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BT142 mut/-

ACS-1018

Product category
Human cells
Organism
Homo sapiens, human
Cell type
neural cell
Morphology
Neurosphere
Tissue
Brain
Disease
Oligoastrocytoma; Grade III
Applications
Cancer research
Neuroscience
Stem cell research
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.

DMEM: F-12 Medium

30-2006

Price: $24.00 ea

L-Glutamine Solution, 200 mM

30-2214

Price: $19.00 ea

Detailed product information

Characteristics

Growth properties
Suspension
Age
38 years
Ethnicity
White
Gender
Male
Comments

Point mutations in isocitrate dehydrogenase I (IDH1) and IDH2 are found in majority of grade II and III gliomas. R132H is the most common IDH1 substitution found in gliomas.

BT142 mut/-  contains a homozygous IDH1 R132H mutation, which originated from a heterozygous IDH1 R132H BT142 cells.

The cells grow as phase-bright, smooth spheres.

The neurospheres should not get too big, ragged or dark as this is a sign of unhealthy, dying cells.

The cells should be passaged when the neurospheres are 200 to 400 µm in size.

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
Media: NeuroCult NS-A Proliferation kit (Catalog No. 5751, Stem Cell Technologies)

Alternate Media: DMEM/F12 (1:1) (Catalog No. 30-2006, ATCC) with an additional 0.9% glucose, 4 mM L-glutamine (Catalog No. 30-2214, ATCC), 25 µg/mL insulin, 100 µg/mL transferrin, 20 nM progesterone, 15 µM putrescine and 30 nM selenite

To make the complete growth medium, add the following supplements to either options of the base medium (see above):
  • 20ng/mL recombinant human Epidermal Growth Factor (EGF, Catalog No. 100-15, PeproTech)
  • 100 ng/mL recombinant human Platelet-Derived Growth Factor-AA (PDGF-AA, Catalog No. 100-13A, PeproTech)
  • 20 ng/mL recombinant human Fibroblast Growth Factor (R&D Systems, Catalog No. 233-FB) 
  • 2 µg/mL heparan sulfate (Catalog No. H3149, Sigma)
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 –80°C. Storage at –80°C will result in loss of viability.

  1. The cells are cryopreserved as neurospheres and should be thawed as clusters.  Do not break apart the neurospheres into a single-cell suspension.
  2. Quickly thaw the vial by gentle agitation in a 37 °C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.
  3. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
  4. Transfer the contents of the vial to a centrifuge tube containing 10 mL of complete culture medium.
  5. Centrifuge the cells at 200 x g for 10 minutes.
  6. Aspirate supernatant and resuspend the cells in 15 mL of complete culture medium and dispense into a 75 cm2 flask.
  7. The cells may take a few weeks to recover from cryopreservation. Viable neurospheres are semi-transparent and phase contrast bright with smooth outer surfaces.

 

Subculturing procedure

The cells grow as phase-bright, smooth spheres. The neurospheres should not get too big, ragged or dark as this is a sign of unhealthy, dying cells. The cells should be passaged when the neurospheres are about 200-400 μm in size.

Volumes used in this protocol are for 75 cm2 flask.

  1. Harvest and collect the entire cell suspension from the culture flask into a 15 mL tube.
  2. Centrifuge at 200 x g for 10 minutes.
  3. Aspirate supernatant, leaving approximately 200 µL to cover the pellet.
  4. Add 1 mL of complete culture medium.
  5. Triturate cells with a P1000 micropipette set to 800 µL by pipetting up and down 40 times or until the cells appear to be in a single cell suspension.
  6. Add 8 mL of complete culture medium and centrifuge at 200 x g for 10 minutes.
  7. Aspirate supernatant and resuspend the cells in 2 mL of complete culture medium.
  8. Count viable cells using trypan blue exclusion assay on a hemacytometer.
  9. Seed single cells between ranges of 8 x 103 to 2 x 104 cells/cm2.

Note: If accurate cell count is necessary, Accumax (Catalog No. AM105, Innovative Cell Technologies) can be used; however, the cells may take some time to recover from an enzymatic dissociation.

 

 

Culture maintenance
Replace medium once per week, or as required if media looks depleted, by replacing 5 mL with fresh complete culture medium (volume for 25 cm2 flask).
Reagents for cryopreservation
Complete growth medium supplemented with 10% (v/v) DMSO (ATCC 4-X)

History

Depositors
This cell line was deposited by Samuel Weiss, Ph.D. and Gregory Cairncross, Ph.D. of the University of Calgary

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

Luchman HA et al. An in vivo patient-derived model of endogenous IDH1-mutant glioma. Neuro Oncol. 14:184-191, 2012. PubMed: 22166263

Frequently Asked Questions

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