Alexa fluor 647 labeled anti cd197

Macrophages were obtained from mouse lungs after sacrifice and prepared for flow cytometry as per the protocol described elsewhere [23] (link). Briefly, the dissected lungs were held in complete RPMI medium supplemented with 25 mM HEPES. Lungs were minced in complete medium and the filtrates were centrifuged (1500 rpm for 5 min). The number of cells in the supernatant were determined and then the solutions passed through magnetic activated cell sorter (MACS) microbeads and through MACS LD or MS columns, as per the manufacturer’s protocol (Miltenyi Biotec, Germany). The eluted unbound and bound cell fractions were first separated, and after a repeat cell count, they were tested for different markers by flow cytometry. The cells were then washed, fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences, NJ, USA), stained with conjugated anti-mouse antibodies as follows: Alexa Fluor 488-labeled anti-CD86, Alexa Fluor 647-labeled anti-CD197, RPE-labeled anti-CD23, and FITC-labeled anti-CD206 (AbD Serotec, UK), and analyzed with BD LSR II flow cytometer using DIVA Software. Data were collected (10,000 events each time) with a FACSCalibur flow cytometry system and analyzed with the CellQuest Software (BD Biosciences, NJ, USA).