使用共同分泌的Luciferase代理物测量相对胰岛素分泌
Summary
该协议描述了如何使用胰岛素相关的高辛比酶作为β细胞胰岛素分泌的代理,在中等通量下执行快速低成本荧光素酶测定。大多数发光板读取器和多通道移液器都可以进行测定。
Abstract
对分泌胰岛素样品后收集进行基于抗体的检测通常需要几个小时到一天的测定时间,并且可能非常昂贵,具体取决于特定的测定。分泌的荧光素酶测定可加快结果,大大降低每个样品的测定成本。在这里,我们提出了一种相对使用不足的方法,通过使用高西亚荧光素酶基因插入C肽中,测量胰腺β细胞的胰岛素分泌活性。在蛋白酶处理蛋白酶胰岛素过程中,将C-肽切除释放胰岛素分泌囊泡内的荧光素酶,与胰岛素共同分泌。由于荧光素酶测定的速度,在样品收集后几分钟内即可得到结果。测定的一个限制是,它是胰岛素分泌的相对测量,而不是绝对定量。但是,此协议经济、可扩展,可以使用大多数标准发光板读取器执行。模拟和数字多通道移液器有助于多个步骤的测定。可以同时测试许多不同的实验变异。一旦确定了一组聚焦条件,应直接使用具有标准曲线的基于抗体的测定法测量胰岛素浓度,以确认荧光素酶测定结果。
Introduction
这里介绍的方法允许从转基因β细胞系的胰岛素分泌快速和负担得起的96孔板格式测定。该协议的关键是胰岛素的改良版本,将自然分泌的高斯卢西酶(GLuc,#18 kDa)插入C肽(见图1),以产生胰岛素高西亚(InsGLuc)1,2。其他较大的蛋白质,如GFP(+25 kDa),已成功插入胰岛素的C-肽中,并表现出从蛋白酶-GFP到胰岛素和GFP-C-肽3、4的预期转化后处理。对于该协议中的测定,GLuc已经针对哺乳动物的表达进行了柯顿优化,并引入了两个突变来增强发光动力学5,6。治疗条件的多种组合和复制可以很容易地以96孔板格式进行测试,并在实验后立即获得分泌结果。
如前所述,如前所述,一个主要优势是这种基于荧光酶的分泌测量( $2/油井) 和同质时间解荧光 (HTRF) 或其他 Fürster 共振能量转移 (FRET) 为基础的抗体 (> $1/well) 测定。与通过参照标准曲线测量胰岛素浓度的基于抗体的测定相比,InsGLuc 测定测量分泌活性,作为对板上控制井的相对比较。因此,每个实验都需要包含适当的控件。这种区别是一种权衡,允许快速和廉价的测量。然而,InsGLuc分泌已被证明与ELISA1,2测量的胰岛素分泌高度相关。该技术已扩展至高通量筛选1、2 、7,并确定了胰岛素分泌的新型调制器,包括电压门状钾通道抑制剂7以及天然产物抑制剂β细胞功能,色霉素A28。InsGLuc 的使用最适合那些计划持续测试许多不同的治疗条件以测试其对胰岛素分泌的影响的研究人员。在后续实验中,有必要在亲子细胞系中重复关键发现,在小鼠或人类胰岛中以最佳方式重复关键发现,并使用基于抗体的测定测量胰岛素分泌。
Protocol
1. 试剂、介质和缓冲液的制备(表1) 用以下添加剂制备500 mL高葡萄糖(4.5克/升)Dulbeco改性鹰培养基(DMEM)的完整介质:15%胎儿牛血清(FBS)、100单位/mL青霉素、100微克/mL链霉素、292微克/mL L-谷氨酰胺和50μMβ-麦卡托托乙醇。注:本例中稳定的细胞系在G418抗生素的250μg/mL中保持。 通过制造含有 5 mM KCl、120 mM NaCl、15 mM HEPES (pH 7.4)、24 mM NaHCO 3、1mM MgCl2、2 mM CaCl2和 1 mg/mL …
Representative Results
为了测量在控制条件下的测定性能,可以完成一个简单的葡萄糖剂量反应曲线或使用二氧化二氮范式的刺激。在前者的情况下,在无葡萄糖条件下预孵化细胞1小时,然后以葡萄糖浓度增加治疗1小时,在5 mM及以下的分泌活性应极小,而观察到8以上增加的分泌mM 葡萄糖 (图 2)35 mM KCl 的刺激也可用作刺激分泌的阳性控制。在刺激期间纳入分泌调节药物应给予分?…
Discussion
在这里,我们提出一种方法,快速评估来自MIN6+细胞的葡萄糖刺激胰岛素分泌反应。为了在测定中获得最佳反应,重要的是将MIN6细胞播种在适当的密度,并允许它们成为85-95%的汇合物。这改善β细胞对葡萄糖的反应,因为改善的细胞-细胞接触和同步,这发生在主小岛17,18,19,20,21以及MIN6</sup…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者感谢科布实验室的所有现任和前任成员所做的宝贵工作和讨论,感谢迪奥内·沃提供行政援助。Michael Kalwat 得到青少年糖尿病研究基金会 SRA-2019-702-Q-R 的支持。这项工作是通过NIH R37 DK34128和韦尔奇基金会授予I1243梅兰妮·科布。这项工作的早期部分也得到了NIH F32 DK100113对迈克尔·卡尔瓦特的支持。
Materials
| Cell culture materials | |||
| rIns-GLuc stable MIN6 cells | Parental MIN6 cell line stably expressing pcDNA3.1+rInsp-Ins-eGLuc and maintained in 250 ug/ml G418 | ||
| DMEM | Sigma | D6429 | 4.5 g/L glucose media |
| fetal bovine serum, heat-inactivated | Sigma | F4135 | |
| Penicillin/Streptomycin | Thermo-Fisher Scientific | SV30010 | |
| beta-mercaptoethanol | Thermo-Fisher Scientific | BP 176-100 | |
| glutamine | Thermo-Fisher Scientific | BP379-100 | |
| Trypsin-EDTA | Sigma | T3924-500 | |
| G418 | Gold Biotechnology | G418-10 | Stock solution 250 mg/mL in water. Freeze aliquots at -20C. |
