</td>
			<td>Fisher Scientific</td>
			<td>14-959-49A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps premium for tissues, 1 x 2 teeth 5 in, German Steel</td>
			<td>Fisher Scientific</td>
			<td>13-820-074</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose urine test strip</td>
			<td>California Pet Pharmacy</td>
			<td>u-tsg100</td>
			<td>Or use similar test strip for glucose measurments in urine/blood</td>
		</tr>
		<tr>
			<td>GlutaMAX&#8211;1 (100x)</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Infrared heating lamp</td>
			<td>Cole Parmer</td>
			<td>03057-00</td>
			<td>Or use similar infrared lamp&#160;</td>
		</tr>
		<tr>
			<td>Insulin syringe 0.5 mL, U-100 29 G 0.5 in</td>
			<td>Becton Dickinson</td>
			<td>309306</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane, USP</td>
			<td>Piramal Critical Care</td>
			<td>6679401725</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Spectrum in vivo imaging system</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td>Instrument for non-invasively collecting bioluminescent images of transplanted cells</td>
		</tr>
		<tr>
			<td>Living Image Analysis Software</td>
			<td>Perkin Elmer</td>
			<td>128113</td>
			<td>Software for collecting and quantifying bioluminescent signal</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes seal-rite, 1.5 mL</td>
			<td>USA Scientific</td>
			<td>1615-5510</td>
			<td>Or use similar sterile microcentrifuge tubes</td>
		</tr>
		<tr>
			<td>NIT-1</td>
			<td>ATCC</td>
			<td>CRL-2055</td>
			<td>Pancreatic beta-celll line derived from NOD/Lt mice</td>
		</tr>
		<tr>
			<td>NOD.Cg-Prkdcscid/J</td>
			<td>The Jackson Laboratory</td>
			<td>001303</td>
			<td>Mice homozygous for the severe combined immune deficiency spontaneous mutation&#160;Prkdcscid, commonly referred to as&#160;scid, are characterized by an absence of functional T cells and B cells, lymphopenia, hypogammaglobulinemia, and a normal hematopoietic microenvironment.</td>
		</tr>
		<tr>
			<td>NOD/ShiLtJ</td>
			<td>The Jackson Laboratory</td>
			<td>001976</td>
			<td>The NOD/ShiLtJ strain of mice (commonly called NOD) is a polygenic model for autoimmune type 1 diabetes</td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010031</td>
			<td>No calcium, no magnesium, no phenol red</td>
		</tr>
		<tr>
			<td>pCMV-VSV-G</td>
			<td>Addgene</td>
			<td>8454</td>
			<td></td>
		</tr>
		<tr>
			<td>pLenti-luciferase-blast</td>
			<td>Made in-house</td>
			<td>Plasmid available upon request</td>
			<td>See Supplemental File 1</td>
		</tr>
		<tr>
			<td>pMD2.G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr>
			<td>pMDLg/pRRE</td>
			<td>Addgene</td>
			<td>12251</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine, Linear, MW 25,000, Transfection Grade (PEI 25K)</td>
			<td>Fisher Scientific</td>
			<td>NC1014320</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSV-Rev</td>
			<td>Addgene</td>
			<td>12253</td>
			<td></td>
		</tr>
		<tr>
			<td>Restrainer for rodents, broome-style round 1 in</td>
			<td>Fisher Scientific</td>
			<td>01-288-32A</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors, sharp-pointed</td>
			<td>Fisher Scientific</td>
			<td>08-940</td>
			<td>Or use other scissors made of surgical-grade stainless steel</td>
		</tr>
		<tr>
			<td>Tissue-culture treated culture dishes</td>
			<td>Millipore Sigma</td>
			<td>CLS430167-20EA</td>
			<td>Or use other sterile cell culture-treated Petri dishes</td>
		</tr>
		<tr>
			<td>Tweezers/Forceps, fine precision medium tipped</td>
			<td>Fisher Scientific</td>
			<td>12-000-157</td>
			<td></td>
		</tr>
	</tbody>
</table>

bioluminescent monitoring of graft survival in an adoptive transfer model of autoimmune diabetes in mice

