Transfect HEK-293T cells with LNP-(EGFP)mRNA
- HEK-293T cells between P3 and P15 were recommended.
- All of the operations should be carried out under strict aseptic conditions.
- Take HEK-293T cells from a 10cm-dish as an example
Day1:
1) Digestion:
Rinse the cell layer with PBS after discarding supernatant. Digest the cells with 2ml Trypsin(0.25%)-EDTA(0.02%) solution for 1 minute at 37℃. Digestion was terminated with 2 ml DMEM containing 10% FBS. Gently blow the cells to collect cell suspension.
2) Centrifugation:
Transfer the contents to a centrifuge tube(15ml). Spin at approximately 125 x g for 5 to 10 minutes.
3) Cell counting
Resuspend the cell pellet with 4ml culture medium without PS. Count the cell number and add 5 x 104 cells into each well of the 24-well plates. Add a total of 1ml noPS culture medium in each well.
4) Culture
Culture the cells at 37°C in a suitable incubator with 5% CO2 overnight.
5) LNP-mRNA assembly
LNP-mRNA can be assembled with Smart LNP Generator S2.
Take EGFP mRNA as an example:
Total flow rate = 16 ml/min, Flow rate ration = 1 : 3, 12mM compound phospholipid and N/P = 6 are recommended.
LNP-mRNA can be detected by Dynamic light scattering (DLS, like Zetasizer Nano ZS produced by Malvern Panalytical). A good result can be like Figure1.
Figure 1: Particle Size Distribution of LNP-(EGFP)mRNA after dilution (Z-average size=65.65; PDI=0.058).
6) Post-processing after LNP-mRNA assembly
LNP-mRNA need to be dialyzed with PBS containing 10% sucrose (overnight). The product should be filtered with a 0.22μm filter membrane to ensure sterility. Dynamic light scattering is recommended.
Day2:
7) Transfection
Detect the LNP-mRNA encapsulation rate, so that the mRNA encapsulated in LNP can be accurately calculated. Add 500ng encapsulated LNP-mRNA in each well (of 24-well palate) directly.
8) Observation
GFP signals can be observed within 24 hours after transfection. FACS can be used to detect transfection efficiency after 48 hours. Good transfection efficiency will be like Figure2.
Figure 2: Images of LNP-(EGFP)mRNA transfected HEK-293T Cells (16 hours after transfection)
