人小脑颗粒细胞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 to be segregated during early
cerebellum formation and produces only granule cells. Cerebellar granule cells (CGC) are the
most abundant neurons of the brain; about 101 billion in man [2]. Their axons run as parallel
fibres along the coronal axis, and the one-dimensional spread of excitation that is expected to
result 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.
HCGC from ScienCell Research Laboratories are isolated from human cerebellum. HCGC are
cryopreserved at primary culture and delivered frozen. Each vial contains >1 x 10^6 cells in 1 ml
volume. HCGC are characterized by immunofluorescent method with antibodies to
neurafilament, MAP2, and beta-tubulin 3. HCGC are negative for HIV-1, HBV, HCV,
mycoplasma, bacteria, yeast and fungi. HCGC are guaranteed to further culture in the conditions
provided by ScienCell Research Laboratories.
人小脑颗粒细胞Recommended Medium
It is recommended to use neuronal medium (NM, Cat. No. 1521) for the culture of human
cerebellar granule cells in vitro.
Product Use
HCGC are for research use only. They are not approved for human or animal use, or for
application in in vitro diagnostic procedures.
Storage
Transfer cells directly and immediately from dry ice to liquid nitrogen upon receiving and keep
the cells in liquid nitrogen until cell culture is needed for experiments.
Shipping
Dry ice.
Reference
[1] Hatten, M. E. (1999) Central nervous system neuronal migration. Annu. Rev. Neurosci. 22, pp. 511–539.
[2] Andersen, B.B., Korbo, L. and Pakkenberg, B. (1992) A quantitative study of the human cerebellum with
unbiased stereological techniques. J. Comp. Neurol., 326:549-560.
[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.
Instruction for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37o
C waterbath
and return them to culture as quickly as possible with minimal handling!
Unpacking
1. For crypreserved cells: If there is dry ice in the package and you are not going to culture
cells right way, place cryovial(s) immediately into liquid nitrogen. If there is no dry ice
left in the package, thaw and culture the cells immediately.
2. For proliferating cells: Spray the culture vessel (flask, plate or slide) with 70% ethanol
for disinfection. Transfer the cells into 37o
C, 5% CO2 incubator and allow equilibrating
for 2 hours. After cells have equilibrated, remove shipping medium from the culture
vessel and replace with fresh medium.
Set up culture after receiving the ordering
人小脑颗粒细胞1. Coat culture vessel with laminin or poly-L-lysine.
Note: It is important that neurons are plated in laminin or poly-L-lysine coated culture
vessels that promote cell attachment and neurites outgrowth (poly-L-lysine coating: coat
flask or plate with poly-L-lysine at 2 μg/ml concentration for one hour and wash the flask
or plate with sterile water three times).
2. Medium preparation: Decontaminate the external surfaces of medium and medium
supplements with 70% ethanol and transfer them to sterile field. Aseptically open each
supplement tube and add them to the basal medium with a pipette. Rinse each tube with
the medium to recover the entire volume.
3. Set up culture: Prepare one T-75 flask for each cryovial. Add the appropriate amount of
medium to the vessel (recommend for 20 ml/T-75 flask) and allow the flask to equilibrate
in 37o
C, 5% CO2 incubator for at least 30 min.
4. Thawing of cells: Place the vial in a 37o
C waterbath, hold and rotate the vial gently until
the contents are completely thawed. Remove the vial from the waterbath immediately,
wipe it dry, and transfer it to a sterile field. Rinse the vial with 70% ethanol, and then
wipe to remove excess. Remove the cap, being careful not to touch the interior threads
with fingers.
5. Using 1 ml eppendorf pipette gently resuspend the cells in the vial and transfer them to
equilibrated culture vessels (a T-75 flask). A high seeding density (>10,000/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 DMSO residue in the culture.
6. Replace the cap or cover, and gently rock the vessel to distribute the cells evenly. Loosen
caps if necessary to permit gas exchange. Return the culture vessels to the incubator.
7. For best result, do not disturb the culture for at least 16 hours after the culture has been
initiated. Change the growth medium the next day to remove the residual DMSO and
unattached cells, then every other day thereafter. A health culture will display normal
neuron morphology, and nonvacuole cytoplasm with multiple processes.
人小脑颗粒细胞Handling human derived products is potentially bioharzadous. Although each cell strain testes negative for HIV,
HBV and HCV DNA, proper precautions mush be taken to avoid inadvertent exposure. 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, W. E., and Polt, S. S. (1988) Guidelines to avoid personal contamination by infective agents in research
laboratories that use human tissues. J Tissue Culture Methods. 11(4).