1型糖尿病的特征是胰腺产生胰岛素的β细胞的自身免疫性破坏。这种疾病的有希望的治疗方法是移植干细胞衍生的β细胞。然而，为了保护移植的细胞免受持续的自身免疫，可能需要进行基因改造。糖尿病小鼠模型是初步评估保护移植细胞免受自身免疫攻击的策略的有用工具。这里描述的是一种在小鼠糖尿病过继转移模型中移植和成像细胞移植物的微创方法。在该协议中，来自表达萤火虫荧光素酶转基因 luc2 的鼠胰腺β细胞系NIT-1的细胞被皮下移植到免疫缺陷的非肥胖糖尿病（NOD）严重联合免疫缺陷（scid）小鼠中。同时向这些小鼠静脉注射来自自发糖尿病NOD小鼠的脾细胞以转移自身免疫。通过非侵入性生物发光成像 定期 对移植物进行成像，以监测细胞存活。将突变细胞的存活率与移植到同一只小鼠中的对照细胞的存活率进行比较。

图 1 概述了该协议的概述。可以比较两个细胞系的存活率，例如突变体和非靶向对照，或者可以在多组小鼠中测量一个细胞系的存活率，例如药物治疗的小鼠与载体处理的对照。图3A显示了三只8周龄的雌性NOD-scid小鼠移植非靶向对照（左）和突变（右）细胞系。小鼠还静脉注射自身反应性脾细胞以转移糖尿病。
小鼠在注射后第0天?...

Watch this Scientific Journal Video about Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice at JoVE.com

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice

该协议描述了一种直接和微创的方法，用于在非肥胖糖尿病（NOD）严重联合免疫缺陷小鼠中移植和成像NIT-1细胞，这些小鼠受到从自发糖尿病NOD小鼠纯化的脾细胞的挑战。

小鼠自身免疫性糖尿病过继转移模型中移植物存活的生物发光监测

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease ...

该协议可以帮助了解预防自身免疫攻击的机制，并确定一型糖尿病β细胞替代的治疗靶点。该技术的主要优点是成本相对较低，细胞培养，移植和成像方法的简单性，以及小鼠技术的最小侵入性。以前从未进行过尾静脉注射的人可能难以将针头插入静脉。在第一次注射之前要求专家演示。首先，取出麻醉的小鼠，并使用带防护装置的电动剃须刀从其背侧去除毛发，露出大约一英寸乘两英寸的皮肤。将剃光的鼠标放回击倒室。在移植之前，一次将一只小鼠从腔室转移到配备有异氟醚鼻锥的干净表面上，并轻轻地将小鼠头部引导到鼻锥中。用异丙醇准备垫擦拭剃光区域，以去除松散的头发并对皮肤进行消毒。接下来，将超过 300 微升的细胞悬液吸入装有 26 号针头的一毫升无菌注射器中。从注射器中取出任何气泡，并保留300微升细胞悬液，同时推出多余的气泡。使用非惯用手握住的一对弯曲的镊子，轻轻抬起小鼠背部一侧的皮肤，以便更容易地进入皮下空间。然后使用惯用手将注射器平行于小鼠身体的冠状和矢状平原。将针头指向小鼠头部，将其插入后躯附近的皮肤中，并将其引导到皮下空间，同时确保整个针头留在皮肤下并且不会戳穿。调整镊子以保持针根周围的皮肤。慢慢分配少于50微升的细胞悬液，并确认在注射部位的皮肤下形成一个小凸起。继续皮下注射多达200微升的细胞悬液。然后将镊子固定到位并保持针头与小鼠身体平行，取出针头。拔出针头后，用镊子闭合皮肤几秒钟，以防止细胞从穿刺伤口泄漏。移植后，将每只小鼠转移到新鲜的笼子中，让动物从麻醉中完全恢复，然后再将其他小鼠添加到笼子中。将安乐死的小鼠放在背上，使用手术剪刀在皮肤上做一个大约两英寸长的垂直切口。切开鼠标的右侧，找到鲜红色的脾脏。轻轻地将脾脏从粉红色胰腺上切开，并将其转移到含有五毫升无菌PBS的无菌10厘米培养皿中。用无菌注射器柱塞的平顶捣碎脾脏。将 40 或 70 微米的过滤器放在 50 毫升的锥形管上，并用 5 毫升的 PBS 灌注。将脾脏悬浮液转移到过滤器中并轻轻捣碎。用10毫升PBS清洗盘子，然后将洗液转移到过滤器中。继续捣碎，直到过滤器上没有红色。然后在室温下以500G的速度旋转试管5分钟，并用吸液管除去上清液，然后将细胞沉淀重悬于预热至室温的5毫升ACK裂解缓冲液中，以在室温下进行红细胞裂解四分钟。每5毫升裂解缓冲液用5毫升NIT-1细胞培养基淬灭反应，并将细胞悬液通过新鲜过滤器进入50毫升离心管以去除团块。在室温下以500G旋转管5分钟。接下来，在计数细胞之前，将细胞沉淀重悬于20毫升PBS中。计数后，在室温下以500G旋转细胞五分钟。将细胞沉淀重悬于无菌PBS中的1.5毫升安全锁定反应管中。立即注射或将细胞储存在冰上最多一小时。通过使用加热灯加热5至10分钟来加热成年受体NOD SCID小鼠的身体以血管扩张静脉。每次注射前重悬脾细胞溶液。对于每只小鼠，将100微升预热的脾细胞悬浮液吸入注射器中，确保不存在气泡。接下来，将鼠标放在约束装置中，然后用非惯用手捕获其尾巴。找到两条侧尾脉之一。如果需要，轻轻旋转尾巴。用含有70%异丙醇的消毒湿巾擦拭尾部。使用惯用手，将针以锐角插入尾巴的中心区域。将针头的斜面朝上，将针头在皮肤上滑动几毫米。然后轻轻按压注射器以注射脾细胞悬浮液。注射时不要让针头进一步向内或向外移动。成功的注射在注射过程中不会产生背压，并且在注射后立即由透明或白色血流指示。注射后，轻轻地将针头从静脉中释放出来，并用消毒湿巾对尾部施加轻微的压力，直到出血停止。将鼠标从约束装置中松开，轻轻地将其转移到新鲜准备好的笼子中。在成像前至少五分钟，使用一毫升注射器和26号针头腹膜内注射适当剂量的D-荧光素溶液。登录到成像软件后，在控制面板中选择初始化。要自动保存图像数据，请单击采集，然后单击自动保存到并在计算机中创建相应的文件夹。仪器完成初始化后，将曝光时间设置为一分钟。将鼠标放在成像仪器上，俯卧姿势，四肢张开，并将其头部引导到鼻锥中。用双手按压鼠标背部中心，然后将双手向外分开，轻轻压平鼠标。然后在成像软件中，选择获取并在弹出窗口中记录实验的任何相关详细信息。在研究的三只小鼠中的两只中，对照移植物在第18天被破坏，这从各自生物发光信号的丢失中可以明显看出。然而，在另一只小鼠中，突变移植物被破坏，而对照移植物存活下来，突出了动物之间的生物学变异。对生物发光信号进行量化，并报告移植存活率为残留生物发光信号与第一个时间点信号的百分比。使用来自突变移植物的发光信号与每只小鼠的对照移植物的比率来可视化动物之间的变异。在疾病转移之前，移植的细胞可能会复制，导致通过皮肤可见的扩张移植物。在成像时，这些移植物显示出无法准确量化的饱和生物发光信号。可以在不同的时间点分离移植物，以表征浸润免疫细胞，例如通过流式细胞术来研究自身免疫攻击和移植物保护的机制。

Research

JoVE Journal

Biology

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

收养大鼠迟发型超敏反应的感应和监测

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

科研

生物学

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

使用IVIS频谱的乳腺肿瘤在体内生物发光成像

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

Quantification of ÃƒÅ½Ã‚Â³H2AX Foci in Response to Ionising Radiation

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

Evaluation of the Spatial Distribution of ÃƒÅ½Ã‚Â³H2AX following Ionizing Radiation

评价以下电离辐射的空间分布的γH2AX

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

功能成像在小鼠体内的棕色脂肪与FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

自主的生物发光哺乳动物细胞连续和实时监测细胞毒性

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

生存时间的测量<em&gt;臂</em&gt;轮虫：产妇条件同步

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

用异体皮肤移植重建的烧伤伤口的穆林模型

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...