Apc conjugated ly6c

Bone marrow (BM) cells were isolated by flushing long bones with PBS and filtering through a 70-μm cell strainer (BD, Inc. Cat.#352350). After red blood cell lysis, BM cells were analysed using PE-conjugated CD11b (BD, Inc. Cat.#553311), FITC-conjugated Gr-1 (eBioscience, Inc. Cat.#RB6-8C5) and APC-conjugated Ly6C (BD, Inc. Cat.#557661) antibodies with Gallios flow cytometer (Beckman Coulter, Inc. Brea, CA, USA).

150 μL peripheral blood and the hind ankles were collected on day 5 and 13 post KBx/N serum transfer, respectively. For the peripheral blood samples, red blood cells were lysed and cells were prepared as single cell solution. The hind ankles were cut into 1 mm² pieces, and then digested in 10% DMEM containing Collagen IV (1 mg/ml) for 1 h at 37°C. Single cell solution was then prepared after passing through 40 μm cell strainer. 1×10⁶ cells were stained with FITC-conjugated CD11b (BD, United States, 1:500), PE-conjugated F4/80 (BD, United States, 1:400), PerCP-Cy5.5-conjugated Ly6G (BD, United States, 1:400) and APC-conjugated Ly6C (BD, United States, 1:200) to detect macrophage, neutrophil and monocyte. Data were acquired using a BD FACSCalibur flow cytometer (BD Biosciences) and were analyzed by using FlowJo software (Version 7.6.1).