Usable as a characterized control in your experiments
To create the stable LifeAct-TagGFP2 expressing cell line, human fibrosarcoma cells were stably transfected with a plasmid coding for TagGFP2-labeled LifeAct.
The HT-1080 LifeAct-TagGFP2 cells present highly dynamic filamentous actin. As a result of stable LifeAct expression, perfect visualization of filamentous actin with very low background fluorescence is achieved without any interference to cellular dynamics. Moreover, full characterization of the cells revealed that they displayed identical behavior to wild type (wt) cells.
Due to the cell line’s fast growth and the absence of contact inhibition, it constitutes an ideal tumor model. These characteristics combined with bright and specific F-actin staining lead to a powerful tool with versatile applications in live cell imaging and high-content approaches.
Homogeneous cell pool
Assured cell line identity
Optimal transgene expression for microscopy assays
Genomic authentication of human cell lines by means of short tandem repeat (STR) analysis is widely used to assure cell line identity and to investigate cell line purity. Short tandem repeat polymorphisms in the human genome are used to differentiate between individuals. By identifying the short repeats of a specific sequence at typical locations in the genome, it is possible to create a genetic profile of an individual human being (as used in forensics), or of a cell line.
ibidi’s HT-1080 LifeAct-TagGFP2 cells were analyzed using the above mentioned method. The analysis was performed by DSMZ, an ISO-certified Leibniz institute. The analysis revealed that HT-1080 LifeAct-TagGFP2 cells are identical to HT-1080 wild type cells.
Cell Proliferation Analysis:
HT-1080 LifeAct-TagGFP2 and wild type cells were cultured for 8 days using the same defined conditions as the cultures’ start points, and then analyzed for cell number each day.
Results from this experiment showed that both cell lines grew at equal rates over the predefined period of time. This result was confirmed using statistical tests.
Fluorescence Intensity in Cell Population:
HT-1080 LifeAct-TagGFP2 and wild type cells were analyzed for fluorescence intensity and number of positive cells using flow cytometry.
At least 95 % of the cells were fluorescent with on average a two log shift in fluorescence intensity compared to wild type cells.
Functional Characterization of HT-1080 LifeAct-TagGFP2
Chemotaxis assays of HT-1080 LifeAct-TagGFP2 and wild type cells were analyzed in the µ-Slide Chemotaxis, using fetal calf serum (FCS) as a chemoattractant (see Application Note 17 for further details).
Results from these assays showed that HT-1080 LifeAct-TagGFP2 cells migrate with the same speed and forward migration index (FMI) as wild type cells. These results were confirmed using the Student’s t-test. p < 0.05 was considered significant.
HT-1080 LifeAct-TagGFP2 and wild type cells were seeded in a µ-Slide 8 well combined with a Culture-Insert. After 24 hours of video microscopy, wound closure was analyzed using the WimScratch automated software solution.
HT-1080 LifeAct-TagGFP2 and wild type cells showed comparable cell front velocities. These results were confirmed using the Student’s t-test. p < 0.05 was considered significant.
More stable LifeAct transfected cell lines will be available in the future. Please contact ibidi for detailed information.