C8-B4 (ATCC® CRL-2540)

Organism: Mus musculus, mouse  /  Cell Type: monocyte/macrophage microglia; spontaneously transformed  /  Tissue: brain/cerebellum  / 

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Organism Mus musculus, mouse
Tissue brain/cerebellum
Cell Type monocyte/macrophage microglia; spontaneously transformed
Product Format frozen
Morphology neuronal
Culture Properties adherent
Biosafety Level 1
Age 8 days juvenile
Strain C57BL/6
Storage Conditions liquid nitrogen vapor phase
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Derivation
Clonal permanent cell lines with astrocytic or microglial properties have been established from explant cultures of 8-day postnatal mouse cerebella after in vitro spontaneous transformation.

The cell lines were derived in a multistage process. Slowly proliferating foci with several morphologies appeared 4 months after initiation of the cultures and became progressively enriched by cells with a homogeneous appearance.
Antigen Expression
CD4
Genes Expressed
glutamate
Cellular Products
glutamate
Comments
Sister flasks of lethally irradiated astrocytes, which are known to synthesize the macrophage-microglia growth factor, M-CSF, were used as a substrate for cloning the cells.

The astrocyte type I cloned cell line named C8-D1A is available as ATCC CRL-2541, the astrocyte type II cloned cell line named C8-S is available as ATCC CRL-2535 and the astrocyte type III cloned cell line named C8-D30 is available as ATCC CRL-2534.

The C8-B4 clone expresses classical microglial markers (MAC1, F4/80, 2-4G2) and appears to be derived from a committed microglial precursor since it does not express differentiation antigens present during the early stage of the monocytic lineage.

C8-B4 cells synthesize the CD4 molecule and produce and release large amounts of glutamate.


Complete Growth Medium The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.
Subculturing Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
Note: Cells do not become confluent.

  1. Remove and discard culture medium. Keep floating cells, if they are numerous and if they are viable.
  2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53% (w/v) 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 and aspirate cells by gently pipetting.
  5. Transfer cell suspension to a centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes. Discard supernatant.
  6. Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of cell suspension to new culture vessels
  7. Incubate cultures at 37°C.

Subculture Ratio: 1:3 to 1:4
Medium Renewal: 2 to 3 times a week.
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.

Cryopreservation
Freeze medium: Complete growth medium 95%; DMSO, 5%
Storage temperature: liquid nitrogen vapor phase
Culture Conditions
Temperature: 37°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Population Doubling Time 48 to 72 hrs
Name of Depositor B Pessac, D Trisler
References

Alliot F, Pessac B. Astrocytic cell clones derived from established cultures of 8-day postnatal mouse cerebella. Brain Res. 306: 283-291, 1984. PubMed: 6466977

Alliot F, et al. A spontaneously immortalized mouse microglial cell line expressing CD4. Brain Res. Dev. Brain Res. 95: 140-143, 1996. PubMed: 8873987

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation
Other Documentation
References

Alliot F, Pessac B. Astrocytic cell clones derived from established cultures of 8-day postnatal mouse cerebella. Brain Res. 306: 283-291, 1984. PubMed: 6466977

Alliot F, et al. A spontaneously immortalized mouse microglial cell line expressing CD4. Brain Res. Dev. Brain Res. 95: 140-143, 1996. PubMed: 8873987