小鼠颗粒细胞Cell Specification
The development of the cerebellum involves a set of coordinated cell movements and two
separate proliferation zones: the ventricular zone and the external granule cell layer (EGL), a
rhombic-lip-derived progenitor pool [1]. The EGL appears segregated during early cerebellum
formation and produces only granule cells. Cerebellar granule cells (CGC) are the most abundant
neurons of the brain [2]. Their axons run as parallel fibres along the coronal axis, and the onedimensional
spread of excitation that results from this arrangement is a key assumption in
theories of cerebellar function. CGC receive inhibitory synaptic input from Golgi cells, which
are mediated by gamma-aminobutyric acid (GABA). During both in vivo and in vitro
development, CGC depend on the activity of the NMDA glutamate receptor subtype for survival
and full differentiation [3]. Cultured CGC are widely used as a model system for studying
neuronal apoptosis.
MCGC from ScienCell Research Laboratories are isolated from neonatal day 8 C57BL/6 mouse
cerebellum. MCGC are cryopreserved at primary culture and delivered frozen. Each vial contains
>1 x 106 cells in 1 ml volume. MCGC are characterized by immunofluorescence with antibodies
specific to neurafilament, MAP2, and beta-tubulin 3. MCGC are negative for mycoplasma,
bacteria, yeast, and fungi. MCGC are guaranteed to further culture under the conditions provided
by ScienCell Research Laboratories.
小鼠颗粒细胞Recommended Medium
It is recommended to use Neuronal Medium (NM, Cat. #1521) for culturing MCGC in vitro.
Product Use
MCGC are for research use only. They are not approved for human or animal use, or for
application in in vitro diagnostic procedures.
Storage
Upon receiving, directly and immediately transfer the cells from dry ice to liquid nitrogen and
keep the cells in liquid nitrogen until they are needed for experiments.
Shipping
Dry ice.
References
[1] Hatten ME. (1999) “Central nervous system neuronal migration.” Annu. Rev. Neurosci. 22, pp. 511-39.
[2] Andersen BB, Korbo L, Pakkenberg B. (1992) “A quantitative study of the human cerebellum with unbiased
stereological techniques.” J Comp Neurol. 326: 549-60.
[3] Monti B, Marri L, Contestabile A. (2002) “NMDA receptor-dependent CREB activation in survival of cerebellar
granule cells during in vivo and in vitro development.” Eur J Neurosci. 16: 1490-8.
Instructions for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37o
C water bath
and return the cells to culture as quickly as possible with minimal handling!
Initiating the culture:
1. Prepare a poly-L-lysine-coated culture vessel (2 μg/cm2
). It is recommended to use a T-25
flask (alternatively, 3 wells of a 6-well plate or 12 wells of a 24-well plate can be used).
Add 5 ml of sterile water to a T-25 flask and then add 5 μl of poly-L-lysine stock solution
(10 mg/ml, Cat. #0413). Leave the vessel in a 37o
C incubator overnight (or for a
minimum of one hour). Rinse the poly-L-lysine-coated vessel twice with sterile water
prior to use.
Note: It is important that these cells are plated in poly lysine coated culture vessels to
promote cell attachment.
2. Prepare complete medium. Decontaminate the external surfaces of medium bottle and
medium supplement tubes with 70% ethanol and transfer them to a sterile field.
Aseptically transfer supplement to the basal medium with a pipette. Rinse the supplement
tube with medium to recover the entire volume.
3. Add complete medium to the culture vessel. Leave the vessel in the sterile field and
proceed to thaw the cryopreserved cells.
4. Place the frozen vial in a 37o
C water bath. Hold and rotate the vial gently until the
contents completely thaw. Promptly remove the vial from the water bath, wipe it down
with 70% ethanol, and transfer it to the sterile field. Carefully remove the cap without
touching the interior threads.
5. Gently resuspend and dispense the contents of the vial into the poly-L-lysine-coated
culture vessel. A seeding density of ≥100,000 cells/cm2 is recommended.
Note: Dilution and centrifugation of cells after thawing are not recommended since these
actions are more harmful to the cells than the effect of residual DMSO in the culture.
6. Replace the cap or lid of the culture vessel and gently rock the vessel to distribute the
cells evenly. Loosen cap, if necessary, to allow gas exchange.
小鼠颗粒细胞7. Return the culture vessel to the incubator.
8. For best results, do not disturb the culture for at least 16 hours after the culture has been
initiated. Refresh culture medium the next day to remove residual DMSO and unattached
cells, then every other day thereafter.
Maintaining the culture:
1. Refresh supplemented culture medium the next morning after establishing a culture from
cryopreserved cells.
2. Change the medium every two to three days thereafter.
It is not recommended that neurons be subcultured beyond their initial plating.
小鼠颗粒细胞Caution: Handling animal derived products is potentially biohazardous. Always wear gloves
and safety glasses when working these materials. Never mouth pipette. We recommend
following the universal procedures for handling products of human origin as the minimum
precaution against contamination [1].
[1] Grizzle WE, Polt S. (1988) “Guidelines to avoid personal contamination by infective agents in research
laboratories that use human tissues.” J Tissue Cult Methods. 11: 191-9.