😊 Product Use
Product Type: Catalog Number:   P10251-IM Immortalized Human Astrocytes Immortalization: Number of cells: SV40 Large T Antigen > 1 x106 cells in Cryopreserved vials Storage: Liquid Nitrogen 😊 Recommended Medium P10251-IM have been obtained immortalizing Human Cortical Astrocytes with Lenti-SV40 Lentivirus. Immortalized cells were controlled passaging side by side with the primary cells. Primary cells go into senescence after the 5th passage while the SV40-tranduced cells go beyond 30 passages. Astrocyte  Medium (Reference: P60101) Astrocytes make up the majority of the cells in the mammalian brain. They are the most variable in type, most intimately associated with all parts of neurons, and thus most functionally interesting in their relationships neurons. They provide structural, with trophic, and metabolic support to neurons and modulate synaptic activity. Astrocytes have been implicated in the pathological processes of many neurological diseases. Long-term recovery after brain injury, through neurite outgrowth, synaptic plasticity, or neuron regeneration, is influenced by astrocyte surface molecule expression and trophic factor release. 😊 Product Characterization Immunofluorescent method o GFAP The cells test negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast and fungi these products are for research use ONLY. Not approved for human or veterinary use, for application to humans or animals, or for use in vitro diagnostic or clinical procedures INNOVATIVE TECHNOLOGIES IN BIOLOGICAL SYSTEMS, S Parque Tecnológico Bizkaia, Edf. 502, 1ª Planta | 48160 | Derio | Bizk Tel.: +34 944005355 | Fax: +34 946579925 innoprot@innoprot.com | www.innoprot.com
IMPORTANT: Cryopreserved cells are very delicate. Thaw the vial in a 37 °C 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). Add 10 ml of sterile water to a T-75 flask and then add 150 μl of poly-L- lysine stock solution (1 mg/ml). Leave the flask in incubator overnight (minimum  one  hour at 37°C incubator). 2. complete Prepare 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 37°C 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-L-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 residual      DMSO remove and unattached cells, then every other day thereafter.
Maintenance of Culture:
1. Refresh supplemented culture medium the next morning after establishing from cryopreserved cells.  2. Change the medium every three days thereafter, until the culture is approximately  70% confluent. 3・ Once culture  reaches the 70% confluency, change medium every other day  until the culture is approximately 90% confluent.
Subculture:
1. Subculture when the culture reaches 90% confluency or above. 2. Prepare poly-L-lysine-coated culture vessels (2 μg/cm²) one day before subculture. 3. Warm complete medium, trypsin/EDTA solution (T/E Solution), T/E neutralization solution (TNS), and DPBS (Ca**- and Mg** -free) to room temperature. We do not recommend warming reagents and medium in a 37°C water bath prior to use. Note: DPBS, trypsin/EDTA solution & trypsin neutralization solution are included in the “Primary Cells Detach Kit provided by Innoprot (Cat. N° P60305). 4. Rinse the cells with DPBS. Add 8 ml of DPBS and then 2 ml of T/E 5. 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 370C 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 370C 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 flask under the a microscope for a successful cell harvest by looking at the number of cells being left behind; there should be less than 5%. 11. Centrifuge the 50 ml centrifuge tube 1000 rpm for 5 minutes. at 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 human derived products is potentially bioharzadous. Although each cell strain testes negative for HIV, HBV and HCV DNA, diagnostic tests are not necessarily 100% 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). innoprot@innoprot.com | www.innoprot.com