Cell Cycle-Chromobody TagRFP U2OS cell line

Description
U2OS cell line stably expressing Cell Cycle-Chromobody® fused to the red fluorescent protein TagRFP.

Specificity
VHH antibody fragment against cell cycle marker PCNA (proliferating cell nuclear antigen)

Applications

Live imaging of endogenous DNA replication
Quantitative assessment of cell cycle progression

Product Size Code Price Buy
Product Cell Cycle-Chromobody TagRFP U2OS cell line Size 5x 10^6 frozen cells Code u-ccc-r Price Please inquire

Specificity
Proliferating cell nuclear antigen (PCNA)

Encoded Nanobody/ VHH
Monoclonal anti-PCNA single domain antibody (sdAb) fragment

Microscopy techniques
Wide-field epifluorescence microscopy; confocal microscopy; super-resolution microscopy e.g. STED

Cells
Cell Cycle-Chromobody TagRFP U2OS-cell line

Format
5 x 106 frozen cells in serum-free cryopreservation medium (Waymouth formulation)

Quality control

All cell lines supplied by ChromoTek undergo comprehensive quality control.
This cell line was tested negative for mycoplasma using Venor® GeM Classic Kit (Minerva biolabs).
Cell viability: >95 % after thawing
Stability of transgene: >95 %  after 3 months without G418 selection

Storage instructions
Shipped on dry ice. Store in liquid nitrogen (vapor phase).

Are Chromobodies constitutively expressed?

Yes, Chromobody expression is regulated by immediate early promotor CMV. This promotor allows constitutive Chromobody expression.

Do Chromobodies only work in live cells?

Yes, the Chromobody plasmid is only expressed in live cells. Cells should be transfected with the Chromobody plasmid at least overnight to observe the Chromobody location signal. Alternatively, cells can be fixed prior to imaging.
Note: We don't recommend fixation of cells for the Histone-Chromobody.

When should I image my cells after transfection with the Chromobody plasmid?

The Chromobody signal is maintained up to 3 days in the cell. However, this also depends strongly on the cell type.
We recommend to image the cells 16-24 hours after transfection.

Do the Chromobodies diffuse through the cell membrane into growth medium?

No, Chromobodies are small proteins being expressed in the cytosol. They are not secreted into the medium and remain in the cell as long as the cell maintains its plasma membrane integrity.

Are Chromobodies fluorogenic or do they only emit fluorescence when bound to a target?

Chromobodies are chimeric proteins consisting of a VHH fused to a fluorescent protein. They maintain their fluorescence regardless of whether they are bound to a target or not.

Can I amplify the Chromobody plasmid in bacteria?

Yes, the Chromobody plasmids can be propagated in E. coli by standard techniques.

Origin information

Source: human
Tissue: bone
Disease: osteosarcoma
Cell type: epithelial
Growth properties: adherent
Virus associated: no
Biosafety Level 1, according to the Central Committee of Biological Safety (ZKBS), Germany

Cell culture medium

Dulbecco's Modified Eagle's Medium with 4.5 g/L glucose
110 mg/L sodium pyruvate and L-glutamine
10 % FCS (fetal bovine serum)
50 µg/mL Gentamycin
Optional: 1 mg/mL G418

 

Assay medium

For live cell assays we recommend using DMEM without phenol red:
No auto-fluorescence of phenol red improved signal-to-noise ratio

Thawing instructions

Cryopreserved cells can be thawed by the following procedures:

Centrifugation

  • Remove cells from storage and thaw quickly in a 37 °C water bath. We recommend eye protection by using approved safety goggles. We also suggest the use of safety gloves to protect uncovered skin.
  • Place 1 to 2 ml of thawed cells in ~25 ml of complete growth medium. Mix cell suspension gently.
  • Centrifuge the cells at ~80 x g for 2 to 3 min.
  • Check clarity of the supernatant and visibility of a consolidated cell pellet. Discard supernatant without disturbing the cells.
  • Gently resuspend the cells in complete growth medium and count the viable cells.
  • Plate the cells.

Direct plating

  • Remove cells from storage and thaw quickly in a 37 °C water bath. We recommend eye protection by using approved safety goggles. We also suggest the use of safety gloves to protect uncovered skin.
  • Plate cells directly with complete growth medium. Use 10 to 20 ml of complete medium per 1 ml of frozen cells.
  • Culture cells for 2 to 4 h. Replace medium with fresh complete growth medium to remove cryopreservative.

We recommend thawing according to the centrifugation protocol (see above).

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Only for research applications, not for diagnostic or therapeutic use!