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Número de publicaciónCN101587237 B
Tipo de publicaciónConcesión
Número de solicitudCN 200910012155
Fecha de publicación5 Oct 2011
Fecha de presentación22 Jun 2009
Fecha de prioridad22 Jun 2009
También publicado comoCN101587237A
Número de publicación200910012155.6, CN 101587237 B, CN 101587237B, CN 200910012155, CN-B-101587237, CN101587237 B, CN101587237B, CN200910012155, CN200910012155.6
Inventores陈洪铎, 高兴华, 齐瑞群
Solicitante中国医科大学附属第一医院
Exportar citaBiBTeX, EndNote, RefMan
Enlaces externos:  SIPO, Espacenet
Method for cleaning and reusing microdissection sample-collecting tubes
CN 101587237 B
Resumen
The invention discloses a method for cleaning and reusing microdissection sample-collecting tubes. The method can be implemented according to the following steps in turn: (1) adding xylene to a collecting tube, capping and oscillating the collecting tube and discarding the xylene; (2) adding suspension A to the collecting tube, oscillating and then emptying the collecting tube; (3) adding double-distilled water to the collecting tube while adding silica particles, oscillating and then emptying the collecting tube; (4) repeating the step (2) and the step (3) twice in turn; (5) adopting the double-distilled water to wash the collecting tube; (6) uncapping the collecting tube and placing the collecting tube in a refrigerator till liquid completely evaporates; (7) taking the collecting tube out, smearing polylysine on the surface of a viscous module on the inner surface of a tube cap, putting the collecting tube back into the refrigerator and keeping the collecting tube standing; and (8) repeating the step (7). The method can effectively maintain and repair the adhesion property of the viscous module on the inner surface of the tube cap of the collecting tube, and can effectively remove superfluous substances remaining in the collecting tube so as to repeatedly recycle the collecting tube and save a large amount of experiment funding.
Reclamaciones(2)  traducido del chino
1.显微切割标本收集管清洗再利用方法,其特征在于,按如下步骤依次实施:(1)向收集管中加入占收集管容积1/2〜2/3的二甲苯,盖上收集管管盖,并振荡2〜 5分钟,打开管盖,弃去二甲苯;(2)向步骤(1)所述收集管中加入占收集管容积1/2〜2/3的混悬液A,盖上管盖,并振荡2〜3分钟,打开管盖,倒空收集管;所述混悬液A由盐酸、酒精及EDTA混配,并加入50mg 直径小于0. Imm的二氧化硅颗粒制成;所述盐酸的浓度为1. 5% ;所述酒精的浓度为30% ; 所述EDTA的浓度为0. 25mol/L ;所述盐酸、酒精及EDTA的体积比为0. 5〜1. 5 : (96. 5〜 99) : 0. 5 〜2 ;(3)向步骤(2)所述收集管中加入占收集管容积〜2/3的双蒸水,同时加入50mg 直径小于0. Imm的二氧化硅颗粒,盖上管盖,并振荡2〜3分钟,打开管盖,倒空收集管;(4)依次重复步骤(¾及步骤(¾两次;(5)采用双蒸水对步骤(4)所得收集管进行冲洗3〜5分钟;(6)将收集管开盖,置入2〜8°C冰箱内,直至收集管盖及管内液体完全蒸发;(7)取出收集管,在超净台内,往收集管管盖内表面黏性模块表面涂抹浓度为4〜8% 的多聚赖氨酸,迅速将收集管放回冰箱,静置;(8)重复步骤(7) 一次。 1. microdissected specimen collection tube cleaning method of recycling, characterized in that the implementation of the following steps in sequence: (1) was added to the collection tube collecting tube volume accounted xylene 1 / 2~2 / 3, the cover collection tube lid and shaken 2 ~ 5 minutes, open the lid, discarded xylene; (2) to the step (1) was added to the collection tube collection tubes volume accounted for suspension A 1 / 2~2 / 3, and Cap the tube and shaken 2 to 3 minutes, open the lid, empty the collection tube; the suspension of A mixed with hydrochloric acid, alcohol and EDTA, and adding 50mg diameter silica particles smaller than 0. Imm system a; the concentration of hydrochloric acid was 1.5%; the concentration of alcohol is 30%; the concentration of EDTA is 0. 25mol / L; the hydrochloric acid, the volume ratio of alcohol and EDTA is 0. 5~1 . 5: (-5 to 96. 99): 0.5 ~ 2; (3) to the step (2) was added to the collection tube collection tubes volume accounted for ~ 2/3 of distilled water, while adding a smaller diameter than 50mg 0 . Imm silica particles, covered with lid and shaken 2 to 3 minutes, open the lid, empty the collection tube; (4) sequentially repeating steps (¾ and step (¾ twice; (5) the use of double-distilled water for step (4) collecting the resulting tube rinsed 3 to 5 minutes; (6) the collection tube openings, placed within 2~8 ° C freezer until collection tube cap and completely evaporate the liquid within the tube; (7) Remove the collection tube, in a clean desk, to cover the collection tube coated surface of the module surface stickiness concentration of 4 to 8% of the poly-lysine, quickly collection tube back in the fridge, standing; (8) Repeat steps ( 7) a.
2.根据权利要求1所述的显微切割标本收集管清洗再利用方法,其特征在于:所述步骤(7)中静置时间t彡24h。 Microstructure according to claim 1, wherein the cutting specimen collection tube cleaning method of recycling, characterized in that: said step (7) of the standing time t San 24h.
Descripción  traducido del chino

