CN111579538A - 一种应用于循环肿瘤细胞检测用凋亡试剂盒及其检测方法 - Google Patents
一种应用于循环肿瘤细胞检测用凋亡试剂盒及其检测方法 Download PDFInfo
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Abstract
本发明属于生物检测技术领域,具体涉及一种应用于循环肿瘤细胞检测用凋亡试剂盒及其检测方法。该试剂盒含有:1%Triton X‑100 200μL、TRITC标记链霉亲和素100μL、脱氧核糖核酸酶I 200μL、脱氧核糖核酸酶I缓冲液200μL、平衡缓冲液1mL、TdT酶80μL、生物素‑11‑dUTP 20μL、标记缓冲液1.0 mL、细菌脂多糖缓冲液50μL。本发明提供的试剂盒只需要通过一步染色,可以进行凋亡细胞的检测,同时,本发明特定的应用于凋亡循环肿瘤细胞的检测,灵敏度、特异性好、检测效果好。
Description
技术领域
本发明属于生物检测技术领域,具体涉及一种应用于循环肿瘤细胞检测用凋亡试剂盒及其检测方法。
背景技术
循环肿瘤细胞(Circulating tumor cell,CTC)是从实体肿瘤脱落进入外周血液循环的肿瘤细胞,自1989年被发现以来,目前已有多种方法用于外周血循环肿瘤细胞的检测。近期研究表明,其检测对于评估肿瘤患者尤其是晚期肿瘤患者的预后以及选择合适的个体化治疗具有重要的临床意义。因CTC检测具有微创、实时检测等特点,被称为肿瘤的“液态活检”。
细胞凋亡试剂盒是用来检测细胞凋亡时出现在细胞膜表面的磷酯酰丝氨酸的一种检测用试剂盒。在细胞发生凋亡的早期,细胞会把磷酯酰丝氨酸外翻到细胞膜外侧,磷酯酰丝氨酸暴露到细胞表面后,会促进凝血和炎症反应,采用带有绿色荧光的荧光探针FITC标记的Annexin V,可以简单而直接的检测到磷酯酰丝氨酸的外翻这一细胞凋亡的重要特征。循环肿瘤细胞由于其本身的特性,会自身演化出一套反凋亡机理来抑制细胞的凋亡,从而使得其凋亡的细胞采用传统的细胞凋亡试剂盒进行检测时,易出现假阳性。
发明内容
针对现有技术中存在的尚未有针对凋亡的循环肿瘤细胞的检测用试剂盒,本发明提供了一种应用于循环肿瘤细胞检测用凋亡试剂盒。
本发明还提供了一种利用上述试剂盒进行检测的方法。
本发明为了实现上述目的所采用的技术方案为:
本发明提供了一种应用于循环肿瘤细胞检测用凋亡试剂盒,该试剂盒含有:1%TritonX-100 200μL、TRITC标记链霉亲和素100μL、脱氧核糖核酸酶I 200μL、脱氧核糖核酸酶I缓冲液 200μL、平衡缓冲液1mL、TdT酶 80μL、生物素-11-dUTP 20μL、标记缓冲液 1.0 mL、细菌脂多糖缓冲液50μL。
进一步的,所述脱氧核糖核酸酶I的浓度为50U/μL;所述细菌脂多糖缓冲液的浓度为30 U/μL。
本发明还提供了一种利用上述凋亡试剂盒检测循环肿瘤细胞的方法,包括以下步骤:
(1)将含有循环肿瘤细胞样品经紫杉醇处理,用PBS洗涤两次,每次5min,然后加入100μL 1%Triton X-100溶液中处理5min;
(2)将样本中加入20μL脱氧核糖核酸酶I和60μL脱氧核糖核酸酶I缓冲液,37℃下处理30min,然后浸入PBS中漂洗三次,每次5min;
(3)然后在样本中添加50μLTdT酶反应液,放入温盒中,37℃下避光反应60min,反应后的样本浸入PBS中漂洗3次,每次5min,期间注意避光;
(4)每个样本上滴加细菌脂多糖缓冲液50μL ,静置10min后,再滴加50μLTRITC标记链霉亲和素标记液,放入湿盒,37℃下避光反应30min;反应后的样本浸入PBS中漂洗3次,每次5min;
