Cd206 alexa flour 488 clone 19

After macrophages were co-cultured with exosomes for 24 h, 48 h, or 72 h, macrophages co-cultured with stained exosomes were then stained with CD64 Percp-cy5 (clone 10.1, BD Pharmingen, San Jose, CA, USA), CD206 Alexa Flour 488 (clone 19.2, eBioscience, Waltham, MA, USA), CD163 Pecy7 (clone eBioGHI/61, Thermo Fisher Scientific, Waltham, MA, USA) and CD11b APCcy7 (clone M1/70, BD Bioscience, San Jose, CA, USA). After staining, the cells were washed twice with PBS. Flow cytometry data were acquired using a BD LSRII (Franklin Lakes, NJ, USA) and data analysis performed with FlowJo X.

Macrophages were stained with CD64 Percp-cy5 (clone 10.1, BD Pharmingen), CD206 Alexa Flour 488 (clone 19.2, eBioscience), and CD163 Pecy7 (clone eBioGHI/61, Thermo Fisher Scientific) for ImageStream analyses. For Image Stream, samples were imaged at 60× magnification while acquiring data on different channels. The machine was calibrated using single color stained and unstained controls, as well as calibration beads to adjust the noise levels and spectral compensation. Brightfield data were collected on channels Ch01 and Ch09, and side-scatter was collected on Ch12. 5000 events were acquired and used for analysis in IDEAS software version 6.2 (EMD Millipore, Billerica, MA, USA).