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ZFL [ZF-L]

CRL-2643

Product category
Animal cells
Organism
Danio rerio, zebrafish
Morphology
epithelial
Tissue
Liver
Disease
Normal
Applications
3D cell culture
High-throughput screening
Toxicology
Product format
Frozen
Storage conditions
Vapor phase of liquid nitrogen
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Documentation

Biosafety Icon BSL 1

ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.

Required Products

These products are vital for the proper use of this item and have been confirmed as effective in supporting functionality. If you use alternative products, the quality and effectiveness of the item may be affected.

Leibovitz's L-15 Medium

30-2008

Price: $25.00 ea

Dimethylsulfoxide (DMSO)

4-X

Price: $49.00 ea

Detailed product information

General

Specific applications
The cells are useful for studies of liver cell metabolism and xenobiotic formation. They may be used for in vitro toxicology studies.

Characteristics

Growth properties
Adherent
Derivation
The ZFL cell line was established in 1992 from a pool of approximately 10 normal adult zebrafish livers. It exhibits some properties characteristic of liver parenchymal cells.
Age
adult
Karyotype
hypodiploid; modal number = 47
Comments
The cells synthesize and release several proteins into the culture medium, including a 70 kDa protein recognized by anti-bovine serum albumin IgG.

A culture submitted to the ATCC in August of 2001 was found to be contaminated with mycoplasma. Progeny were cured by a 21-day treatment with BM Cycline.

The cells were assayed for mycoplasma, by the Hoechst stain, PCR and the standard culture test, after a six-week period following treatment. All tests were negative.

The dioxin, TCDD, induces one or possibly two forms of cytochrome P450 immunochemically and functionally related to rainbow trout P4501A1.

ZFL cell homogenates exhibit alanine and aspartate aminotransferase, glucose-6-phosphatase and alkaline phosphatase enzyme activities.

Handling information

Unpacking and storage instructions
  1. Check all containers for leakage or breakage.
  2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.
Complete medium
These cells are grown in
  • 50 % L-15 (ATCC 30-2008)
  • 35 % DMEM HG (GIBCO 12100)
  • 15 % Ham's F12 (GIBCO 21700)
All without sodium bicarbonate Supplemented with:
  • 0.15 g/L sodium bicarbonate
  • 15 mM HEPES
  • 0.01mg/ml bovine insulin
  • 50 ng/ml mouse EGF
  • 5% heat-inactivated fetal bovine serum
  • 0.5% Trout Serum
Do not filter complete medium.
Temperature
28°C (26-29°C)
Atmosphere
100% Air
Handling procedure
To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C.  Storage at -70°C will result in loss of viability.

  1. Thaw the vial by gentle agitation in a 28°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.  Thawing should be rapid (approximately 2 minutes).
  2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
  3. It is recommended that the cryoprotective agent be removed immediately. Resuspend content of ampule in 9 mL of complete growth medium containing an additional 5% heat-inactivated FBS.  Centrifuge the cell suspension at approximately 125 x g for 5 to 10 minutes.  Discard the supernatant and resuspend the cell pellet in an appropriate amount of serum-free growth medium.
  4. Transfer the vial contents to an appropriate size vessel.  Incubate the culture at 28°C in a suitable incubator for 30 mins. If using the medium described on this product sheet, the medium formulation was devised for use in a free gas exchange with atmospheric air. A CO2 and air mixture is detrimental to cells when using this medium for cultivation.
  5. Examine to ensure attachment and then add heat-inactivated FBS for a final concentration of 5% and Trout serum for a final concentration of 0.5%.

Subculturing procedure
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
  3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
    Note: To avoid clumping, do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
  4. Add 6.0 to 8.0 mL of complete growth medium containing 10% heat-inactivated FBS and aspirate cells by pipetting gently.
  5. To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximately  125 x g for 5 to 10 minutes.
  6. Discard supernatant and resuspend cells in fresh serum-free growth medium.  Add appropriate aliquots of cell suspension to new culture vessels. 
  7. Place culture vessels in incubators at 28°C for 30 minutes.
  8. Examine to ensure attachment, and then add heat-inactivated FBS for a final concentration of 5% and Trout serum for a final concentration of 0.5%.

Subcultivation Ratio: 1:2 to 1:3
Medium Renewal: Every 2 to 3 days.
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells: a Manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.

Quality control specifications

Mycoplasma contamination
Not detected
Population doubling time
Approximately 72 hrs

History

Deposited as
zebrafish
Depositors
DW Barnes
Year of origin
1992

Legal disclaimers

Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
Warranty

The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

Disclaimers

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.

Permits & Restrictions

Import Permit for the State of Hawaii

If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

References

Curated Citations

Ghosh C, et al. Derivation and characterization of a zebrafish liver cell line. Cell Biol. Toxicol. 10: 167-176, 1994. PubMed: 7994634

Miranda CL, et al. Regulation of cytochrome P450 expression in a novel liver cell line from zebrafish (Brachydanio rerio). Arch. Biochem. Biophys. 305: 320-327, 1993. PubMed: 8373170

Collodi P, et al. Induction of zebrafish (Brachydanio rerio) P450 in vivo and in cell culture. Xenobiotica 24: 487-493, 1994. PubMed: 7975714

Frequently Asked Questions

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