|Species||Rat (R. norvegicus)|
|Caution||For Research Use Only, not for therapeutic or diagnostic purposes.|
|Cell Type||Ready-to-Use Stem Cells|
|Propagation Requirements||The optimized medium for this cell line is Rat iPSC Culture Medium available from ABM (TM021). This cell line needs to be cultured on inactivated REF feeder cells (T2017). Atmosphere:air: 95%, CO2: 5%; Temperature: 37.0°C.|
|Subculture Protocol||1. Remove and discard culture medium. 2. Add 2.0mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed. Note: Cells that are difficult to detach may be placed at 37°C to facilitate dispersal. 3. Centrifuge cells at 1500rpm for 3 minutes to pellet. 4. Aspirate out trypsin, leaving pellet undisturbed. 5. Resuspend pellet in fresh culture medium and plate in new feeder cell culture vessel. 6. Incubate cultures at 37°C. Be sure to use inactivated REF feeder cells (T2017).|
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Can you confirm if these cells have been generated with lentiviral or episomal vectors?
The cells have been generated with lentiviral vectors. There is no fluorescent reporter expressed.
What genes were used for the iPSC generation?
Lentiviral vectors expressing the 4 Yamanaka factors were used.
What is the protocol for freezing iPSCs?
1) 4-5 days after cell split, when iPSCs reach a confluency of 30-60%, aspirate out spend medium and add 1ml 0.5mM EDTA/PBS. Incubate cells at 37C for ~5min. 2) When colonies start to detach, gently harvest all the cells into a 5-ml or 15-ml or 50-ml tube by pippeting 1-2 times. Avoid multiple pippetting. Breaking down clumps into single cells may substantially decrease cell survival. May add iPSC medium to harvest all the cells. 3) Spin down at 200g for 2-3 minutes. 4) Carefully aspirate out supernatent and gently resuspend cells in 0.5ml iPSC medium. 5) Add 0.5ml freezing medium (TM023) and ROCK inhibitor. Mix well by flipping the vial and transfer the medium to a cryovial. 6) Finally transfer the vial to -80C freezer for short-term storage (days to weeks). After cells are frozen, you may then transfer the vial to liquid nitrogen tank for long-term storage (years).
What is the recommended seeding density for iPSC?
IPSCs require seeder cells before thawing and plating the IPSC. Seed ~3×10^5 feeder cells in each well of a 6-well plate. After thawing the iPSCs, put all the cells from the vial into 1 well. After several days, you may start splitting the iPSCs into multiple wells depending on how many cells survived. The density mentioned above is for the seeder cells which should be at 3×10^5. Afterwards, please plate the entire IPSC into one well at 10^6 concentration.
Which rat strain is this derived from?
Which rat strain is this derived from?