LoxP-STOP-loxP-mCherry HeLa Cells

LoxP-STOP-loxP-mCherry HeLa 细胞

LoxP-STOP-loxP-mCherry HeLa Cells

L752168-1EA

1个

-80℃储存

超低温冰袋运输

-80℃保存，2个月有效。

LoxP-STOP-loxP-mCherry HeLa Cells，即LoxP-STOP-loxP-mCherry HeLa细胞，简称LSL-mCherry HeLa Cells或LSL-mCherryHela细胞，是阿拉丁通过把含有loxP-STOP-loxP-mCherry元件和G418抗性的质粒转染HeLa细胞，经G418筛选最终获得的单克隆细胞株。本细胞株可以用于检测Cre重组酶的活性，也可以用于检测能表达Cre重组酶的质粒或病毒的效果。LoxP (locus of X (cross)-over inP1)位点是一段34bp的DNA序列，其两端为两个13bp的反向重复序列(invertedrepeats)，中间是8bp的间隔区(图1)。Cre重组酶是来源于大肠杆菌噬菌体P1的一种类型I的拓朴异构酶(Type I topoisomerase)，也是一种酪氨酸重组酶(tyrosinerecombinase)，它可识别34bp的loxP位点并催化loxP位点之间的DNA发生重组；重组产物根据loxP位点的位置和相对方向的不同而不同，两个含单个loxP位点的DNA将发生融合：两个正向重复的loxP位点间的DNA将以环状形式被切割，而两个反向loxP位点间的DNA序列将被翻转(图2) 1,2]。图1.loxP位点序列图。Cre重组酶与两端13bp反向重复序列(小写字母)结合，中间是8bp不对称中心间隔区(大写字母)，箭头所示为Cre重组酶的酶切位点。图2. Cre-loxP位点特异性重组示意图。本细胞株中含有loxP-STOP-loxP-mCherry元件，在没有Cre重组酶存在的情况下，mCherry的表达会被阻断，观察不到红色荧光。在细胞中转染表达Cre重组酶的质粒(例如D2608 pCMV-Cre-EGFP)、与可以直接穿透细胞膜的D0512 TAT-Cre Recombinase孵育、或直接转染Cre重组酶(例如D0509 Cre Recombinase)时，Cre重组酶可以剪切并重组loxP位点，去除两个loxP位点之间的3×SV40poly(A)这个STOP元件，从而激活mCherry的表达，随后可以通过荧光显微镜等荧光检测设备进行观察和检测。本细胞株中的loxP-STOP-loxP-mCherry元件通过PCR扩增和测序验证。本细胞株经过支原体检测(Mycoplasma Test)，检测结果为阴性。本单克隆细胞株构建之前通过STR (short tandem repeats)鉴定。Cre重组酶(Cre Recombinase)作用于本细胞株后，细胞的明场和荧光图片请参考图3。图3. 阿拉丁LoxP-STOP-loxP-mCherry HeLa Cells 功能鉴定图。按照每孔约10万LoxP-STOP-loxP-mCherry HeLaCells接种到24孔板进行培养过夜，使细胞密度能达到约60%。每孔用 PBS洗一次，每孔加入0.5ml含4μM TAT-CreRecombinase的完全培养液(含有血清和链霉素\\青霉素)。继续培养6h后，更换为不含TAT-Cre Recombinase的完全培养液，24h后通过荧光显微镜下可以观察到细胞内红色荧光。实际结果会因实验条件、检测仪器等的不同而存在差异，图中效果仅供参考。本细胞株的培养条件可以参考HeLa细胞的培养条件，可以同时在培养液中添加终浓度为200μg/ml的G418，以维持适当的筛选压力。进行Cre重组酶测试时，可以考虑去除G418，以减少G418可能产生的干扰。HeLa细胞的基本信息如下：HeLa细胞的详细信息如下:* Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility ofthe customer to ensure that their facilities comply with biosafety regulations for their own country.对于细胞培养瓶或离心管运输的活细胞，室温3-5天有效。对于干冰运输的冻存细胞，液氮保存，长期有效； 使用说明 ： 1.细胞株的复苏a.将冻存管在37℃水浴锅中迅速完全融化(保持冻存管的盖子在液面以上以防止污染)，并适当轻轻摇晃促融，切勿vortex。快速、完全融化可以提高细胞的复苏效果。b.打开冻存管前时用70%酒精擦拭细胞冻存管外壁，注意某些记号笔不耐酒精，小心标注的记号被擦拭掉。c.将完全融化的细胞直接离心，或者转移至无菌1.5ml或其它合适无菌离心管中，500g离心2-5min，吸除上清，注意不要吸走细胞沉淀，然后用新鲜完全培养液重悬后转移至培养器皿，混匀，置于CO2培养箱37℃培养。d.第二天视贴壁或生长状态，更换培养液。2.贴壁细胞的常规传代流程a.将细胞培养液、PBS等放入37℃水浴锅内预热。b.以10cm细胞培养皿为例。吸出原培养皿中的培养液，用2-5ml无菌PBS润洗细胞1-2次以去除残留的血清(如果细胞贴壁较差，润洗时要轻柔以避免细胞飘起)，然后加1-2ml胰酶细胞消化液(含EDTA)室温消化，注意消化时间，通常为1-5分钟。如果细胞比较难消化，可以置于37℃细胞培养箱一定时间以加速消化。注意：消化时间过长，会导致传代后细胞出现生长状态不良的情况。c.每30秒-1分钟用显微镜观察细胞消化情况，贴壁细胞明显收缩、细胞间间隙变大、细胞趋于圆形但还未漂起，并用移液器吹打细胞发现细胞刚好可以被吹打下来时，吸除胰酶细胞消化液，再加入1-2ml新鲜完全培养液，适当晃动细胞皿以终止胰酶作用，用移液器轻轻吹打贴壁的细胞，获取细胞悬液。吹打时需控制力度，避免产生大量气泡，将细胞悬液分别接种到另外的2~5个细胞培养皿内，加入新鲜培养液，置于CO2培养箱37℃培养，第2天观察细胞贴壁生长情况。d.也可以在消化后，加3-5ml完全培养液终止消化，用移液器轻轻吹打细胞悬液，尽量把细胞全部吹落、吹散，然后将全部细胞悬液500g离心2-5min，离心后去上清，再用完全培养液重悬后转移到新的培养皿中，添加适量完全培养液，于CO2培养箱37℃培养。e.注意培养液的酚红颜色变化或根据细胞的换液要求定期换液，待细胞密度达到80-90%时需要传代或者冻存。如果没有及时传代导致细胞过密，传代后细胞容易出现生长状态不良的情况。3.悬浮细胞的常规传代流程a.将细胞悬液转移到无菌离心管内，500g离心2-5min，弃去上清，加入新鲜的培养液，用吸管小心吹散沉淀，获取细胞悬液，将细胞悬液分别接种到另外的2-5个细胞瓶内，加入新鲜完全培养液，置于CO2培养箱37℃培养。b.也可以取少量悬浮细胞直接转移到新的培养瓶中，添加适当的新鲜完全培养液，置于CO2培养箱37℃培养。c.注意培养液的酚红颜色变化或根据细胞的换液要求定期换液，待细胞密度达到80-90%时可以考虑传代或者冻存。4.半贴壁半悬浮细胞的培养a.若悬浮细胞较多且折光率良好，可离心收集，继续培养。b.若仅有少量细胞悬浮，也可不用收集，传代操作按常规贴壁细胞操作流程处理。