小鼠海马趾星形胶质细胞Cell Specification
Astrocytes are the majority cell type of the mammalian brain. Astrocytes have been implicated in
a variety of supportive functions for their partner neurons in the CNS, such as neuronal guidance
during development, and nutritional and metabolic support throughout life [1]. The functions of
astroyctes are also complicated during pathological processes [2]. Numerous studies have
demonstrated that astrocytes are among the most functionally diverse group of cells in the CNS
[3]. Much of what we have learned about astrocytes is from in vitro studies and astrocyte culture
is continually providing a useful tool in exploring the diverse property of astrocytes.
MA-h from ScienCell Research Laboratories are isolated from neonate day two mouse brain
(cerebral cortex). MA-h are cryopreserved at primary culture and delivered frozen. Each vial
contains >5 x 10^5 cells in 1 ml volume. MA-h are characterized by immunofluorescent method
with antibody to GFAP. MA-h are negative for mycoplasma, bacteria, yeast and fungi. MA-h are
guaranteed to further expand for 5 population doublings in the condition provided by ScienCell
Research Laboratories.
小鼠海马趾星形胶质细胞Recommended Medium
It is recommended to use Astrocyte Medium-animal (AM-a, Cat. No. 1831) for the culturing of
MA-h in vitro.
Product Use
MA-h 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] Astrocytes, pharmacology and function. Edited by Sean Murphy. 1993 by Academic press, Inc.
[2] Van der Laan, L. J. W., De Groot, C. J. A., Elices, M. J. and Dijkstran, C. D. (1997) Extracellular matrix proteins
expressed by human adult astrocytes in vivo and in vitro: an astrocyte surface protein containing the CS1 domain
contributes to binding of lymphoblasts. J. Neurosci. Res. 50:539-548.
[3] Shao, Y. and McCarhy, K. D. (1994) Plasticity of astrocytes. Glia 11:147-155.
Instruction for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37o
and return them to culture as quickly as possible with minimal handling!
C waterbath
Set up culture after receiving the order:
1. Prepare a poly-L-lysine coated flask (2 μg/cm2
, T-75 flask is recommended). Add 10 ml
of sterile water to a T-75 flask and then add 15 μl of poly-L-lysine stock solution (10
mg/ml, ScienCell cat. no. 0413). Leave the flask in incubator overnight (minimum one
hour at 37o
2. Prepare complete medium: 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
medium to recover the entire volume.
C incubator).
3. Rinse the poly-L-lysine coated flask with sterile water twice and add 20 ml of complete
medium to the flask. Leave the flask in the hood and go to thaw the cells.
4. Place the vial in a 37o
5. Dispense the contents of the vial into the equilibrated, poly-L-lysine coated culture
vessels. A seeding density of 5,000 cells/cm
C waterbath, hold and rotate the vial gently until the contents are
completely thawed. Remove the vial from the waterbath immediately, wipe it dry, rinse
the vial with 70% ethanol and transfer it to a sterile field. Remove the cap, being careful
not to touch the interior threads with fingers. Using a 1 ml eppendorf pipette gently resuspend
the contents of the vial.
2
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. It is
also important that the c lls are plated in poly lysine coated flask that promotes cell
attachment and growth
is recommended.
6. Replace the cap or cover, and gently rock the vessel to distribute the cells evenly. Loosen
cap if necessary to permit gas exchange.
7. Return the culture vessels to the incubator.
8. 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 polygonal
shaped, sheets of contiguous cells and the cell number will be double after two to three
days in culture.
Maintenance of Culture:
1. Change the medium to fresh supplemented medium the next morning after establishing a
culture from cryopreserved cells.
2. Change the medium every three days thereafter, until the culture is approximately 70%
confluent.
3. Once the culture reaches 70% confluence, change medium every other day until the
culture is approximately 90% confluent.
Subculture:
1. Subculture the cells when they are over 90% confluent.
2. Prepare poly-L-lysine coated cell culture flasks (2 μg/cm2
3. Warm medium, trypsin/EDTA solution (T/E, cat. no. 0103), trypsin neutralization
solution (TNS, cat. no. 0113), and DPBS (Ca
).
++ and Mg++ free, cat. no. 0303) to room
temperature. We do not recommend warming the reagents and medium at 37o
4. Rinse the cells with DPBS.
C
小鼠海马趾星形胶质细胞waterbath prior to use.
5. Add 8 ml of DPBS first and then 2 ml of trypsin/EDTA solution into flask (in the case of
T-75 flask); gently rock the flask to make sure cells are covered by trypsin/EDTA
solution; incubate the flask at 37o
C incubator for 2 minutes or until cells are completely
rounded up (monitored with inverted microscope). During incubation, prepare a 50 ml
conical centrifuge tube with 5 ml of fetal bovine serum (FBS, cat. no. 0500); transfer
trypsin/EDTA solution from the flask to the 50 ml centrifuge tube (a few percent of cells
may detached); continue incubate the flask at 37o
Note: Use ScienCell Research Laboratories’ trypsin/EDTA solution that is optimized to
minimize the killing of the cells by over trypsinization.
C for 1 or 2 minutes more (no solution
in the flask at this moment); at the end of trypsinization, one hand hold one side of flask
and the other hand gently tap the other side of the flask to detach cells from attachment;
check the flask under inverted microscope to make sure all cells are detached, add 5 ml of
trypsin neutralization solution to the flask and transfer detached cells to the 50 ml
centrifuge tube; add another 5 ml of TNS to harvest the residue cells and transfer it to the
50 ml centrifuge tube. Examine the flask under inverted microscope to make sure the cell
harvesting is successful by looking at the number of cells left behind. There should be
less than 5%.
6. Centrifuge the 50 ml centrifuge tube (harvested cell suspension) at 1000 rpm (Beckman
Coulter Allegra 6R centrifuge or similar) for 5 min; re-suspend cells in growth medium.
7. Count cells and plate cells in a new, poly-L-lysine coated flask with cell density as
recommended.
小鼠海马趾星形胶质细胞Caution: Handling animal derived products is potentially biohaza dous. Always wear gloves and safety glasses
when working these materials. Never mouth pipette. We recommend following the universal procedures for handling
products of animal 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).