1. Transfection: I need a double knockdown protocol because breeding is too slow.
2. Lipofectamine: I used RNAiMAX.
3. Cell density: I used 6-well plates, 160K cells per plate (the true macrophage count is lower due to including red blood cell count at initial cell culture). All but 20 cells died by 48h.
4. Is there a better way than lipofectamine? It seems to inevitably activate the cells, thus they don't respond to LPS.
Hi Leah,
I used Lipofectamine 2000 (Invitrogen) several times for silencing gene expression in bronchial epithelial cells and I definitely say yes, Lipofectamine show cytotoxicity after 24h incubation with pro-inflammatory pathways activation. I have never incubated Lipofectamine for more than 6-10 hours because of poor cell viability after that period. I washed twice with medium and left cells in complete medium futher 16 hours to restore homeostasis and complete gene silencing.
I also recently generated knock-down of SBDS gene in lymphoblasts, which are actually tough to transfect. In this case, I used SiPORT NeoFX (Thermofisher) which shows less cytotoxicity in our cell model. Anyway, I would transfect cells (50% confluence) in a minimal volume (almost 50% of usual) of serum-free medium for 6 hours and finally restore the final volume adding further medium to force the transfection in a short time, thus avoiding lipofectamine-mediated cell death. Also, I would try other vectors (including siPORT), using similar protocols.
I do agree that lipofectamin exposure for more than 24 hours is toxic for the cells, I used lipofectamin 3000 for 6,9 and 24 hours maximum with no significant cell killing.
Hi Leah. Someone from Dr. Subash Sad's lab should be able to help you out. They have a lot of experience knocking genes down in BMDMs prior to LPS stimulation. Just give them a call and ask for Ardeshir.
https://www.subashsadlab.com/contact-me
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