c.若悬浮细胞较多，离心收集，原瓶中贴壁细胞按照常规贴壁细胞操作流程进行消化、终止消化、吹打，并与之前收集的悬浮细胞混合，接种到新的细胞培养皿中。5.细胞株的冻存a.按照细胞传代方法收集细胞。b.细胞计数：一般要求冻存的细胞，每毫升的细胞数量为1×106-107个细胞。c.取适当细胞悬液，500g离心2-5min，弃上清，加入细胞冻存液，重悬，转移到冻存管中，用记号笔标记好细胞株名称、冻存日期、代数等信息，并记录在相应表格中以便管理和快速查找细胞位置。d.将冻存管放入专用的细胞冻存盒中，-80℃过夜，然后转移至液氮灌中保存。如果没有专用的细胞冻存盒，可以按下面程序进行冻存：4℃ 1h，-20℃ 2h，-80℃过夜，然后转移至液氮灌中保存。冻存细胞储存在-80℃中通常不建议超过半年，时间太长会影响复苏效率。推荐使用阿拉丁的细胞冻存盒。e.为保持细胞的良好状态，每隔1年，取出1-2支冻存的细胞复苏一次，并冻存新的细胞。6.本细胞株的检测a.对于细胞株中正常情况下被阻断表达的mCherry荧光蛋白，可以通过在细胞中转染表达Cre重组酶的质粒(例如D2608 pCMV-Cre-EGFP)或直接转染Cre重组酶(例如D0509 Cre Recombinase)，以剪切并重组loxP位点，从而激活mCherry的表达，随后可以通过荧光显微镜等荧光检测设备进行观察和检测。b.对于细胞株中是否存在loxP-STOP-loxP-mCherry元件相应的DNA序列的检测，可以通过抽提细胞基因组DNA后，使用5’-CGCAAATGGGCGGTAGGCGTG-3’和5’-CTCCTCGCCCTTGCTCACCAT-3’作为引物，通过PCR扩增插入到基因组中的相应DNA序列，PCR扩增产物长度约1085bp，并对PCR扩增产物进行直接测序或插入到T载体中后进行测序鉴定。推荐使用无需基因组DNA抽提的阿拉丁生产的D7281 动物组织直接PCR试剂盒或D7285 Easy-Load™ Blood Direct PCR Master Mix (2X)进行PCR扩增。参考文献：1.Pinkney JN, Zawadzki P, Mazuryk J, Arciszewska L K, Sherratt D J, Kapanidis A N. Proc Natl Acad Sci USA. 2012. 109(51):20871-6.2.Kühn R, Torres R M. Methods Mol Biol. 2002. 180:175-204. Aladdin's LoxP-STOP-LoxP-mCherry HeLa Cells, abbreviated as LSL-mCherry HeLa Cells, carry the plasmid containing loxP-STOP-loxP-mCherry element and G418 resistance gene. This monoclonal cell line can be used to assay the activity of Cre Recombinase as well as the effect of plasmids or viruses that can express Cre Recombinase.This cell line shall not be used for any commercial purposes without written permission from aladdin, nor may it be transferred to any person or entity outside of the laboratory where it is purchased. Users should indicate the source of the cell line when publishing research papers or results.The loxP (locus of X (cross)-over in P1) site is a 34bp DNA sequence comprised of two 13bp inverted repeats at two ends and an 8bp spacer region in between. Cre Recombinase is a type I topoisomerase from E. coli phage P1 and also a tyrosine recombinase that recognizes the loxP site and catalyzes the recombination of DNA between loxP sites. Recombination products depends on the location and relative orientation of the loxP sites. Two DNAs containing a single loxP site each will be fused. DNA between two directly repeated loxP sites will be excised in circular form, while DNA between two opposing loxP sites will be inverted in terms of external sequences (Figure 2) [1,2].Figure 1. DNA sequence of the loxP site. Cre Recombinase binds to the 13bp inverted repeat sequence (lowercase letters) at both ends with an 8bp spacer region (uppercase letters) in the middle. The arrow shows the cleavage site of Cre Recombinase.Figure 2. Schematic diagram of site-specific recombination of DNA between loxP sites catalyzed by Cre Recombinase.The information of this cell line is provided by combining the information from aladdin and that from ATCC (American Type Culture Collection), DSMZ (German Collection of Microorganisms and Cell Cultures), JCRB (Japanese Collection of Research Bioresources Cell Bank), Cellosaurus (Swiss Institute of Bioinformatics) and other websites. Due