显微切割标本收集管清洗再利用方法 Microdissected specimen collection tube cleaning method of recycling

技术领域 Technical Field

[0001] 本发明涉及显微切割标本耗材清洗技术领域,具体的说是一种显微切割标本收集管清洗再利用方法。 [0001] The present invention relates to microdissected specimens supplies cleaning technology, specifically a microdissected specimen collection tube cleaning method of recycling.

背景技术 Background

[0002] 激光捕获显微切割(Laser Capture Microdissection)技术及其系统的出现,使得相关科研工作者可以精确分离待研究的目的组织标本、细胞(体外培养的活细胞及固定在切片中组织细胞)、细胞器甚至染色体区带,用于后续研究,其控制样本的精确性受到全世界科研工作者的青睐,目前多数有实力的研究机构均已配备此技术及相关设施。 [0002] The laser capture microdissection (Laser Capture Microdissection) a technical and systems, making the relevant researchers can precisely separate organizational purposes be studied specimens, cells (in vitro culture of living cells and cells fixed tissue sections) , organelles and even chromosomal zone, for follow-up study, the accuracy of the control samples favored by researchers around the world, and currently most powerful research institutions have been equipped with this technology and related facilities.

[0003] 用组织切片进行显微切割时,需要用到一种专用的耗材,该耗材为带盖的管形容器(以下简称收集管),其特征为收集管盖的内表面上携带表面光滑的特殊黏性模块,利用模块的黏性吸附力可收集显微切割时膜性载玻片上被激光切割分离的带有目标组织的膜。 [0003] When using tissue sections were microdissected, need to use a special supplies, supplies for the tubular containers with lids (hereinafter referred to as the collection tube), carrying a smooth surface on the inner surface wherein collection tube cap When the special adhesive membrane module, use the module viscous adhesive force can be collected on membrane microdissection slides are laser cut with isolated target tissue. 这种收集管耗材价格非常昂贵;同时,由于用于研究的目的标本需要富集足够的数量,使得研究过程中该耗材使用数量较大,就单个标本而言,DNA、RNA相关的基因水平研究一般需要2-3个,蛋白水平的甚至需要数十个。 This collection tube supplies very expensive; Meanwhile, since the samples used for research purposes requires enrichment sufficient quantity so that in the course of the study using a large number of the supplies, the terms of a single sample, DNA, RNA levels of genes related to the study Usually takes 2-3 protein levels even need dozens. 目前,国内仍没有制造商能够生产这种专用耗材,重复利用该耗材不失为一个好方法。 Currently, domestic manufacturers still do not capable of producing the special supplies, re-use of the supplies is a good idea. 由于实验过程中激光会烧灼黏性模块表面致使出现焦痕, 从而降低其黏附性;同时,粘在盖子上的组织、核酸、蛋白及其他小分子物质会在后续分子生物学实验中造成污染。 Since the laser will burn during the experiment module surface and causes a scorch viscosity, thereby reducing its adhesion; meanwhile, stuck to the lid of the organization, nucleic acids, proteins and other small molecules can cause pollution in the subsequent molecular biology experiments. 因此,如何能有效去除收集管管盖上的组织、核酸、蛋白及其他小分子物质,又不损伤收集管管盖特殊黏性模块的黏附性,且又能修复黏性模块表面在实验过程中被激光烧灼的焦痕,是重复利用该收集管的难题。 So how can effectively remove the collection tube cover tissue, nucleic acids, proteins and other small molecules, do not damage the collection tube cover special adhesive module adhesion, and can repair the sticky surface of the module during the test by laser ablation of scorch marks, it is to reuse the collection tube problems. 常规机械洗涤、高温高压洗涤、强酸强碱洗涤等都会损伤收集管的黏附性,紫外线、钴60照射也只能灭活微生物,不能清除小分子物质。 Adhesion of conventional mechanical washing, high temperature and pressure washing, acid and alkali washing will damage the collection tube, ultraviolet, cobalt 60 irradiation can only inactivate microorganisms can not remove small molecules.

