LoxP-STOP-loxP-mCherry HeLa Cells

Item Number

L752168

• Stability And Storage: For live cells transported in culture flasks or centrifuge tubes, store at room temperature for up to 3-5 days. For cryopreserved cells transported on dry ice, Store in liquid nitrogen for long-term storage, or at -80℃for up to 2 months.
• Storage Temp: Store at -80°C
• Shipped In: Ice chest + Ice pads

Product Description

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.Related Products:产品编号产品名称包装C0201-100mlTrypsin-EDTA Solution100mlC0201-500mlTrypsin-EDTA Solution500mlC0202Trypsin-EDTA Solution100mlC0203-100mlTrypsin-EDTA Solution(Containing phenol red)100mlC0203-500mlTrypsin-EDTA Solution(Containing phenol red)500mlC0204Trypsin-EDTA Solution with Phenol Red100mlC0205Trypsin Solution without EDTA100mlC0207Trypsin Solution with Phenol Red and without EDTA100mlC0210Cell Culture Freezing Medium50mlC0212L-Glutamine (100X)100mlC0218Hanks' Balanced Salt Solution500mlC0219Hanks' Balanced Salt Solution (with Ca2+ & Mg2+)500mlC02207.5% NaHCO₃ Solutioin100mlC0221APBS500mlC0221DD-PBS500mlC0221GD-PBS (with Ca2+ & Mg2+)500mlC0222Penicillin-Streptomycin Solution, 100X100mlC0226Fetal Bovine Serum (FBS, Biowest, South America)500mlC0232Fetal Bovine Serum (FBS, Gibco, South America)500mlC0234Fetal Bovine Serum (FBS, Gibco, Australia)50mlC0235Fetal Bovine Serum (FBS, Gibco, Australia)500mlC0251Fetal Bovine Serum (FBS, South America)50mlC0252Fetal Bovine Serum (FBS, South America)500mlC0258Newborn Calf Serum (NCS, New Zealand)50mlC0265Goat Serum50mlC0288MMycoplasma Removal Agent100mgC0288SMycoplasma Removal Agent20mgC0290MMycoplasma Removal Agent Plus50mgC0290SMycoplasma Removal Agent Plus10mgC0296Mycoplasma Stain Assay Kit＞100TD0509LCre Recombinase1000UD0509MCre Recombinase250UD0509SCre Recombinase50UD2607-100μgpCMV-Cre-mCherry100µgD2607-1μgpCMV-Cre-mCherry1µgFBX08181-well Cryobox for Liquid Nitrogen Tank1pcs/PackageFBX082100-well Cryobox for Liquid Nitrogen Tank1pcs/PackageFCD035 35mm Cell Culture Dishes (TC-treated)10pcs/Package, 50Packages/caseFCD060 60mm Cell Culture Dishes (TC-treated)10pcs/Package, 50Packages/caseFCD100 100mm Cell Culture Dishes (TC-treated)10pcs/Package, 20Packages/caseFCFC012 Cell Freezing Container(2ml)1pcFCN11010ml Pipet 50/bag FCN12525ml Pipet 25/bag FCP060 6 Well Cell Culture Plates50pcs/caseFCP126 12 Well Cell Culture Plates (TC-treated)50pcs/caseFCP243 24 Well Cell Culture Plates (TC-treated)50pcs/caseFCP485 48 Well Cell Culture Plates (TC-treated)50pcs/caseFCP962 96 Well Cell Culture Plates (TC-treated)50pcs/caseFCP966-320pcs 96-Well Black Opaque Plates (TC-treated)80pcs/box, 320pcs/boxFCP966-80pcs 96-Well Black Opaque Plates (TC-treated)80pcs/boxFCP968-320pcs 96-Well White Opaque Plates (TC-treated)80pcs/box, 320pcs/boxFCP968-80pcs 96-Well White Opaque Plates (TC-treated)80pcs/boxFFLK021 25cm2 Cell Culture Flasks with Vented Cap10/Package, 200/caseFFLK023 25cm2 Cell Culture Flasks with Plug Seal Cap10/Package, 200/caseFFLK075 75cm2 Cell Culture Flasks with Vented Cap5/Package, 100/caseFFLK077 75cm2 Cell Culture Flasks with Plug Seal Cap5/Package, 100/caseFFLK176 175cm2 Cell Culture Flasks with Vented Cap5/Package, 40/caseFFLK178 175cm2 Cell Culture Flasks with Plug Seal Cap5/Package, 40/caseFLFT021 Cell Lifters (21cm, Individual Pack, Sterile)100/boxFPIP105 5ml Serological Pipettes (Individually Paper/Plastic Wrapped & Sterile)50/bag, 4bags/cartonFPIP110 10ml Serological Pipettes (Individually Paper/Plastic Wrapped & Sterile)50/bag, 4bags/cartonFPIP125 25ml Serological Pipettes (Individually Paper/Plastic Wrapped & Sterile)25/bag, 8bags/cartonFSCP023 Cell Scrapers (23cm, Individual Pack, Sterile)100/boxFSCP029 Cell Scrapers (29cm, Individual Pack, Sterile)100/boxFSTR040 Cell Strainers (40μm, Individual Pack, Sterile)100/boxFSTR070 Cell Strainers (70μm, Individual Pack, Sterile)100/boxFSTR100 Cell Strainers (100μm, Individual Pack, Sterile)100/boxFTIP610 Pipette Tips (0.1-10μl, Clear, Racked & Sterile)96/box, 50boxes/caseFTIP616 Pipette Tips (0.1-10μl, Low Retention, Clear, Racked & Sterile)96/box,50 boxes/boxFTIP620 Pipette Tips (1-200μl, Yellow, Racked & Sterile)96/box, 50boxes/caseFTIP628 Pipette Tips (100-1000μl, Extended Length, Blue, Racked & Sterile)96/box, 50boxes/caseFTUB306 1.5ml Microcentrifuge Tubes (Clear, Nuclease free)500/box, 10boxes/caseFTUB515 15ml Centrifuge Tubes 25/bag,20bags/caseFTUB550 50ml Centrifuge Tubes 25/bag,20bags/caseST081-1gG418 Sulfate1gST081-1mlG418 Sulfate50mg/ml×1mlST081-5gG418 Sulfate5gST081-5mlG418 Sulfate50mg/ml×5mlST083L-Glutamine100gST476PBS (10X)500mlST875-100ml Ultrapure Water (Suitable for Cell Culture)100mlST875-500ml Ultrapure Water (Suitable for Cell Culture)500ml