Anti cd11c clone im7

Female Ai14 mice were purchased from Jackson Laboratory and housed in the Laboratory Animal Facility of the Stanford University Medical Center. CARTs were complexed with 15 μg Cre mRNA (Trilink L-7211) in PBS 5.5 to a total volume of 100 μl at 10:1 N:P ratio, with the same formulation procedure described above. Spleens were isolated 48 h after transfection to measure Cre-mediated recombination. Spleens were smashed with a 100-μm strainer to make a single-cell suspension. Red blood cells were lysed before staining. Single-cell samples were then stained with Zombie NIR (BUV570, BioLegend 423103), anti-CD45 (clone 145-2C11, BioLegend, 1:300 dilution), anti-CD8α (clone 53-6.7, BioLegend, 1:200 dilution), anti-CD4 (clone RM4-5, BioLegend, 1:200 dilution), anti-CD11c (clone IM7, BioLegend, 1:400 dilution), anti-CD19 (clone 30-F11, BioLegend, 1:200 dilution), and anti-F4/80 (clone H1.2F3, BioLegend, 1:100 dilution). Cells were then washed twice and analyzed by flow cytometry. Data were collected on an Attune Nxt Flow Cytometer.

To determine the cell specificity of mRNA delivery with CART, mice were injected with 7.5 µg of Cy5-labeled mRNA encoding luciferase. After 2 h, spleens were isolated and processed into a single-cell suspension, followed by red blood cell lysis. Single-cell suspensions were stained with Zombie NIR (BUV570, BioLegend 423103) to exclude dead cells and labeled with antibodies against various cell surface markers: anti-CD45 (clone 145-2C11, BioLegend, 1:300 dilution) for total leukocytes, anti-CD8α (clone 53-6.7, BioLegend, 1:200 dilution) for CD8 + T cells, anti-CD4 (clone RM4-5, BioLegend, 1:200 dilution) for CD4 + T cells, anti-CD11c (clone IM7, BioLegend, 1:400 dilution) for dendritic cells, anti-CD19 (clone 30-F11, BioLegend, 1:200 dilution) for B cells, and anti-F4/80 (clone H1.2F3, BioLegend) for macrophages. After staining, cells were washed twice and subjected to flow cytometry analysis. Data acquisition was performed using an Attune Nxt Flow Cytometer. The percentage of Cy5+ cells per cell type was then calculated.