(5)DAPI染色液复染细胞核,室温避光反应10min,洗去DAPI染液,加入封片剂,然后进行荧光显微镜检测。
进一步的,所述TdT酶反应液具体配置过程为:将1.0μL生物素-11-dUTP和4.0μLTdT酶,加入45μL平衡缓冲液,搅拌均匀即可。
进一步的,所述TRITC标记链霉亲和素标记液具体配置过程为;将5μL TRITC标记链霉亲和素同45μL标记缓冲液,混合均匀即可。
进一步的,所述封片剂是由三亚乙基二胺、甘油及PBS按照体积比2:4:5组成。
本发明的保存条件为:-20℃下避光进行保存。
本发明的有益效果为:本发明提供的试剂盒只需要通过一步染色,可以进行凋亡细胞的检测,同时,本发明特定的应用于凋亡循环肿瘤细胞的检测,灵敏度、特异性好、检测效果好。
附图说明
图1为实施例1检测得到的染色细胞图片。
具体实施方式
下面通过具体的实施例对本发明的技术方案作进一步的解释和说明。
本发明试剂盒具体的组成如表1所示。
表1
实施例1
利用上述试剂盒具体的检测方法为:
(1)将含有循环肿瘤细胞样品经紫杉醇处理,用PBS洗涤两次,每次5min,然后加入100μL 1%Triton X-100溶液中处理5min;
(2)将样本中加入20μL脱氧核糖核酸酶I和60μL脱氧核糖核酸酶I缓冲液,37℃下处理30min,然后浸入PBS中漂洗三次,每次5min;
(3)然后在样本中添加50μLTdT酶反应液,放入温盒中,37℃下避光反应60min,反应后的样本浸入PBS中漂洗3次,每次5min,期间注意避光;
所述TdT酶反应液具体配置过程为:将1.0μL生物素-11-dUTP和4.0μL TdT酶,加入45μL平衡缓冲液,搅拌均匀即可;
(4)每个样本上滴加细菌脂多糖缓冲液50μL ,静置10min后,再滴加50μLTRITC标记链霉亲和素标记液,放入湿盒,37℃下避光反应30min;反应后的样本浸入PBS中漂洗3次,每次5min;
所述TRITC标记链霉亲和素标记液具体配置过程为;将5μL TRITC标记链霉亲和素同45μL标记缓冲液,混合均匀即可;
(5)DAPI染色液复染细胞核,室温避光反应10min,洗去DAPI染液,加入封片剂,然后进行荧光显微镜检测;所述封片剂是由三亚乙基二胺、甘油及PBS按照体积比2:4:5组成。
图1为荧光显微镜下染色图片,图片中可以看出,凋亡的CTC其细胞核或细胞质内可见浓染致密的荧光,具有明显的核形态变化,产生凋亡小体,具有3个或者3个以上的DNA荧光碎片。
对比例1
(1)将含有循环肿瘤细胞样品经紫杉醇处理,用PBS洗涤两次,每次5min,然后加入100μL 1%Triton X-100溶液中处理5min;
(2)将样本中加入20μL脱氧核糖核酸酶I和60μL脱氧核糖核酸酶I缓冲液,37℃下处理30min,然后浸入PBS中漂洗三次,每次5min;
(3)然后在样本中添加50μLTdT酶反应液,放入温盒中,37℃下避光反应60min,反应后的样本浸入PBS中漂洗3次,每次5min,期间注意避光;
所述TdT酶反应液具体配置过程为:将1.0μL生物素-11-dUTP和4.0μL TdT酶,加入45μL平衡缓冲液,搅拌均匀即可;
(4)每个样本滴加50μLTRITC标记链霉亲和素标记液,放入湿盒,37℃下避光反应30min;反应后的样本浸入PBS中漂洗3次,每次5min;
所述TRITC标记链霉亲和素标记液具体配置过程为;将5μL TRITC标记链霉亲和素同45μL标记缓冲液,混合均匀即可;
(5)DAPI染色液复染细胞核,室温避光反应10min,洗去DAPI染液,加入封片剂,然后进行荧光显微镜检测。
利用该方法检测出的凋亡细胞,假阳性率高,部分为坏死细胞而非凋亡CTC细胞。
