Anti human igg apc isotype

After treatment with 200 ng/ml CCL7 recombinant protein (#282-P3, R&D, Minneapolis, MN, USA) for 1, 3, 6, and 12 hours, cells were suspended in PBS and incubated with FcR blocking reagent (Miltenyi Biotec, Gladbach, Germany) for 10 min. Cells were stained for 30-40 min at 4°C with the following directly conjugated antibodies: anti-human E-Cadherin-APC (#180224, R&D, Minneapolis, MN, USA), anti-human CCR1 (#PA-141062, Thermo), anti-human CCR2 (#ab32144, Abcam, Cambridge, MA), anti-human CCR3 (#ab32512, Abcam, Cambridge, MA), anti-human CCR5 (#ab32048, Abcam, Cambridge, MA), and anti-human IgG-APC isotype (Miltenyi Biotec, Miltenyi, Germany). Secondary antibody for CCR1, -2, -3, and -5 was conjugated to PE (#12-4739-81, Ebioscience). IgG isotype antibodies were used in parallel as control. Cells were analyzed on an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA) with CFlow software (BD Biosciences, San Jose, CA, USA).

Cells were suspended in phosphate buffered saline (PBS) and incubated with an FcR blocking reagent (Miltenyi Biotec, Germany) for 10 minutes. Then, cells were stained with the directly conjugated monoclonal antibodies anti-human CD166-PE, CD44-APC, anti-human IgG-PE isotype, and anti-human IgG-APC isotype (Miltenyi Biotec, Germany) for 30–40 minutes at 4 °C. The IgG isotype control was incubated in parallel. Flow cytometry was performed with an Accuri C6 (BD Biosciences, CA) using the CFlow software (BD Biosciences, CA).