大鼠星形膠質細胞

【資料簡介】

                   大鼠星形膠質細胞

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 process [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 from ScienCell Research Laboratories are isolated from day 2 rat cerebral cortex. 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 ten population doublings in the condition provided by ScienCell Research Laboratories. 
Recommended Medium 
It is recommended to use Astrocyte Medium (AM, Cat. No. 1801) for the culturing of rat astrocyte in vitro. 
Product Use 
RA 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 immediay 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.
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37oC waterbath 
and return them to culture as quickly as possible with minimal handling! 
Set up culture after receiving the order: 
1. Prepare a poly-L-lysine coated flask (2 μg/cm2, T-75 flask is recommended) and leave the flask in incubator overnight (minimum one hour at 37oC incubator). 
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. 
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 37oC waterbath, hold and rotate the vial gently until the contents are compley thawed. Remove the vial from the waterbath immediay, 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 1 ml eppendorf pipette gently resuspend the contents of the vial. 
5. Dispense the contents of the vial into the equilibrated, poly-L-lysine coated culture vessels. 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 DMSO residue in the culture. It is also important that astrocytes are plated in poly-L-lysine coated culture vessels that promote cell attachment. 
6. Replace the cap or cover of flask, and gently rock the vessel to distribute the cells evenly. Loosen caps 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 healthy culture will display slar morphology, nongranular cytoplasm, and the cell number will be doubled 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. For subsequent subcultures, change medium 48 hours after establishing the subculture. 
2. Change the medium every other day thereafter, until the culture is approximay 50% confluent. 
3. Once the culture reaches 50% confluence, change medium every day until the culture is approximay 80% 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, trypsin neutralization solution, and DPBS to room temperature. We do not recommend warming the reagents and medium at 37oC waterbath prior to use. 
4. Rinse the cells with DPBS. 
5. Incubate cells with 10 ml of trypsin/EDTA solution (in the case of T-75 flask) until 80% of cells are rounded up (monitored with microscope). Add 10 ml of trypsin neutralization solution to the digestion immediay and gently rock the culture vessel. 
Note: Use ScienCell Research Laboratories’ trypsin/EDTA solution that is optimized to minimize the killing of the cells by over trypsinization. 
6. Transfer the released cells into a 50 ml centrifuge tube. Rinse the flask with another 10 ml of growth medium to collect the residue cells. Examine the flask under microscope to make sure the harvesting is successful by looking at the number of cells left behind. There should be less than 5%. 
7. Centrifuge the harvested cell suspension at 1000 rpm for 5 min and resuspend cells in growth medium. 
8. Count cells and plate them in a new, poly-L-lysine coated flask with cell density as recommended. 

Caution: Handlinghuman derived products is potentially bioharzadous. Although each cell strain testes negative for HIV, HBV and HCV DNA, diagnostic tests are not necessarily 99% accurate, therefore, 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).