小鼠结肠癌细胞荧光素酶标记

培养方法

传代方法 将旧培养液吸除，PBS清洗两遍后，加入6mL（/100mm皿）胰酶，在显微镜下观察，期间禁止摇晃培养皿，细胞刚有脱落时，则吸除大部分胰酶，留约0.5mL，移至培养箱消化，约2min取出。传代用12mL CM1-1培养液终止消化，轻轻吹打均匀细胞，后可分3~6皿培养；

生长条件 37℃，5%CO2，CM1-1培养液。CM1-1培养液：90%DMEM-H+10%FBS。DMEM-H：DMEM高糖培养液，含谷氨酰胺，含丙酮酸钠。

存储条件 冻存则用6mL冻存液（90%FBS+10%DMSO）终止消化，吹打均匀，分为6支冻存管，用程序降温盒于-80℃冻存，过夜转移至液氮中保存。

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培养操作步骤 ：

1．用盖片镊将盖玻片自75%乙醇中取出，用无菌丝绸布擦拭干净，不要用纱布； 

2．将盖玻片轻轻放入6孔培养板（每孔一片）或培养皿中（每个平皿可放置2-3片）； 

3．在距离紫外灯直射范围内20-30 厘米处照射2-3小时； 

4．将经过计数的细胞悬浮液移入培养板中，使盖玻片完全浸在培养液中； 

5．将培养板在5% CO2水浴孵箱中37℃孵育2-3天，当贴壁细胞生长至覆盖培养板底部2/3面积时，将培养板取出，用盖片镊轻轻取出盖玻片，用蒸馏水漂洗后即可进行快速固定以及细胞化学检测。 

细胞培养方法：

1、细胞传代：细胞密度达到80-90%时即可传代

①弃去培养上清，用PBS或生理盐水清洗1-2次；

②加入2ml0.25%胰酶（T25瓶），使胰酶覆盖整个瓶或皿，盖好放入培养箱消化；

③1-2min后，显微镜下观察细胞，若大部分细胞回缩且有少量细胞脱落，轻轻吹打下确认消化情况后加入完全培养基终止消化；若细胞还是贴壁，放回培养箱继续消化至可以轻轻吹打下为止；

④将细胞悬液1000RPM左右条件下离心4min，弃上清；

⑤用新鲜培养基重悬后加入培养瓶或皿中，T25培养瓶加6-8ml培养基；

⑥悬浮细胞直接离心收集，细胞沉淀重悬后分到新培养瓶中。

2、细胞复苏：

①将冻存管在37℃温水中快速摇晃融化，时间1min左右，加入4-5ml培养基混匀。

②在1000RPM左右条件下离心4min，弃上清,加1-2ml培养基吹匀，将细胞悬液加入培养瓶中，补加适量培养基。

3、细胞冻存：待细胞生长状态良好时进行细胞冻存保种

①弃去培养上清，用PBS或生理盐水清洗1-2次，加入1mL 0.25%胰蛋白酶（T25瓶）

②1-2min后，显微镜下观察细胞，大部分细胞回缩且有少量细胞脱落，轻轻吹打下确认消化情况后加入完全培养基终止消化；

③将细胞悬液1000RPM左右条件下离心4min，弃上清，加1ml冻存液重悬细胞；

④将冻存管放入程序降温盒，放入-80℃冰箱，4小时后将冻存管转入液氮罐储存。

（石蜡切片需做抗原修复）

 not yet tested in other applications.

 optimal dilutions/concentrations should be determined by the end user.  

保存条件  Store at -20 °C for one year. Avoid repeated freeze/thaw cycles. The lyophilized antibody is stable at room temperature for at least one month and for greater than a year when kept at -20°C. When reconstituted in sterile pH 7.4 0.01M PBS or diluent of antibody the antibody is stable for at least two weeks at 2-4 °C.

Important Note  This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications.

产品介绍 Otoancorin, also known as OTOA, CT108 or DFNB22, is a 1,153 amino acid protein belonging to the stereocilin family. Expressed in the inner ear and restricted to the interface between the apical surface of sensory epithelia, otoancorin is suggested to act as an adhesion molecule. Otoancorin ensures the attachment of the inner ear acellular gels to the apical surface of the underlying nonsensory cells. Mutations in the gene encoding otoancorin leads to deafness autosomal recessive type 22 (DFNB22), which is a form of sensorineural hearing loss. Sensorineural deafness results from damage to the neural receptors of the inner ear, the nerve pathways to the brain or the area of the brain that receives sound information. Existing as three alternatively spliced isoforms, otoancorin is encoded by a gene located on human chromosome 16p12.2.

Function : May act as an adhesion molecule.

Subcellular Location : Apical cell membrane. Secreted > extracellular space > extracellular matrix. At the interface between the apical surface of the epithelia and the overlying acellular gel of the tectorial and otoconial membranes.

DISEASE : Defects in OTOA are the cause of deafness autosomal recessive type 22 (DFNB22) [MIM:607039]. DFNB22 is a form of sensorineural hearing loss. Sensorineural deafness results from damage to the neural receptors of the inner ear, the nerve pathways to the brain, or the area of the brain that receives sound information.

Similarity : Belongs to the stereocilin family.

Database links : UniProtKB/Swiss-Prot: Q7RTW8.1

英文名称  Anti-Osteocalcin

中文名称  骨钙蛋白/骨钙素抗体

别    名  Osteocalcin Propeptide; Osteocalcin; Osteocalcin precursor; Gamma-carboxyglutamic acid-containing protein; Bone Gla-protein; BGLAP; Bone gamma carboxyglutamate protein; BGLAP; BGP; Bone gamma carboxyglutamate gla protein osteocalcin; Bone gamma carboxyglutamate protein; Bone Gla protein; Gamma carboxyglutamic acid containing protein; OC; PMF1; OSTCN_HUMAN.

浓    度  1mg/1ml

规 格  0.1ml/100μg  0.2ml/200μg  1ml/1mg

抗体来源  Rabbit  

克隆类型  polyclonal
小鼠结肠癌细胞荧光素酶标记骨桥素检测试剂盒脱氢乙酸钠 99%DL-乳酸 ACS, ≥85%

骨特异性碱性磷酸酶检测试剂盒脱氢乙酸 98%L-乳酸钠  AR，60%水溶液

骨特异性碱性磷酸酶B检测试剂盒乙酰乙酸乙酯  >99.0%(GC)L-乳酸钠 98%

骨退化特异标志物检测试剂盒乙酰乙酸甲酯  99%6-氨基吲唑 98%

骨涎蛋白检测试剂盒3-基吡啶 99%间羟基苯甲酸 98%

瓜氨酸检测试剂盒邻苯二甲 98%5-硝基吲唑 98%

寡克隆区带检测试剂盒山梨酸 >99.0%(T)6-硝基吲唑 98%

实验要点及说明：

1．本方法适用于贴壁细胞培养，而不适用于悬浮细胞培养，悬浮细胞可使用滴片法； 

2．所使用的盖玻片应该为玻璃**，并经过铬酸洗液处理； 

3．盖玻片非常薄，易碎，取放盖玻片时动作要轻； 

4．如果需要更多生长状态一致的细胞，可以使用较大的培养皿，但不宜过大，以避免培养液的浪费和增加污染机率； 

5．如果细胞贴壁生长能力较差，可将盖玻片在0.5%多聚赖氨酸溶液中浸泡5-10分钟并自然晾干。