LNP transfection

LNP transfection-

1.    Count live cells using trypan blue manually or by automatic cell counter.

2.    Seed 0.1 × 10⁶ cells in tissue culture 12-well plates with 1 ml of growing medium and place for overnight incubation in 37°c, 5% CO₂ incubator.

3.    Add LNPs to the growth medium at RNA amounts of 0.1 to 2 μg (Need to be calibrated to each cell line).

a.    For 005 GBM cells, preincubated LNPs with ApoE3 (0.001 mg/ml) for 20 minutes at 37°c before the addition to the growth medium.

4.    Incubate cells for 24 to 120 hours (according to the specific output) in 37°c, 5% CO₂ incubator.

5.    Wash cells X 3 with PBS.

6.    Collect cells for flow cytometry (72 to 96 hours) or cell cycle assays (24 to 48 hours) using Trypsin-EDTA 0.25%.

7.    Wash cells X 3 with PBS.

8.    Resuspend cell pellets in Flow cytometry buffer (PBS supplemented with 2% BSA) for flow cytometry or PBS for cell cycle analysis.

Materials:

PBS- Biological industries, Israel.

BSA- Fraction V, MP biomedical, USA. Cat:0216006980X

Recombinant Human ApoE3- PeproTech, USA. Cat:350-02