to cell culture and passaging conditions, the provided cells may differ slightly from the information provided in this manual, and the actual cells shall prevail.This cell line contains a loxP-STOP-loxP-mCherry element. In the absence of Cre Recombinase, the expression of red fluorescent protein (mCherry) is blocked in cells. However, in cells transfected with Cre Recombinase-expression plasmids such as pCMV-Cre-EGFP , incubated with cell-permeable TAT-Cre Recombinase , or directly transfected with Cre Recombinase , the 3×SV40 poly(A) STOP element is removed through recombination between two loxp sites catalyzed by Cre Recombinase, thereby activating the expression of mCherry that can be examined by fluorescence microscopy or other fluorescence detection equipment.STR results can be compared to the databases in ATCC, DSMZ and other websites. A match of 80% or more is considered correct for the cell line.The loxP-STOP-loxP-mCherry element in this cell line has been validated by PCR amplification and sequencing.The shipping method for this product varies depending on whether the cells are being cultured and distance to the destination, etc. The cell line can be shipped as cryopreserved cells on dry ice, adherent or suspension cells in small vials at room temperature. To better tolerate long-distance transport and changes of temperature during transport, cells that are cultured adherently may be transported in suspension culture in culture flasks or centrifuge tubes.This cell line has been tested negative for mycoplasma.For frozen cells transported on dry ice, if cells have completely thawed upon receipt, please perform cell recovery immediately and do not freeze them again. Cells in cryopreserved state can be recovered immediately upon receipt or stored in liquid nitrogen immediately for long-term storage.This monoclonal cell line was identified by STR (short tandem repeats) prior to construction.We recommend users to perform cell recovery as soon as possible after receipt to confirm cell viability, status and to preserve cells. The frozen cells can be stored at -80℃ for up to 2 months.Please refer to Figure 3 for the performance of this cell line after treatment with Cre Recombinase.Figure 3. Functional characterization of Aladdin's LoxP-STOP-LoxP-mCherry HeLa Cells . Approximately 100,000 LoxP-STOP-LoxP-mCherry HeLa Cells were seeded per well of 24-well plates and cultured overnight until cell density reached 60% approximately. After washing with PBS, cells were cultured for 6h in 0.5 ml of complete culture medium containing 4μM TAT-Cre Recombinase , followed by culture in new complete culture medium without Cre Recombinase for another 24h before examination by fluorescence microscopy. This figure is for reference only, which may vary due to experimental conditions.Each tube of this product contains approximately 1×106 cells in a volume of 0.5-1ml, with an expected survival rate of 60-90%. It is recommended to be resuscitated in a 6cm culture dish. If the survival rate is low, the cells can be digested and transferred to a 3.5cm dish