Dear all,
I am trying to optimize siRNA transfection of primary sorted satellite cells from mice. Previously I had a lot of succes with transfecting murine myoblast cell lines with JetPrime (Polyplus), and now we want to bring our research into the primary isolated cells. However, collegues of mine have had very poor results transfecting SCs with JetPrime, Lipofectamine, siPort Neofx, and PEI - low efficiency or high toxicity made succesful transfection previously impossible.
Does anyone have any experience with successful primary sattelite cell transfection? which protocol you used? Do you have any suggestions for further optimization?
Thank you all in advance!
Dear Bart,
It looks like your cells are simply very hard-to-transfect. Maybe you can also try our Viromers BLUE and GREEN, but I am afraid that it's not the transfection reagent the problem.
Another option could be the siRNA you use. Are you aware of potential background effects and other issues? I would recommend to have a look at siTOOLs oligos.
The link to their website is below.
For very challenging cells, our Viromers in combination with siTOOLs material give sometimes very surprising good results.
Anyway, if you need free test samples of Viromers, please let me know.
Our manual gives also useful guidelines for optimization, that are applicable to other reagents (see the link below)
Good luck!
Bests,
Sandra
https://www.sitoolsbiotech.com/sipools.php
https://viromer-transfection.com/wp-content/uploads/2015/10/transfection-protocol-Viromer-BLUE-06-15.pdf
Thank you Sandra, I will have a look on those links you have send me and let you know :)
Hi,
i've done siRNA transfections in murine satellite cells using the following protocol:
Satellite cells are plated on matrigel coated dishes
Transfection was done when they were at ~50-60% confluency (6wells)
Add 1.75ml fresh growth media without antibiotics (still contained serum, that didn't matter)
Transfection mix A: 150ul OptiMem, 2ul SiRNA (10uM)
Transfection mix B: 150ul OptiMem, 9ul RNAiMax
Incubate both at room temperature for 5 mins
Mix solution and wait another 5 minutes
Add 250ul of it to a 6well
Media was changed back to growth media with antibiotics after 6h (to avoid toxicity)
Cells were then split for further experiments after 24hours
This normally resulted in about 70% of knockdown. Hope that is helpful, and let me know if you need any more info.
Cheers,
Johanna
Thank you Johanna!
Yo had a satellite cell line I guess? (as you are talking about 6 well plates...)
we are sorting primary murine satellite cells and we obtain only a very low number of SCs...
Anyway, e might give your protool a try! :)
Best,
Bart
Hi,
no, in our protocol we isolate the whole EDL from a mouse (tendon to tendon), seperate the single fibres and allow the satellite cells to migrate from the muscle fibres on matrigel coated dishes, confirming that they are satellite cells (rather than fibroblasts) by Pax7 staining.
Hope it works!
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