Cd11c microbeads ultrapure mouse kit

C57BL/6J mice were euthanized and the tibias and femurs were removed under sterile conditions. Monocytes were then isolated from the bone marrow cell suspension, suspended in RPMI-1640 medium supplemented with 1% Pen-Strep, 10% FBS, 50 ng ml⁻¹ GM-CSF, and 50 ng mL⁻¹ IL-4, and incubated at 37 °C in 5% CO₂ incubator for 8 days to generate immature DCs. The immature DCs were then isolated using CD11c MicroBeads UltraPure, mouse kit (Miltenyi Biotec, Cat: 130–108-338) following the manufacturer’s instructions. 0.8 × 10⁶ immature BMDCs were seeded in a 6-well plate in 1.6 mL RPMI-1640 medium supplemented with 25 ng mL⁻¹ GM-CSF, 25 ng mL⁻¹ IL4 for 24 h before treatment. For treatment, 125 μL of formulated RAL2 and RAL2-CD40 LNPs (RAL2-CD40 mRNA concentration: 0.01 mg mL⁻¹) to each well and cultured for 12 h before adding anti-mouse CD40 monoclonal antibody (BioXCell, Cat: BP0016–2). The cells were cultured for an additional 12 h and stained with fluorescent labeled anti-CD40 antibodies or fluorescent labeled DC activation markers (CD80, CD86, MHCII) for 30 min at 4 °C before flow cytometry. IL-12 and TNF-α cytokine production were detected by ELISA.

In vitro CFSE proliferation assays were performed as previously described (Beltra et al., 2016 (link)). Sorted Tex subsets were re-suspended in 1ml of PBS and stained for 6min with CFSE by addition of an equal volume of PBS containing 4μM of CFSE (2μM final). The reaction was stopped by addition of an equal volume of cold FBS and cells were subsequently washed two times in cRPMI. Dendritic cells (DCs) were enriched from the spleen of naive C57B/L6 mice using CD11c+ MicroBeads Ultra pure mouse kit (Miltenyi) and loaded with gp_33-41 peptide (0.1 μM) for 30min at 37°C 5% CO2 in cRPMI. Each Tex subset was co-cultured with peptide loaded DCs (10⁴ each) for three days in 96-round bottom well plates.