BT142 mut/- (ATCC^® ACS-1018^™)

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.

There are two options for the base medium for this cell line.

Option 1: NeuroCult NS-A Proliferation kit (Catalog No. 5751, Stem Cell Technologies)

Option 2: 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 2 (bFGF, Catalog No. ACS-3025, ATCC)
• 2 µg/mL heparan sulfate (Catalog No. H9902, Sigma)

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.

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

1. ATCC ACS-1018 BT142 Homozygous seeding density
   The ATCC® ACS-1018 ™ BT142 Homozygous cells grow slowly.  If you split the cells at a very low seeding density, they may take longer than the usual 2 to 3 weeks before the cells ar...
   Date Updated: 3/29/2013
2. ACS-1018 BT142 Homozygous neurosphere diameter
   It is important to prevent the ATCC® ACS-1018 ™ BT142 Homozygous neurospheres from growing too large.  This can lead to necrosis as a result of a lack of oxygen and nutrient exchan...
   Date Updated: 3/29/2013
3. ACS-1018 BT142 Homozygous neurosphere passaging
   We recommend mechanically dissociating the ATCC® ACS-1018 ™ BT142 Homozygous neurospheres into a single cell suspension. Perform a cell count and dilute the cells to an appropriate...
   Date Updated: 3/29/2013
4. ACS-1018 BT142 Homozygous neurospheres are dark and ragged
   Dark, ragged neurospheres are a sign of overgrown, unhealthy or dying spheres. If your neurospheres are dark and ragged, we recommend passaging the neurospheres right away.
   Date Updated: 3/29/2013
5. ACS-1018 BT142 Homozygous recommended seeding density
   

   The ATCC® ACS-1018 ™ BT142 Homozygous cells should be seeded at a cell density of 8 x 10³ to 2 x 10⁴ single cells/cm².

   
   Date Updated: 3/29/2013
6. Significance of IDH1 mutation in BT142 cells
   Mutations in IDH1 are found in approximately 80% of grade II-III gliomas and secondary glioblastomas in humans. A single amino acid residue of IDH1, arginine 132, is most commonly mutated to...
   Date Updated: 3/29/2013
7. BT142 Homozygous neurospheres stick to plastic
   The ATCC® ACS-1018 ™ BT142 Homozygous neurospheres may stick to plastic disposable tips and some plastic tubes.  To prevent cell loss, we recommend that you pre-wet the pipett...
   Date Updated: 3/29/2013
8. Changing the medium for BT142 Homozygous cells
   The medium for the ATCC® ACS-1018 ™ BT142 Homozygous cells should be changed prior to becoming acidic (orange/yellow in color).  It is important to monitor the culture regular...
   Date Updated: 3/29/2013
9. When to change media for BT142 Homozygous cells
   If the ATCC® ACS-1018 ™ BT142 Homozygous cell neurospheres have not reached the appropriate size range, but the medium has become acidic, we recommend that you change half of the m...
   Date Updated: 3/29/2013
10. Difference between BT142 Homozygous and original BT142 cells
    ATCC® ACS-1018 ™ BT142 mut/- (Homozygous) cells have lost the wild-type IDH1 allele but retains the mutant allele.  The loss of the wild-type allele has been reported
    Date Updated: 3/29/2013

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