for better cell growth.This cell line can be cultured under the same conditions as HeLa cells, and G418 at a final concentration of 200 μg/ml can be used to maintain selective pressure. Removal of G418 can be considered when performing Cre Recombinase tests to reduce the possible interference from G418.If the product is received as adherent cells in culture flasks with complete culture medium at room temperature, please observe the cell state under the microscope. If the cell density is more than 85%, cells should be passaged as soon as possible. If there are more cells in suspension, place the culture flask in a cell incubator overnight to make the suspended cells adhere again. If suspension cells are received in centrifuge tubes shipped at room temperature, transfer cells directly to culture dishes or culture flasks for cultivation. If the color of the culture medium is normal, continue to culture in the culture medium and keep half of original culture medium when changing the culture medium for the first time to avoid the maladaptation of cells.Basic information of HeLa cells is as follows Instructions for Use ： 1. Cell recoverya. Thaw the fozen cells rapidly and completely in a 37℃ water bath and shake the tube gently to promote thawing. Rapid and complete thawing will improve the recovery rate of cells.b. Wipe the outer surface of the tube with 70% alcohol before opening.c. Centrifuge cells at 500×g for 2-5min directly or after transferring cells to a sterile 1.5ml centrifuge tube. Discard the supernatant, carefully, resuspend cells in fresh complete culture medium, then transfer to a culture vessel. Mix well and incubate at 37℃ in a CO2 incubator.d. After overnight incubation, replace with new culture medium depending on the state of adherence or growth.2. Routine passaging of adherent cellsa. This protocol is provided for passaging cells cultured in 10cm culture dishes.b. Preheat the cell culture medium and PBS in a 37℃ water bath.c. Aspirate culture medium from the culture dish, wash cells 1-2 times with 2-5ml of sterile PBS (If cells are poorly adhered, be gentle when washing to avoid cell suspension), then add 1-2ml of trypsin solution (containing EDTA) for cell digestion at room temperature. It takes 1-5 minutes usually for cell digestion. If the cells are difficult to digest, perform the cell digestion in a 37℃ cell incubator for a certain period of time to promote digestion. Note: Too long digestion time may cause poor growth of cells after passaging.d. Observe cells under microscope every 30 seconds-1 minute and stop digestion when adherent cells shrink obviously, tend to be round but not yet floating up, and can just be blowing off by pipetting. Dicard the trypsin solution, add 1-2ml of fresh complete culture medium, and blow off the adherent cells gently by pipetting to obtain the cell suspension. Inoculate the cell suspension into 2-5 culture dishes with fresh culture medium, incubate at 37℃ in CO2 incubator, then observe the cell growth state after overnight incubation.e. Alternatively, after digestion, add 3-5ml