Lipofectamine® LTX Reagent

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	Do not add antibiotics to the Transfection Medium

Protocol tips
Do not add antibiotics to the Transfection Medium

Publication protocol

TRANSFECTION AND TRANSDUCTION
Transfection was performed 24 h after plating. The DNA and transfection reagent were pre-incubated for 30 min in 100 µl of Opti-MEM (GIBCO) with 2.5 µl of transfection reagents per 500 µl of culture medium in a 24 well plate (or with 400 µl of Opti-MEM with 10 µl of transfection reagents per 10 ml of culture medium in 9 cm petri dishes). Lipofectamine (Invitrogen), Lipofectamine 2000, Lipofectamine LTX (Invitrogen), and Fugene HD (Promega; Southampton, UK) were used as received from manufacturer. Cells were incubated with proteins or transfection reagents at 37oC in cell culture incubator for 18 h, washed with PBS, the medium was replace, and cell were left for 42 h or as otherwise indicated. Sampled medium was gently taken directly from the petri dishes or wells at the indicated time points and maintained at 4oC until needed and were kept for a maximum of 14 days. 200 µl of this medium was added to 400 µl of pre-plated Neuro2A cells in 24 well culture dishes. The sampled medium, when indicated, was also centrifuged at 20 000 rcf for 10 min to separate soluble and suspended fractions. Supernatant was added as described while precipitated pellet was subsequently resuspended in the same volume of culture medium before being added onto pre-plated Neuro2A cells. Peptides were added to LTX treated or untreated Neuro2A at 10 µg/ml and incubated for 30 min at 37oC as described elsewhere [Arsenault et al., 2014].


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