I control infinite m1000

One day prior to transfection, cells were seeded in a 96-well plate at a density of 20,000 cells per well. After 24 h, complete medium was removed and replaced by a 100 µL mix of formulation and medium without serum, allowing the transfection of 90 ng of Fluc mRNA or equivalent volumes of noncomplexed LP and LP_auto. Supernatants were removed 3 h post transfection, and 100 μL of complete medium was added to each well. Finally, cells were incubated at 37 °C and 5% CO₂ until the analysis 24 h later. Lipofectamine 2000^TM transfection reagent was used as a positive control following the manufacturer’s instructions. Non-transfected cells were used as a negative control and were labeled as ‘untreated’.
A luciferase assay was performed using the Bright-Glo™ Luciferase Assay System (Promega, Charbonnières-les-Bains, France) according to the manufacturer’s recommendations. Briefly, the luciferine substrate was added (v/v) to the well and left to incubate for 5 min at RT. Luminescence was then measured on a Tecan i-control Infinite M1000 (Integration time 1 s) (Tecan, Männedorf, Switzerland) and was determined as the mean of three replicates and three independent experiments.

The HeLa cell line (Invivogen, Toulouse, France) was maintained in a complete medium composed of DMEM culture supplemented with 10% heat-inactivated FBS. One day before transfection, 2 × 10⁴ cells/well were seeded on white 96-well plates (Greiner Bio-One, Courtaboeuf, France) with completed medium and incubated at 37 °C under 5% CO₂. After 24 h, cells were transfected with LNP at 100 ng of encapsulated FLuc-mRNA/well in completed medium. TransIT^® transfection reagent (#MIR2225, Mirius) was used as a positive control to transfect FLuc mRNA, following the manufacturer’s instructions. Non-transfected cells were used as a negative control. The bioluminescence assay was performed, 24 h post-transfection, using the Bright-Glo^TM Luciferase assay system (Promega, Charbonnières-les-bains, France). Briefly, 100 µL of reconstituted reagent was added to each well and incubated for 5 min at room temperature. Luminescence intensity was measured on a Tecan i-control Infinite M1000 (Tecan, Männedorf, Switzerland).

Micelles were prepared as previously reported [37 (link)]. In brief, 5 mL of a copolymer solution (10 mg/mL) in acetonitrile (Carlo Erba Reagents, Peypin, France) was added to 10 mL of milli-Q water under agitation (200 rpm), allowing the formation of micelles. acetonitrile and a part of water were removed by evaporation under reduced pressure using a Rotavapor R-300 (Buchi, Villebon sur Yvette, France). The micelle concentration was determined by measuring the solid content, after heating a known volume of the micellar solution to a constant weight in an oven at 70 °C for 24 h. The micelle solution (500 µL, 5 mg/mL) was further incubated with DiR (0.26 wt%/micelle, 1 µL of a 6.5 mg/mL of DiR solution in ethanol) overnight, protected from the light. DiR encapsulation was directly evidenced on the micellar solution by fluorescence (750 nm/780 nm) and visible spectrometry using a Tecan i-control Infinite M1000 (Tecan, Männedorf, Switzerland). DiR loading efficiency was determined by fluorescence intensity on a micellar solution after dialysis (3500 Da cut-off) and 10-fold dilution in acetonitrile, using a calibration curve established under the same conditions. The micelles were diluted 2-fold in Phosphate Buffer Saline (PBS) at pH 7.4 (Gibco, Dublin, Ireland) and incubated overnight before further biological studies.