发明内容 DISCLOSURE

[0004] 本发明旨在克服现有技术的不足之处而提供一种能有效保持和修复收集管管盖内表面黏性模块黏附性,且能有效去除残留在收集管内的组织、核酸、蛋白及其他小分子物质,从而使收集管得以多次循环使用,节约大量实验经费的显微切割标本收集管清洗再利用方法。 [0004] The present invention is directed to overcoming the deficiencies of the prior art and to provide an effective collection tube to maintain and repair the module cover surface adhesion adhesive, and can effectively remove residual tissue in the collection tube, nucleic acids, proteins and other small molecules, so that the collection tube to be reused many times, saving a lot of experiments funded cutting microscopic specimen collection tube cleaning method of recycling.

[0005] 为达到上述目的,本发明是这样实现的: [0005] To achieve the above object, the present invention is achieved by:

[0006] 显微切割标本收集管清洗再利用方法,可按如下步骤依次实施: [0006] microdissected specimen collection tube cleaning method of recycling, follow these steps in order to implement:

[0007] (1)向收集管中加入占收集管容积1/2〜2/3的二甲苯,盖上收集管管盖,并振荡2〜5分钟,打开管盖,弃去二甲苯; [0007] (1) was added to the collection tube collecting tube volume accounted for 1 / 2~2 / 3 of xylene, the cover collection tube cap, and shaken 2~5 minutes, open the lid, discarded xylene;

[0008] (2)向步骤(1)所述收集管中加入占收集管容积1/2〜2/3的混悬液A,盖上管盖, 并振荡2〜3分钟,打开管盖,倒空收集管; [0008] (2) to step (1) was added to the collection tube collection tubes volume accounts for 1 / 2~2 / 3 of suspension A, covered with lid and shaken 2 to 3 minutes, open the lid, Empty the collection tube;

[0009] (3)向步骤(¾所述收集管中加入占收集管容积1/2〜2/3的双蒸水,同时加入50mg直径小于0. Imm的二氧化硅颗粒,盖上管盖,并振荡2〜3分钟,打开管盖,倒空收集管; [0009] (3) to step (¾ representing the collection tube collection tubes was added 1 volume / 2~2 / 3 of distilled water, while adding 50mg diameter of less than 0. Imm silica particles, covered with lid and shaken 2 to 3 minutes, open the lid, empty the collection tube;

[0010] (4)依次重复步骤⑵及步骤⑶两次; [0010] (4) in order to repeat the steps and step ⑵ ⑶ twice;

[0011] (5)采用双蒸水对步骤(4)所得收集管进行冲洗3〜5分钟; [0011] (5) double-distilled water in step (4) collecting the resulting tube rinsed 3 to 5 minutes;

[0012] (6)将收集管开盖,置入2〜8°C冰箱内,直至收集管盖及管内液体完全蒸发; [0012] (6) The collection tube openings, placed within 2~8 ° C freezer until collection tube cap and completely evaporate the liquid within the tube;

[0013] (7)取出收集管,在超净台内,往收集管管盖内表面黏性模块表面涂抹浓度为4〜 8%的多聚赖氨酸,迅速将收集管放回冰箱,静置; [0013] (7) Remove the collection tube in a clean desk, to cover the inner surface of the collection tube coated adhesive surface of the module 4 ~ 8% concentration of polylysine, quickly collection tube back in the fridge, quiet home;

[0014] (8)重复步骤(7) —次。 [0014] (8) repeating steps (7) - times.

