Mouse cd11c microbeads ultrapure

THP-1 Dual cells (InvivoGen, San Diego, CA, USA) were cultured in RPMI 1640 medium (ATCC, Manassas, VA, USA) supplemented with glutamine, penicillin (100 U/mL), streptomycin (100 µg/mL), normocin (100 µg/mL), and 10% heat-inactivated fetal bovine serum. To maintain the selection pressure, blasticidin (10 µg/mL) and zeocin (100 µg/mL) were added to the growth medium every other passage.
Bone marrow cells were isolated from femurs and tibias of 8–12-week-old C57BL/6 mice and seeded in a 10-cm petri dish at a density of 2 × 10⁶ viable cells in 10 mL RPMI 1640 medium supplemented with glutamine, penicillin (100 U/mL), streptomycin (100 µg/mL), 2-mercaptoethanol (55 µM), 10% heat-inactivated fetal bovine serum, and 20 ng/mL of murine granulocyte-macrophage colony-stimulating factor (GM-CSF, Peprotech). An additional 10 mL of fresh warm medium with GM-CSF (40 ng/mL) was added on day 3 and a half volume of fresh medium was exchanged for fresh warm medium with GM-CSF (40 ng/mL) on day 6. On day 8, non-adherent and loosely adherent cells in the culture supernatant were harvested. The percentage of CD11c⁺ bone marrow-derived dendritic cells (BMDCs) was approximately 70%, as determined by flow cytometry. More concentrated CD11c^high BMDCs were positively selected by mouse CD11c⁺ microbeads ultrapure (Miltenyi Biotec, Bergisch Gladbach, Germany).

CD11c⁺ DCs were positively enriched from splenocytes using mouse CD11c MicroBeads UltraPure and LS separation columns (Miltenyi Biotec., San Diego, CA, USA) according to the manufacturer’s instructions. The purity of CD11c-positive DCs was above 80% after staining with FITC-conjugated mouse anti-CD11c antibody. The MFI of cells was calculated using BD Accuri™ C6 system software.

At day 10 after CpG-ODN footpad injection, single cell suspensions from draining popliteal lymph nodes were enriched for CD11c⁺ cells using mouse CD11c MicroBeads UltraPure according to the manufacturer's instructions (Miltenyi Biotec).

Eighteen-month-old (aged) female C57BL/6 mice were maintained at the Laboratory Animal Resource Center of the Korea Research Institute of Bioscience and Biotechnology (KRIBB). Six-week-old female C57BL/6 mice and OT-I transgenic mice were purchased from OrientBio (Gyeonggido, Republic of Korea) and Jackson Laboratory (ME, USA), respectively. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of KRIBB (Approval number: KRIBB-AEC-20042) and performed under the guidelines in a specific pathogen-free facility. Splenocytes were cultured in the RPMI 1640 medium with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco, NY, USA). Splenic DCs were isolated using mouse CD11c MicroBeads UltraPure (Miltenyi Biotec, Bergisch Gladbach, Germany), and the purity was > 80%. Madin-Darby canine kidney (MDCK) cells (ATCC, VA, USA) were maintained in EMEM medium (Lonza, MD, USA) supplemented with 5% heat-inactivated FBS, 1% MEM vitamin solution (Sigma-Aldrich Chemical Co., MO, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco).