Cancer assays - (Apr/16/2007 )
Hey everyone!
I infected a human cancer cell line with my protein of interest. Currently I am trying to figure out, if these cells now are any different from the uninfected (protein of interest lacking) cells regarding their tumorigenicity.
I tried the following assays:
- anchorage independent growth:
1) soft agar for 21+ days
2) growth in suspension on ultra low attachment plates (time course over 5 days, MTT assay)
(On that note: has anyone ever seen a difference between ultra low attachment plates and poly(HEMA) coated plates???)
- growth curve in starvation media (MTT assay)
- matrigel invasion assay
- transwell migration assay
These assays yielded either inconsistent results or showed no difference between protein of interest expressing and -lacking cells. And I know that this could all be due to the fact, that this protein has nothing to do with tumorigenicity.
However, if anyone can think of any other cancer assay, I really would like to try it out, before I wrap up the project on my protein.
Please let me know, if you have done or just heard about any assays, which I could use to further analyze my cells.
Thanks a lot!
Hazel
Is your transfection stable - do you use selective media or some more elegant confirmation (e.g. PCR) that the transgene stays >21 days.
Do you know if your inserted protein is toxic - can you do a live/dead or other cytotoxicity assay.
Tumorigenicity? - Could you inject the cells into mice (nude or SCID)?
Is your transfection stable - do you use selective media or some more elegant confirmation (e.g. PCR) that the transgene stays >21 days.
Do you know if your inserted protein is toxic - can you do a live/dead or other cytotoxicity assay.
Tumorigenicity? - Could you inject the cells into mice (nude or SCID)?
The cells were stably infected with lenti-virus a few months ago. I checked for the presence of my gene by Western blot and by a functional assay. The cells still are expressing the protein after several weeks.
The protein is not toxic to the cells - at least not that I know of. The infected cells look the same as the control cells.
I was thinking about nude mice injection, but was hoping to find some other in vitro assays before going to mice. Does every cancer cell line form tumors in those mice? The cell lines, which I currently use for my studies all are able to grow on ultra low attachment plates.
Thanks,
Hazel
Is your transfection stable - do you use selective media or some more elegant confirmation (e.g. PCR) that the transgene stays >21 days.
Do you know if your inserted protein is toxic - can you do a live/dead or other cytotoxicity assay.
Tumorigenicity? - Could you inject the cells into mice (nude or SCID)?
The cells were stably infected with lenti-virus a few months ago. I checked for the presence of my gene by Western blot and by a functional assay. The cells still are expressing the protein after several weeks.
The protein is not toxic to the cells - at least not that I know of. The infected cells look the same as the control cells.
I was thinking about nude mice injection, but was hoping to find some other in vitro assays before going to mice. Does every cancer cell line form tumors in those mice? The cell lines, which I currently use for my studies all are able to grow on ultra low attachment plates.
Thanks,
Hazel
To help you we need to know a little bit about functional activity of your protein to give your appropriate advice !
I infected a human cancer cell line with my protein of interest. Currently I am trying to figure out, if these cells now are any different from the uninfected (protein of interest lacking) cells regarding their tumorigenicity.
I tried the following assays:
- anchorage independent growth:
1) soft agar for 21+ days
2) growth in suspension on ultra low attachment plates (time course over 5 days, MTT assay)
(On that note: has anyone ever seen a difference between ultra low attachment plates and poly(HEMA) coated plates???)
- growth curve in starvation media (MTT assay)
- matrigel invasion assay
- transwell migration assay
These assays yielded either inconsistent results or showed no difference between protein of interest expressing and -lacking cells. And I know that this could all be due to the fact, that this protein has nothing to do with tumorigenicity.
However, if anyone can think of any other cancer assay, I really would like to try it out, before I wrap up the project on my protein.
Please let me know, if you have done or just heard about any assays, which I could use to further analyze my cells.
Thanks a lot!
Hazel
one may also check cell cycle, cell morphology, or molecular network integrity depending on your kind of protein f.i. to check by microarray for mRNA expression