[0015] 作为一种优选方案,本发明所述混悬液A由盐酸、酒精及EDTA混配,并加入50mg 直径小于0. Imm的二氧化硅颗粒制成。 [0015] As a preferred embodiment, the present invention A suspension mixed with hydrochloric acid, alcohol and EDTA, and adding 50mg diameter of less than 0. Imm made of silica particles.

[0016] 作为另一种优选方案,本发明所述盐酸的浓度为1. 5% ;所述酒精的浓度为30% ; 所述EDTA的浓度为0. 25mol/L。 [0016] As another preferred embodiment, the concentration of hydrochloric acid of the present invention is 1.5%; the concentration of alcohol is 30%; the concentration of EDTA is 0. 25mol / L.

[0017] 进一步地,本发明所述盐酸、酒精及EDTA的体积比为0.5〜1.5 : (96. 5〜 99) : 0. 5 〜2。 [0017] Further, the present invention is hydrochloric acid, alcohol and EDTA in a volume ratio of 0.5~1.5: (96. -5 to 99): 0.5 ~ 2.

[0018] 更进一步地,本发明所述步骤(7)中静置时间t彡24h。 [0018] Furthermore, the steps of the present invention (7), the standing time t San 24h.

[0019] 本发明与现有技术相比具有如下特点: [0019] The present invention over the prior art has the following characteristics:

[0020] 1、本发明整个操作流程中不涉及损伤暴力、高温高压、强酸碱等对膜有损害的因素; [0020] 1, the present invention does not involve the entire operation process damage violence, high temperature and high pressure, strong acid and other factors that damage the membrane;

[0021] 2、本发明通过使用混悬液A,使黏性模块得到充分清洗,同时充分保护了模块的黏附性; [0021] 2, the present invention through the use of suspensions A, so that the viscous module is fully cleaned, while fully protecting the adhesion module;

[0022] 3、本发明通过往表面涂抹多聚赖氨酸,可以很好的修复激光在黏性模块表面造成的烧痕,并在一定程度上恢复烧痕部位的黏附性; [0022] 3, the present invention applied to the surface of the poly-lysine, can be a good fix laser module surface stickiness caused burn marks, burn marks and restore the site to a certain extent adhesion;

[0023] 4、本发明清洗后的收集管不含有对实验有影响的成分; [0023] 4, collecting test tube does not contain ingredients that affect the present invention after cleaning;

[0024] 5、本发明所需试剂均为生物实验室常规试剂,且价格低廉,可批量操作; [0024] 5, the present invention is desired biological laboratory reagents are conventional reagents, and low prices, batch operations;

[0025] 6、本发明重复利用率高,可节省10-20倍的收集管数量,节省大量科研经费。 [0025] 6, the present invention effectively reused, saving 10 to 20 times the number of collection tubes, save a lot of research funding.

附图说明 Brief Description

[0026] 下面结合附图和具体实施方式对本发明作进一步说明。 [0026] below in conjunction with the accompanying drawings and specific embodiments of the present invention will be further described below. 本发明的保护范围不仅局限于下列内容的表述。 The scope of the present invention is not limited to the following statements.

[0027] 图1为验证收集管是否清洗干净而进行的2%的琼脂糖电泳图。 [0027] Figure 1 is verified clean collection tube is carried out by 2% agarose electrophoresis.

具体实施方式 DETAILED DESCRIPTION

[0028] 实施例1 [0028] Example 1

[0029] 显微切割标本收集管清洗再利用方法,可按如下步骤依次实施: [0029] microdissected specimen collection tube cleaning method of recycling, follow these steps in order to implement:

[0030] (1)向收集管中加入占收集管容积1/2的二甲苯,盖上管盖,涡旋器振荡3分钟,打 [0030] (1) was added to the collection tube collecting tube volume accounted for 1/2 of xylene, covered with lid, vortex oscillation 3 minutes, play

开管盖,弃去二甲苯; Open lid, discarded xylene;

