小鼠肾系膜细胞Cell Specification
Astrocytes are the major cell type in the mammalian brain. They provide a variety of supportive
functions to their partner neurons in the central nervous system (CNS), such as neuronal
guidance during development, nutritional and metabolic support throughout life [1]. Astrocytes
have also been implicated in various pathological processes [2]. Impairment of normal astrocyte
functions during stroke and other insults can critically influence neuron survival. Long-term
recovery after brain injury, through neurite outgrowth, synaptic plasticity, or neuron
regeneration, is also influenced by astrocyte surface molecule expression and trophic factor
release [3]. Numerous studies have demonstrated that astrocytes are among the most functionally
diverse group of cells in the CNS [4]. Much of what we have learned about astrocytes is from in
vitro studies and astrocyte culture is a useful tool for exploring the diverse properties of this cell
type.
MA from ScienCell Research Laboratories are isolated from postnatal day 2 C57BL/6 mouse
brain. MA are cryopreserved as primary cultures and delivered frozen. Each vial contains >5 x
105
cells in 1 ml volume. MA are characterized by immunofluorescence with antibody specific to
GFAP. MA are negative for mycoplasma, bacteria, yeast, and fungi. MA are guaranteed to
小鼠肾系膜细胞further expand for 5 population doublings under the conditions provided by ScienCell Research
Laboratories.
Recommended Medium
It is recommended to use Astrocyte Medium-animal (AM-a, Cat. #1831) for culturing MA in
vitro.
Product Use
MA 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
小鼠肾系膜细胞Dry ice.
References
[1] Rudge JS. (1993) “Astrocyte-derived neurotrophic factors.” In Murphy S, Astrocytes: harmacology and
unction (pp 267-94). San Diego: Academic Press, Inc.
[2] van der Laan LJ, De Groot CJ, Elices MJ, Dijkstra CD. (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: 539-48.
[3] Chen Y, Swanson RA. (2003) “Astrocytes and brain injury.” J Cereb Blood Flow Metab. 23: 137-49.
[4] Shao Y, McCarhy KD. (1994) “Plasticity of astrocytes.” Glia. 11: 147-55.
Instructions for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37oC 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
, 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, Cat. #0413). Leave the vessel in a 37oC incubator overnight (or for a
minimum of one hour).
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. Rinse the poly-L-lysine-coated vessel twice with sterile water and then add 15 ml of
complete medium. Leave the vessel in the sterile field and proceed to thaw the
cryopreserved cells.
4. Place the frozen vial in a 37oC 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.
5. Carefully remove the cap without touching the interior threads. Gently resuspend and
dispense the contents of the vial into the equilibrated, poly-L-lysine-coated culture vessel.
A seeding density of 5,000 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. It is
also important that cells are plated in poly lysine coated culture vessels to promote cell
attachment.
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.
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 three days thereafter, until the culture is approximately 70%
confluent.
3. Once the culture reaches 70% confluency, change medium every other day until the
culture is approximately 90% confluent.
Subculturing:
1. Subculture when the culture reaches 90% confluency or above.
小鼠肾系膜细胞2. Prepare poly-L-lysine-coated culture vessels (2 μg/cm2
) one day before subculture.
3. Warm complete medium, trypsin/EDTA solution (T/E, Cat. #0103), T/E neutralization
solution (TNS, Cat. #0113), and DPBS (Ca++
- and Mg++
-free, Cat. #0303) to room
temperature. We do not recommend warming reagents and medium in a 37oC water bath
prior to use.
4. Rinse the cells with DPBS.
5. Add 8 ml of DPBS and then 2 ml of T/E solution into flask (in the case of a T-75 flask).
Gently rock the flask to ensure complete coverage of cells by T/E solution. Incubate the
flask in a 37oC incubator for 1 to 2 minutes or until cells completely round up. Use a
microscope to monitor the change in cell morphology.
6. During incubation, prepare a 50 ml conical centrifuge tube with 5 ml of fetal bovine
serum (FBS, Cat. #0500).
7. Transfer T/E solution from the flask to the 50 ml centrifuge tube (a small percent of cells
may detach) and continue to incubate the flask at 37oC for another 1 to 2 minutes (no
solution in the flask at this moment).
8. At the end of incubation, gently tap the side of the flask to dislodge cells from the
surface. Check under a microscope to make sure that all cells detach.
9. Add 5 ml of TNS solution to the flask and transfer detached cells to the 50 ml centrifuge
tube. Rinse the flask with another 5 ml of TNS to collect the residual cells.
10. Examine the flask under a microscope for a successful cell harvest by looking at the
number of cells being left behind; there should be less than 5%.
Note: Use ScienCell T/E solution that is optimized to minimize cell damages due to over
trypsinization.
小鼠肾系膜细胞11. Centrifuge the 50 ml centrifuge tube at 1000 rpm for 5 minutes. Resuspend cells in
culture medium.
12. Count and plate cells in a new poly-L-lysine-coated culture vessel with the recommended
cell density.
Caution: Handling animal derived products is potentially biohazardous. Always wear gloves
and safety glasses when working with 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.