对比例2
利用上述试剂盒具体的检测方法为:
(1)将含有循环肿瘤细胞样品经紫杉醇处理,用PBS洗涤两次,每次5min,然后加入100μL 1%Triton X-100溶液中处理5min;
(2)将样本中加入20μL脱氧核糖核酸酶I和60μL脱氧核糖核酸酶I缓冲液,37℃下处理30min,然后浸入PBS中漂洗三次,每次5min;
(3)然后在样本中添加50μLTdT酶反应液,放入温盒中,37℃下避光反应60min,反应后的样本浸入PBS中漂洗3次,每次5min,期间注意避光;
所述TdT酶反应液具体配置过程为:将1.0μL生物素-11-dUTP和4.0μL TdT酶,加入45μL平衡缓冲液,搅拌均匀即可;
(4)每个样本上滴加细菌脂多糖缓冲液50μL ,静置10min后,再滴加50μLTRITC标记链霉亲和素标记液,放入湿盒,37℃下避光反应30min;反应后的样本浸入PBS中漂洗3次,每次5min;
所述TRITC标记链霉亲和素标记液具体配置过程为;将5μL TRITC标记链霉亲和素同45μL标记缓冲液,混合均匀即可;
(5)DAPI染色液复染细胞核,室温避光反应10min,洗去DAPI染液,加入封片剂,然后进行荧光显微镜检测;所述封片剂是由甘油及PBS按照体积比6:5组成。
该对比例中,荧光物质发生淬灭的时间较实施例1和对比例1明显缩短,本发明提供的封片剂能够明显的减缓淬灭时间。
Claims (6)
1. 一种应用于循环肿瘤细胞检测用凋亡试剂盒,其特征在于,该试剂盒含有:1%Triton X-100 200μL、TRITC标记链霉亲和素100μL、脱氧核糖核酸酶I 200μL、脱氧核糖核酸酶I缓冲液 200μL、平衡缓冲液1mL、TdT酶 80μL、生物素-11-dUTP 20μL、标记缓冲液 1.0mL、细菌脂多糖缓冲液50μL。
2.根据权利要求1所述的凋亡试剂盒,其特征在于,所述脱氧核糖核酸酶I的浓度为50U/μL;所述细菌脂多糖缓冲液的浓度为30 U/μL。
3.一种利用权利要求1或2所述的凋亡试剂盒检测循环肿瘤细胞的方法,其特征在于,包括以下步骤:
(1)将含有循环肿瘤细胞样品经紫杉醇处理,用PBS洗涤两次,每次5min,然后加入100μL 1%Triton X-100溶液中处理5min;
(2)将样本中加入20μL脱氧核糖核酸酶I和60μL脱氧核糖核酸酶I缓冲液,37℃下处理30min,然后浸入PBS中漂洗三次,每次5min;
(3)然后在样本中添加50μLTdT酶反应液,放入温盒中,37℃下避光反应60min,反应后的样本浸入PBS中漂洗3次,每次5min,期间注意避光;
(4)每个样本上滴加细菌脂多糖缓冲液50μL ,静置10min后,再滴加50μLTRITC标记链霉亲和素标记液,放入湿盒,37℃下避光反应30min;反应后的样本浸入PBS中漂洗3次,每次5min;
(5)DAPI染色液复染细胞核,室温避光反应10min,洗去DAPI染液,加入封片剂,然后进行荧光显微镜检测。
4.根据权利要求3所述的方法,其特征在于,所述TdT酶反应液具体配置过程为:将1.0μL生物素-11-dUTP和4.0μL TdT酶,加入45μL平衡缓冲液,搅拌均匀即可。
5.根据权利要求3所述的方法,其特征在于,所述TRITC标记链霉亲和素标记液具体配置过程为;将5μL TRITC标记链霉亲和素同45μL标记缓冲液,混合均匀即可。
6.根据权利要求3-5任一项所述的方法,其特征在于,所述封片剂是由三亚乙基二胺、甘油及PBS按照体积比2:4:5组成。
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