LoxP-STOP-loxP-EGFP HeLa Cells

Item Number

L752167

• Stability And Storage: 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-EGFP HeLa Cells, abbreviated as LSL-EGFP HeLa Cells, carry the plasmid containing loxP-STOP-loxP-EGFP 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.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.This cell line contains a loxP-STOP-loxP-EGFP element. In the absence of Cre Recombinase, the expression of enhanced green fluorescent protein (EGFP) 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 EGFP that can be examined by fluorescence microscopy or other fluorescence detection equipment.The loxP-STOP-loxP-EGFP element in this cell line has been validated by PCR amplification and sequencing.This cell line has been tested negative for mycoplasma.This monoclonal cell line was identified by STR (short tandem repeats) prior to construction.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-EGFP HeLa Cells . Approximately 100,000 LoxP-STOP-LoxP-EGFP 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.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.Basic information of HeLa cells is as follows

Precautions：

This cell line shall not be used for any commercial purposes without written permission from , 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 information of this cell line is provided by combining the information from 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.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 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.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.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.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.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.The cell culture should be operated aseptically in a biosafety cabinet.Please add an appropriate amount of penicillin-streptomycin solution (, C0222) to the culture medium to prevent bacterial contamination.Theoretically immortalized cells can be passaged indefinitely, but to ensure that cells are in good condition, we recommend freezing cells in the earliest passage and recovering cells after every period of culture.please comply with relevant laws and regulations for receiving, handling, preserving and discarding cells, fully consider the possible risks and liabilities, and take appropriate measures to minimize health or environmental hazards.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.

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 cellsThis protocol is provided for passaging cells cultured in 10cm culture dishes.a. Preheat the cell culture medium and PBS in a 37℃water bath.b. 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.c. 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.d. 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.e. 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. 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