Conditionally Immortalized Human Glomerular Podocytes (Fabry Patient, CI-HGlPod Cells-F)
|Name of Products
|Conditionally Immortalized Human Glomerular Podocytes (Fabry Patient, CI-HGlPod Cells-F)
|GENERAL INFORMATION: Fabary Patient glomerular Podocytes were initially collected from the urine. Conditionally immortalized Fabry Patient Glomerular Podocytes (CI-HGlPod Cells-F) are selected by puromycin after primary Fabry patient podocytes are infected with the lentiviral particles expressing SV40 under the control of CMV promoter with the Ten-on system. Podocyte Growth Medium (UBP-47) is recommended for cell culture and these cells have an average minimum population doubling levels > 50, when cultured following the detailed protocol described below).
|CHARACTERIZATION OF THE CELLS
|CD2AP, and Nephrin Positive
|HGlPod Cells-F are tested negative for HIV-1, HBV, HCV, and mycoplasma.
|PRODUCT USAGE: The cells are offered for Research Use Only.
|SHIPPING: Frozen Vials in a Dry Ice Package.
|HANDLING OF ARRIVING CELLS: When you receive the cells in a frozen vial, you can transfer the vial of cells into a -80C freezer for short period storage or a liquid nitrogen tank for long term storage. Thaw the cells in a 37C water bath, and then transfer the cells into a T25 flask pre-coated with Universal Coating Solution (UBP-01) as described in detail in Subculture Protocol, in 9 ml Human Podocytes Growth Medium (UBP-47). (Make sure to add 2.0ug/ml of Doxycycline, UBP-45).
|PROTOCOLS FOR THAWING THE CELLS AND SUBCULTURE
|Pre-coating of T25 flasks- Add 2ml each Universal Coating Solution (UBP-01) into a T25 flask to cover the whole surface of the flask, 5 mins later, dispose the excessive coating solution by aspiration and the flask is ready to be used (although solution containing other extracellular matrix, i.e. gelatin, collagen, and fibronectin, can be used, make sure to optimize the conditions in advance).
|Thaw the frozen cell vial in a 37C water bath first, and then transfer the cells into the pre-coated T25 flask with 10ml of UBP-47 medium, cells usually become confluent with 5-7 days.
|To passage the cells, rinse the cells in a T25 flask with 5ml HBSS (RT) twice; then add 2ml Universal Detachment Solution (RT) (UBP-23) into one T25 flask; gently dispose the excessive Universal Detachment Solution within 20 seconds by aspiration.
|Leave the T25 flask with the cells at RT or 37C for 1 min (most cells usually will detach from the surface within 1-2 mins; or monitor the cells under a microscope until most of cells become rounded up, and then gently tap the flask against the bench surface, and the cells will move on the surface of the flask when monitoring under microscope.
|Add 5ml Trypsin Neutralization Buffer and spin down the cells with 800g centrifugation for 5 mins.
|Re-suspend the cell pellet with 10 or 15ml Full medium and transfer 5 ml each into 2 or 3 pre-coated T25 flasks (for 1/2 to 1/3 subculture ratio). (Make sure to add 2.0ug/ml of Doxycycline).
|Change medium every 2 or 3days and the cells usually become confluent within 7 days (when split at a 1/3 ratio).
|The cells conditionally immortalized are proliferative better if the cells are maintained at 33-35C rather than 37C
|To induce cellular quiescence, withdrawing DOX and maintain the cells at 37C for 3-5 days.
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