of complete culture medium to terminate the digestion, gently blow off and disperse cells by pipetting, then centrifuge at 500×g for 2-5min. Discard the supernatant, resuspend cells in complete culture medium, then transfer to a new culture dish with an appropriate amount of complete culture medium for incubation at 37℃ in a CO2 incubator.f. Pay attention to the phenol red color change of the culture medium or change the medium regularly according to the requirements of cells. Cells should be passaged or cryopreserved when the cell density reaches 80-90%. Otherwise, cells will grow poorly after passaging.3. Routine passaging of suspension cellsa. Transfer the cell suspension to a sterile centrifuge tube, centrifuge at 500×g for 2-5min. Discard the supernatant and resuspend cells in fresh culture medium by carefully dispersing the cell pellet with a pipette. Inoculate the cell suspension into 2-5 cell bottles with fresh complete culture medium and incubate at 37℃ in a CO2 incubator.b. A small amount of suspended cells can also be taken and transferred directly to a new culture flask with an appropriate amount of fresh complete culture medium, then incubated at 37℃ in a CO2 incubator.c. Pay attention to the phenol red color change of the culture medium or change the medium regularly according to the requirements of cells. Cells should be passaged or cryopreserved when the cell density reaches 80-90%.4. Culture of semi-adherent and semi-suspension cellsa. If there are relatively more suspension cells with good refractive index, they can be collected by centrifugation and continue to be cultured.b. If there is only a small number of suspension cells, they can be passaged directly without collection according to the conventional procedures for adherent cells.c. If there are more suspended cells, collect suspension cells by centrifugation. Digest the adherent cells, suspend cells after stopping digestion, and then combine with the original suspension cells collected. Finally, inoculate them into new culture dishes.5. Cryopreservation of cellsa. Collect cells according to the cell passaging method.b. Cell counting: Cell density should be 1×106-107 cells per ml for cryopreservation.c. Take an appropriate amount of cell suspension, centrifuge at 500×g for 2-5min. Discard the supernatant, resuspend cells in cell freezing medium, then transfer to cryovials labled with the name of cell line, date of freezing, passage number and other information as desired. Record the location of cells. d. Place the cryovials in a special cell freezing container at -80℃ overnight and then transfer to liquid nitrogen for storage. If a special cell freezing container is not available, the following procedure can be used: 4℃ for 1h, -20℃ for 2h, -80℃ overnight, and then transfer to liquid nitrogen for storage. Freezing cells at -80℃ for more than six months is usually not recommended, as too long a period of time may affect the recovery efficiency. We recommend using Blood Direct PCR Master Mix (2X) for PCR amplification without the need of genomic DNA preparation.