大鼠小脑星形胶质细胞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 astrocytes
culture is continually providing a useful tool in exploring the diverse property of astrocytes.
RA rom ScienCell Research Laboratories are isolated from day 8 rat cerebellum. RA are
cryopreserved at secondary culture and delivered frozen. Each vial contains >5 x 10^5 cells in 1
ml volume. RA are characterized by immunofluorescent method with antibody to GFAP. RA
are negative for mycoplasma, bacteria, yeast and fungi. RA are guaranteed to further expand
for 5 population doublings in the condition provided by ScienCell Research Laboratories.
Recommended Medium
大鼠小脑星形胶质细胞It is recommended to use strocyte edium animal (AM , Cat. No. 183 for the culturing of
RA in vitro.
Product Use
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 37
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 lysine coated flask (2 μg/cm , 75 flask is recommended). Add 10 ml
of sterile water to a T 75 flask and then add 15 μl of poly lysine stock solution (10
mg/ml, ScienCell cat. no. 0413). Leave the flask in incubator overnight (minimum one
hour at 37
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 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 37
5. Dispense the contents of the vial into the equilibrated, poly 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 re
suspend the contents of the vial.
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 cells 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.
大鼠小脑星形胶质细胞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 lysine coated cell culture flasks (2 μg/cm
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 37
4. Rinse the cells with DPBS.
C
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
75 flask); gently rock the flask to make sure cells are covered by trypsin/EDTA
solution; incubate the flask at 37 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 37
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 lysine coated flask with cell density as
recommended.
大鼠小脑星形胶质细胞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 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).