[0031] (2)向收集管中加入占收集管容积2/3的混悬液A(由1.5%盐酸、30%酒精、 0. 25mol/L EDTA按体积比1 : 97 : 1混合,并加入50mg直径小于0. Imm的二氧化硅颗粒配制而成)。 [0031] (2) was added to the collection tube collecting tube volume accounted for 2/3 of suspension A (from 1.5% hydrochloric acid, 30% ethanol, 0. 25mol / L EDTA by volume ratio of 1: 97: 1 mixture, and Join 50mg diameter less than 0. Imm formulated silica particles). 盖上管盖,涡旋器振荡2. 5分钟,打开管盖,倒空收集管; Cap the tube, vortex oscillation 2.5 minutes, open the lid, empty the collection tube;

[0032] (3)向收集管中加入占收集管容积1/2的双蒸水,同时加入50mg直径小于0. Imm [0032] (3) was added 1/2 volume accounts for collection tubes double distilled water, and added to 50mg diameter of less than 0. Imm collection tube

4的二氧化硅颗粒,盖上管盖,涡旋器振荡2. 5分钟,打开管盖,倒空收集管; Silica particles 4 Cap the tube, vortex oscillation 2.5 minutes, open the lid, empty the collection tube;

[0033] (4)重复步骤(2)及步骤(3)两次; [0033] (4) repeating steps (2) and step (3) twice;

[0034] (5)大量双蒸水充分冲洗4分钟; [0034] (5) a large number of double-distilled water thoroughly rinsed four minutes;

[0035] (6)将收集管开盖,放入洁净的5°C冰箱,静置直至收集管盖及管内液体完全蒸发; [0035] (6) The collection tube openings, into a clean 5 ° C refrigerator and let stand until the collection tube cap and completely evaporate the liquid within the tube;

[0036] (7)取出收集管,在超净台内,往收集管管盖内表面黏性模块表面涂抹浓度为4-8%的多聚赖氨酸,迅速将收集管放回冰箱,静置^h ; [0036] (7) Remove the collection tube in a clean desk, to cover the inner surface of the collection tube coated adhesive surface of the module 4-8% concentration of polylysine, quickly collection tube back in the fridge, quiet Set ^ h;

[0037] (8)重复步骤(7) —次。 [0037] (8) Repeat steps (7) - times.

[0038] 实施例2 [0038] Example 2

[0039] 显微切割标本收集管清洗再利用方法,可按如下步骤依次实施: [0039] microdissected specimen collection tube cleaning method of recycling, follow these steps in order to implement:

[0040] (1)向收集管中加入占收集管容积2/3的二甲苯,盖上收集管管盖,并振荡4分钟, 打开管盖,弃去二甲苯; [0040] (1) was added to the collection tube collecting tube volume accounted for 2/3 of the xylene, the cover collection tube cap, and shaken 4 minutes, open the lid, discarded xylene;

[0041] (2)向步骤(1)所述收集管中加入占收集管容积1/2的混悬液A,盖上管盖,并振荡3分钟,打开管盖,倒空收集管;混悬液A由盐酸、酒精及EDTA混配,并加入50mg直径小于0. Imm的二氧化硅颗粒制成;其中盐酸的浓度为1. 5% ;所述酒精的浓度为30% ;所述EDTA 的浓度为0. 25mol/L;盐酸、酒精及EDTA的体积比为0. 5 : 97 : 2。 [0041] (2) to step (1) was added to the collection tube collection tubes volume accounts for 1/2 suspensions A, covered with lid and shaken for 3 minutes, open the lid, emptied collection tube; mix A suspension was mixed with hydrochloric acid, ethanol and EDTA, and a diameter of less than 0. Imm added 50mg of silica particles formed; wherein the concentration of hydrochloric acid was 1.5%; the alcohol concentration of 30%; the EDTA concentration of 0. 25mol / L; hydrochloric acid, the volume ratio of alcohol and EDTA 0. five ninety-seven: 2.

