Hello dear colleagues,
I am experiencing strange problems when performing transDUCTION on my CD4/CD8 cells (separated using Miltenyi beads from total PBMCs from a buffy coat).
For now on it is just simple as that: I cannot see ANY expression of total gfp using flow cytometry (meaning I do not use any antibodies or smth - it's total gfp) - please see the picture attached.
The construct contains IRES and seem to work fine when transfecting/transducing HeLa.
But in primary T-cells (please see the picture) only construct without IRES is expressed well for some reason.
The procedure I use is the following: after 48h activation with CD2/3/28 Miltenyi beads, T-cells are force treated to get rid of the beads (using MACSiMAG) and then, seeded 10E6 cells per well into 24-well plate.
VLPs at certain MOI (on the example shown it's MOI=10) are added to the cells in the wells and then, after addition of IL2 plate is placed into incubator (+37, 5%CO2 as usual).
No additional agents added, no spinoculation...
Here comes the question. May be there's another (more versatile) protocol that may improve the efficiency of transduction --> expression of gfp??
I would be veeery much obliged for any clue...
Thanks a lot in advance for any suggestion.
Dear Mark,
in order to improve the efficiency of infection/transduction in your model I would like to present you the Magnetofection technology. This Method uses a magnetic field to attract and concentrate magnetic complexes onto the cell surface. In brief you first form complexes of viral particles and magnetic nanoparticles, then after incubation (20 min at RT), you add the magnetized virus onto the cells and put the cell plate onto a magnetic plate for 20-30 min.
The magnetic plate will attract the magnetic nanoparticles that carry the virus and the almost dose of the vector comes into contact with the cell membrane within minutes. If the cells present the receptor to virus, then the virus will enter through its classic pathway, if the cells are non-permissive, the vector will be concentrated into cluster and enter through endocytosis pathway.
Moreover infection will be synchronized ; all the cells receive the virus at the same time reducing the risk of a autocrine immune response.
I would recommend:
I hope this will give you some ideas to improve your infection; would you like to try Magnetofection, please do not hesitate to contact me via ResearchGate or directly at [email protected]
best regards,
Cedric
http://www.ozbiosciences.com/module/citationfinder/default
http://www.ozbiosciences.com/content/17-imisct-magneticcellsorting-transfection-transduction
http://www.ncbi.nlm.nih.gov/pubmed/21357765
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