Mice were treated with LNP A-3 or B-4 with encapsulated GFP mRNA as described above. Fetuses were extracted after 24 hours and livers were collected for imaging and flow cytometry. For GFP imaging experiments, individual fetal livers were imaged using a fluorescent stereoscopic microscope (MZ716FA, Leica, Heerburg, Switzerland). Flow cytometry was used to quantify the percentage of cells in the liver that were GFP+ following treatment. To do this, freshly dissected livers were immediately transferred to a dish containing ice-cold PBS, manually homogenized to create a single-cell suspension, and resuspended in FACS (fluorescence-activated cell sorting) staining buffer (1× PBS, 0.5% bovine serum albumin, and 0.2 mM EDTA, prepared in-house). After lysis of RBCs with ACK Lysing Buffer (Lonza Bioscience, Basel, Switzerland), liver cells were stained with anti-CD45APC (e-Bioscience Inc., San Diego, CA) for 30 min, washed three times, and resuspended in FACS buffer before running on a FACSAria flow cytometer (BD Biosciences, San Jose, CA). Gating was conducted to exclude CD45+ lymphohematopoietic cells. Age-matched untreated mice served as a negative control for GFP+ gate placement to determine the percentage of positive cells in livers from fetuses treated with LNPs.

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