[0042] (3)向步骤(2)所述收集管中加入占收集管容积1/2的双蒸水,同时加入50mg直径小于0. Imm的二氧化硅颗粒,盖上管盖,并振荡2〜3分钟,打开管盖,倒空收集管; [0042] (3) to the step (2) was added to the collection tube collection tubes volume accounted for 1/2 of double distilled water, while adding 50mg diameter of less than 0. Imm silica particles, covered with lid and shaken 2 to 3 minutes, open the lid, empty the collection tube;

[0043] (4)依次重复步骤(2)及步骤(3)两次; [0043] (4) sequentially repeating steps (2) and step (3) twice;

[0044] (5)采用双蒸水对步骤(4)所得收集管进行冲洗4分钟; [0044] (5) the use of double-distilled water in step (4) washing the resulting collection tube 4 minutes;

[0045] (6)将收集管开盖,置入6°C冰箱内,直至收集管盖及管内液体完全蒸发; [0045] (6) The collection tube openings, placed within 6 ° C freezer until collection tube cap and completely evaporate the liquid within the tube;

[0046] (7)取出收集管,在超净台内,往收集管管盖内表面黏性模块表面涂抹浓度为4〜 8%的多聚赖氨酸,迅速将收集管放回冰箱,静置48h ; [0046] (7) Remove the collection tube in a clean desk, to cover the inner surface of the collection tube coated adhesive surface of the module 4 ~ 8% concentration of polylysine, quickly collection tube back in the fridge, quiet Set 48h;

[0047] (8)重复步骤(7) —次。 [0047] (8) Repeat steps (7) - times.

[0048] 为了验证本清洗再利用方法的有益效果,取两支收集管,一支经过本方法清洗,一支未经本方法清洗,进行以下实验。 [0048] In order to verify this cleaning method of recycling a beneficial effect, take two collection tubes, one after this cleaning method, a method of cleaning without this, the following test. 分别向两支管中加入80ul双蒸水,37°C孵育1小时。 The two tubes were added to 80ul distilled water, 37 ° C for 1 hour. 分别取出4ul液体作为模板,配制50ul标准体系(dNTP :0. 5ul,Taq酶:0. 5ul,10nmol/ul上下游引物:各0. 5ul,IOXbuffer :5ul,ddH20 :43. 5ul),进行多聚酶链反应(PCR),经40个循环扩增后,分别取6ul液体放入2%琼脂糖凝胶进行电泳如图1,泳道1为Marker标志; 泳道2为经本方法处理后的样本,未扩增出任何条带,表明经本方法清洗后的收集管已经没有核酸污染,该收集管经多聚赖氨酸处理后,有效保持和修复了收集管管盖内表面黏性模块黏附性,可以继续用于显微切割;泳道3为未经处理的样本,明显有核酸模板存在。 4ul liquid were removed as a template to prepare 50ul standards (dNTP:. 0 5ul, Taq enzyme:. 0 5ul, 10nmol / on ul downstream primers: 0. 5ul, IOXbuffer: 5ul, ddH20:. 43 5ul), performed polymerase chain reaction (PCR), after 40 cycles of amplification were taken 6ul liquid into a 2% agarose gel electrophoresis in FIG. 1, lane 1 flag Marker; lane 2 is a sample treated by the present method, not amplify any bands, suggesting the process was collected after cleaning the pipe has no nucleic acid contamination, the collector tube through the poly-lysine treatment, effectively maintain and repair the collection tube cover module adhesion surface stickiness, You can continue to be used microdissection; Lane 3 untreated sample, the presence of the nucleic acid template obvious.

[0049] 可以理解地是,以上关于本发明的具体描述,仅用于说明本发明而并非受限于本发明实施例所描述的技术方案,本领域的普通技术人员应当理解,仍然可以对本发明进行修改或等同替换,以达到相同的技术效果;只要满足使用需要,都在本发明的保护范围之内。 [0049] may be appreciated that the above detailed description of the invention, the present invention is for illustration only and not limited to the technical solution of the present invention described embodiments, one of ordinary skill in the art will appreciate that the present invention can still be modifications or equivalents in order to achieve the same technical effect; as long as meet the needs of all within the scope of the present invention.

5 5

Citas de patentes
Patente citada Fecha de presentación Fecha de publicación Solicitante Título
US681300810 Jun 20022 Nov 2004Palantyr Research, LlcMicrodissection optical system
WO2005/026811A1 Título no disponible
Otras citas
Referencia
1JP特开2005-15642A 2005.01.20
Clasificaciones
Clasificación internacionalG01N1/06, B08B3/12, B08B9/08, C11D7/60
Eventos legales
FechaCódigoEventoDescripción
25 Nov 2009C06Publication
20 Ene 2010C10Request of examination as to substance
5 Oct 2011C14Granted