Eccles:HuADPKD Cell Culture: Difference between revisions
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==Background== | ==Background== | ||
These cells were made from ~0.5g chunks of kidney taken from a male with ADPKD.<br> | These cells were made from ~0.5g chunks of kidney taken from a male with ADPKD.<br> | ||
Refer to ([http://www.openwetware.org/wiki/Eccles: Making_Primary_Cells_From_Surgery_Tissue Making Primary Cells from Surgery Tissue]) | Refer to ([http://www.openwetware.org/wiki/Eccles:Making_Primary_Cells_From_Surgery_Tissue Making Primary Cells from Surgery Tissue]) | ||
There were 9 different tissue samples in total. Which part of the kidney the tissue was taken from is unknown as the kidney was so riddled with cyts and fibrotic tissue that the original kidney anatomy was unrecognisable.<br> | There were 9 different tissue samples in total. Which part of the kidney the tissue was taken from is unknown as the kidney was so riddled with cyts and fibrotic tissue that the original kidney anatomy was unrecognisable.<br> | ||
Six samples successfully grew in culture. Samples are identified as "sample 1" etc.<br> | Six samples successfully grew in culture. Samples are identified as "sample 1" etc.<br> | ||
Revision as of 17:12, 4 October 2007
Background
These cells were made from ~0.5g chunks of kidney taken from a male with ADPKD.
Refer to (Making Primary Cells from Surgery Tissue)
There were 9 different tissue samples in total. Which part of the kidney the tissue was taken from is unknown as the kidney was so riddled with cyts and fibrotic tissue that the original kidney anatomy was unrecognisable.
Six samples successfully grew in culture. Samples are identified as "sample 1" etc.
Grow Cells on Collagen Coated Surfaces
Refer to (Collagen Coating of Cell Culture Vessels) for protocol.
Cell Culture Media used for human ADPKD kidney cells (with additives)
| Reagent | 50mL | 500mL | Final Concentration |
|---|---|---|---|
| DMEM/F-12 (1:1) | 46.85mL | ||
| insulin-transferrin-selenium (100x) | 375uL | 3.75mL | 0.75x |
| dexamethasone (20ug/mL) | 250uL | 2.5mL | 100ng/mL |
| triiodotyronine (20ug/mL) | 15uL | 150uL | 6ng/uL |
| murine EGF (0.1mg/mL) | 5uL | 50uL | 10ng/mL |
| pen/strep | 0.5mL | 5mL | |
| FCS | 2.5mL | 25mL | 5% |
- Grow cells at 37 degrees C, 5% CO2.
- Trypsinise cells as usual.
- Split 1:6 maximum.
Insulin-Transferrin-Selenium (100x)
Insulin-transferrin-selenium, 10mL, Invitrogen #41400-045
Store at 4 degrees C
Ready to use
Dexamethasone (20ug/mL)
Dexamethasone, 1mg, Sigma #D8893
Lyophilised reagent stored at 4 degrees C
- Dissolve 1mg dexamethasone in 1mL of absolute ethanol to give a 1mg/mL solution
- Dilute 1 in 50 by adding 49mL media (DMEM/F-12 1:1 without additives) to give a final stock concentration of 20ug/mL
- Aliquot and store at -20
- Use between 20-200ng/mL final (50-500uL in 50mL media)
Triiodotyronine (20ug/mL)
Triiodotyronine, 1mg, Sigma #T5516
- Dilute 1mg in 1mL sterile 1M NaOH.
- Dilute 1 in 50 by adding 49mL media (DMEM/F-12 1:1 without additives) to give a final stock concentration of 20ug/mL
- Aliquot and store at -20
- Use between 0.6-6ng/mL final (1.5-15ul in 50mL media)
Murine Epidermal Growth Factor
mEGF, 0.1mg, Sigma #E4127
- Add 1mL media containing 10% FCS (0.9mL DMEM/F-12 1:1 without additives + 0.1ml FCS)
- Aliquot into 6uL lots to avoid freeze/thawing
- Store at -20
- 0.1mg/mL stock solution is stable for up to 2 weeks at 4 degrees C once made up
- Use between 2-20ng/mL final (1-10ul in 50mL media)
