Apc cyanine7 anti mouse cd11c antibody

Mouse bone marrow cells were isolated from tibia and femur. For preparations of BMDMs, the bone marrow cells were cultured in 10% M-CSF-containing conditional medium from L929 cells for 3–5 days. For preparations of BMDCs, the bone marrow cells were cultured in medium containing murine GM-CSF (50 ng/ml) for 6–9 days.
To differentiate bone marrow-derived cDCs, bone marrow cells were suspended in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% FBS, 1% Penicillin Streptomycin solution, 1% Sodium Pyruvate, 1% MEM non-essential amino acid, 1% L-glutamine solution, and 55 mM β-mercaptoethanol (complete IMDM) and were cultured with Flt3L (25 ng/ml) conditioned medium for 7 to 8 days. The cells were then stained with fluorescent antibodies before sorting. pDCs were Bst2^– B220⁺, cDC1s were Bst2^– B220^– CD11c⁺ MHCII⁺ CD24⁺ CD172a^–, cDC2s were Bst2^– B220^– CD11c⁺ MHCII⁺ CD172a⁺. The antibodies used in FACS were APC anti-mouse CD317 (BST2, PDCA-1) antibody (0.06 μg/10⁶ cells, Biolegend), PE anti-mouse/human CD45R/B220 antibody (0.25 μg/10⁶ cells, Biolegend), APC/Cyanine7 anti-mouse CD11c antibody (1 μg/10⁶ cells, Biolegend), PE/Cyanine7 anti-mouse CD24 antibody (0.5 μg/10⁶ cells, Biolegend), PerCP/Cyanine5.5 anti-mouse CD172a (SIRPα) antibody (1.5 μg/10⁶ cells, Biolegend) and Pacific Blue™ anti-mouse I-A/I-E antibody (0.25 μg/10⁶ cells, Biolegend).

BAL cells were resuspended in FACS buffer and centrifuged 215 × g for 5 min at 4 °C. BAL cells were stained for 15 min at 4 °C in the dark with the following antibodies used per manufacturer’s instructions The following antibodies were used for flow cytometry analysis (dilutions were 1:100): APC/Cyanine7 anti-mouse CD11c Antibody (117324), PE anti-mouse F4/80 Antibody (123110), Alexa Fluor® 647 anti-mouse CD64 (FcγRI) Antibody (139322), Alexa Fluor® 488 anti-mouse/human CD11b Antibody (101217), and PerCP anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody (108426), which were purchased from BioLegend, San Diego, CA, USA. Cells were gated on lymphocytes or macrophage-sized, singlet, live cells. Flow cytometry analysis was performed using a NovoCyte Flow Cytometer (150014, Agilent Technologies, Inc., CA, USA).