| T75 tissue culture flasks | Fisher Scientific | 07-202-000 | |
| 96 well tissue culture plates | Celltreat | 229196 | |
| Reagent reservoirs (50 mL) | Corning | 4870 | |
| Name | Company | Catalog Number | Comments |
| Secretion assay reagents | |||
| BSA (RIA grade) | Thermo-Fisher Scientific | 50-146-952 | |
| D-(+)-Glucose | Sigma | G8270-1KG | |
| KCl | Thermo-Fisher Scientific | P217-500 | |
| NaCl | Thermo-Fisher Scientific | S271-3 | |
| Hepes, pH 7.4 | Thermo-Fisher Scientific | 50-213-365 | |
| NaHCO3 | Thermo-Fisher Scientific | 15568414 | |
| MgCl2 | Thermo-Fisher Scientific | M9272-500G | |
| CaCl2 | Sigma | C-7902 | |
| Name | Company | Catalog Number | Comments |
| Optional drugs for stimulation experiments | |||
| Diazoxide | Sigma | D9035 | Stock solution: 50 mM in 0.1N NaOH. Add equal amount of 0.1N HCl to any buffer where diazoxide is added. |
| epinephrine (bitartrate salt) | Sigma | E4375 | Stock solution: 5 mM in water |
| PMA (phorbol 12-myristate) | Sigma | P1585 | Stock solution: 100 µM in DMSO |
| Name | Company | Catalog Number | Comments |
| Guassia assay materials | |||
| Disodium phosphate (Na2HPO4) | Thermo-Fisher Scientific | S374-500 | |
| Glycerol | Thermo-Fisher Scientific | G334 | |
| Sodium Bromide | Thermo-Fisher Scientific | AC44680-1000 | |
| EDTA | Thermo-Fisher Scientific | AC44608-5000 | Stock solution: 0.5 M pH 8 |
| Tris base | RPI | T60040-1000.0 | Stock solution: 1 M pH 8 |
| Ascorbic Acid | Fisher Scientific | AAA1775922 | US Patent US7718389 suggested ascorbate can increase coelenterazine stability. |
| Na2SO3 | Sigma | S0505-250G | US Patent US8367357 suggested sulfite may decrease background due to BSA |
| Coelenterazine (native) | Nanolight / Prolume | 3035MG | Stock solution: 1 mg/ml in acidified MeOH (2.36 mM) |
| OptiPlate-96, White Opaque 96-well Microplate | Perkin Elmer | 6005290 | Any opaque white 96 well plate should be sufficient. Clear bottom plates will also work, however some signal will be lost. |
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| Synergy H1 Hybrid plate reader or equivalent | BioTek | 8041000 | A plate reader with luminescence detection and 96-well plate capabilities is required. |
| 8-channel VOYAGER Pipette (50-1250 µL) | Integra | 4724 | An automated multichannel pipette is extremely useful for rapid addition of luciferase reagents and plating cells in 96 well format |
| 8-channel 200 µL pipette | Transferpette S 20-200 µL | 2703710 |
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Citazione di questo articolo
Kalwat, M., Cobb, M. H. Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate. J. Vis. Exp. (148), e59926, doi:10.3791/59926 (2019).