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The cell line can be used as a reporter of proteasome activity and enables the monitoring the function of the ubiquitin-proteasome system in an intact cell.

This line was derived in July, 2000 from the human embryonic kidney line, 293. 293 cells were transfected with a reporter consisting of a short degron, CL1, fused to the COOH-terminus of green fluorescent protein (GFPu).

This line was derived in July, 2000 from the human embryonic kidney line, 293. 293 cells were transfected with a reporter consisting of a short degron, CL1, fused to the COOH-terminus of green fluorescent protein (GFPu). The GFPu plasmid was created by ligating an oligonucleotide encoding ACKNWFSSLSHFVIHL into the GFP-C1 plasmid (Clontech) [PubMed:11375494]. Cells contain unstable green fluorescent protein (GFP). The cell line can be used as a reporter of proteasome activity and enables the monitoring the function of the ubiquitin-proteasome system in an intact cell. Ubiquitin dependent proteolysis has a role in cell division and apoptosis due to protein aggregation.

This line was derived in July, 2000 from the human embryonic kidney line, 293. 293 cells were transfected with a reporter consisting of a short degron, CL1, fused to the COOH-terminus of green fluorescent protein (GFPu). The GFPu plasmid was created by ligating an oligonucleotide encoding ACKNWFSSLSHFVIHL into the GFP-C1 plasmid (Clontech) [PubMed:11375494]. Cells contain unstable green fluorescent protein (GFP). The cell line can be used as a reporter of proteasome activity and enables the monitoring the function of the ubiquitin-proteasome system in an intact cell. Ubiquitin dependent proteolysis has a role in cell division and apoptosis due to protein aggregation.

Protocol:

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
3. Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new culture vessels.
   An inoculum of 5 X 10(3) to 3 X 10(4) viable cells/cm2 is recommended. Maintain cultures at a cell concentration between 5 X 10(4) and 5 X 10(5) cells/cm2. Do not exceed 8 X 10(5) cells/cm2.
6. Incubate cultures at 37°C.

Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:8 is recommended

Medium Renewal: Two to three times weekly

Note: Do not allow cell density to exceed 90%.

Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO

Storage temperature: liquid nitrogen vapor phase

Atmosphere: air, 95%; carbon dioxide (CO₂), 5%

Temperature: 37°C