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Enhanced-Primary-Human-Hepatocytes-\u2013-1-Million

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Enhanced-Primary-Human-Hepatocytes-\u2013-1-Million

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Description	Enhanced Primary Human Hepatocytes are expanded primary cells which retains the physiologically relevant profile and phenotype of primary hepatocytes. These cells are ready-to-use for applications in research and development, such as viral infections, screening, co-cultures, transient transfection, and for metabolism studies. The late expansion cells are tested for CYP induction and inhibition. It is not recommended for further extensive passaging after thawing but can be used for long term culture which is necessary for cell-based assays. Enhanced Primary Human Hepatocytes -1 Million is a lower cell count of our expanded primary hepatocytes offered at abm. We have a higher cell count available as the Enhanced Primary Human Hepatocytes – 5 Million (Cat. T5996).
Procedure Overview
Biosesafety Level	II
Organism	Homo sapiens
Source Organ	Liver
Growth Properties	Adherent
Morphology	Polygonal
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Recommended Seeding Density	10,000 cells/cm²
Applications	For Research Use Only
Propagation	Use of Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. Grow cells in ECM-coated culture vessels with the following conditions. We strongly recommend end users to purchase the Applied Cell Extracellular Matrix (G422) to coat your cell culture vessels. Use the ready-to-use Hepatocyte Thawing Medium (TM102) and the Enhanced Primary Human Hepatocytes Media Kit (TM103) which comes with the Hepatocyte Growth Basal Medium, and Hepatocyte Growth Supplement Mix available from abm . Atmosphere: air: 95%, CO₂: 5%; Temperature: 37.0°C. To make complete Hepatocyte Growth Basal Medium add the entire content of Hepatocyte Growth Supplement Mix into Hepatocyte Growth Basal Medium and mix properly. Addition of the Hepatocyte Growth Supplement Mix may change medium appearance more opaque. To thaw: 1. Pre-warm Hepatocyte Thawing Medium and fully supplemented Hepatocyte Growth Medium to room temperature. 2. Carefully remove cryovial from storage tank. 3. Thaw cells in 37°C water bath until only a small chunk of ice is left. Do not shake the vial, or take it out of the water during thawing, as this will damage the cells. 4. Spray the vial and the tube containing 50 ml of thawing medium with 70% ethanol and transfer to a Biosesafety cabinet. 5. Transfer the now completely thawed cell suspension from the cryovial into 50 ml Hepatocyte Thawing Medium by gently pouring the cells into the medium. 6. Use a 1 ml pipette to transfer 1 ml of the thawing medium back to the cryovial and pour the contents back into the 50 ml tube. Repeat this process twice to completely remove the cells from the cryovial. 7. Pellet the cells by centrifuging at 90×g for 5 min at RT. Important note: Higher g-forces will significantly reduce cell recovery. 8. Aspirate the supernatant without disturbing the pellet. Leave approximately 200-400 μl medium on top of the cells. 9. Gently loosen and re-suspend the cells without adding any extra medium by agitating and rotating the tube. Do not vortex or shake the cells as this will compromise cell survival. 10. Add an appropriate volume of pre-warmed supplemented Hepatocyte Growth Medium to the pellet (~1ml per million cells thawed) and re-suspend the cells. Avoid pipetting the cells up and down. 11. Determine viable cell number by cell count. 12. Dilute hepatocytes in pre-warmed, fully supplemented Hepatocyte Growth Medium and seed at ~10,000 cells/cm² in collagen coated cell culture flasks (e.g. T175) or appropriate cell culture dishes. 13. Incubate the cells at 95% humidity, 37°C and 5% CO2.
Subculturing	To Subculture: 1. Pre-warm PBS, trypsin/EDTA and Hepatocyte Medium to 37°C. 2. Carefully aspirate the culture supernatant. 3. Wash the plate once with ~100 µl PBS/cm². 4. Add ~50 µl/cm² trypsin/EDTA (0.05%/0.02% EDTA). 5. Incubate for 3-4 min at 37°C until most of the cells are rounded up and detached. Avoid incubating the cells for more than to 10 min. 6. Gently tap the cell culture vessel to detach remaining adherent cells. 7. Stop the trypsin activity by adding twice the volume (100 µl/cm²) of supplemented Hepatocyte Growth Medium containing 10% FBS (TM999) or Trypsin Neutralization Solution (Lonza). 8. Rinse the surface with the cell suspension using a pipette. 9. Transfer the complete suspension to a tube and centrifuge at 90xg for 5 min at RT. 10. Aspirate the supernatant without disturbing the pellet. Leave approximately 200-400 μl medium on top of the cells. 11. Gently loosen and re-suspend the cells without adding any extra medium by agitating and rotating the tube. Do not vortex or shake the cells as this will compromise cell survival. 12. Add an appropriate volume of pre-warmed supplemented Hepatocyte Growth Medium to the pellet (~1ml per million cells thawed) and re-suspend the cells. Avoid pipetting the cells up and down. 13. Determine viable cell number by cell count. 14. Dilute hepatocytes in pre-warmed, fully supplemented Hepatocyte Growth Medium and seed at ~10,000 cells/cm² in collagen coated cell culture flasks (e.g. T175) or appropriate cell culture dishes. We strongly recommend end users to purchase the Applied Cell Extracellular Matrix (G422) to coat your cell culture vessels. 15. Incubate the cells at 95% humidity, 37°C and 5% CO2.
Preservation	We do not recommend freezing down the Enhanced Primary Human Hepatocytes.
Quality Control	Each lot has been tested for: >90% plateability Post-thaw viability CMV, HIV, HBV, HCV, and Mycoplasma negative Hepatic markers via immunofluorescence staining: CK8+, AAT+, CK18+, HAS+, and AFP- Basal and inducible CYP activities Glycogen storage via PAS staining
Disclaimer	1. The CoA for this product (provided upon request) verifies the cell-type specific gene expression via RT-PCR only. All test parameters provided in the CoA are conducted using abm's standardized culture system and procedures. The stated values may vary under the end-user's culture conditions. Please verify that the product is suitable for your studies by referencing published papers. All sales are final. 2. We strongly recommend live cell shipments for ease of cell transfer and this option can be requested at the time of ordering. Please note that the end-user will need to evalsuate the feasibility of live cell shipment by taking into account the final destination's temperature variation and its geographical locations. In addition, we thoroughly test our cell lines for freeze-thaw recovery. If frozen cells were received and not recovered in your lab under the exact, specified conditions (using recommended culture vessel, media, additional supplements, and atmospheric conditions), a live cell replacement is possible at a cost (plus shipping). 3. All of abm's cell biology products are for research use ONLY and NOT for therapeutic/diagnostic applications. abm is not liable for any repercussions arising from the use of its cell biology product(s) in therapeutic/diagnostic application(s). Please contact a technical service representative for more information. 4. abm makes no warranties or representations as to the accuracy of the information on this site. Citations from literature and provided for informational purposes only. abm does not warrant that such information has been shown to be accurate.