Anti cd163 antibody

The primary antibodies included anti-IBA1 antibody (1:100, Wako), anti-CD68 antibody (1: 100, Abcam, Cambridge, MA, United States), anti-CD16/32 antibody (1:100, BD Biosciences), anti-CD206 antibody (1:100, R&D Systems, Inc., Minneapolis, MN, United States), anti-CD11b antibody (1: 100, Abcam, Cambridge, MA, United States), anti-CD86 antibody (1: 100, Abcam, Cambridge, MA, United States), anti-CD40 antibody (1:100, Abcam, Cambridge, MA, United States), anti-CD163 antibody (1:100, Santa Cruz, MA, United States), anti-TMEM119 antibody (1: 100, Abcam, Cambridge, MA, United States), anti-Rhodopsin antibody (1:100, Santa Cruz, MA, United States). These primary antibodies and abbreviates were explained in Supplementary Table S1, including the description of the characteristics of M1 and M2 markers. Secondary antibodies included donkey anti-rabbit IgG (H+L) Alexa Fluor^® 555, goat anti-rat IgG (H+L) Alexa Fluor^® 488, donkey anti-rabbit IgG (H+L) Alexa Fluor^® 488, and donkey anti-goat IgG (H+L) Alexa Fluor^® 555 secondary antibodies (1:800, Invitrogen).

J774.1 cells were cultured at 2.5 × 10⁵ cells/dish with 20 μg of F10-EVs, Nanog⁺F10-EVs, or PBS (control) for 48 h. The cells were collected by trypsin/EDTA treatment and suspended in PBS. An aliquot of cell suspension containing 1.0 × 10⁶ cell was collected and washed twice with PBS containing 3% BSA. Then, the cells were reacted with anti-CD163 antibody (1:10, sc-58965, Santa Cruz) at room temperature for 1 h and subsequently with goat anti-mouse IgG (H + L) Alexa Fluor 568 (1:285.7, A-11004, Invitrogen, Waltham, MA, USA) at room temperature for 30 min. The cells were plated on a glass dish and the fluorescent images were analyzed using ImageJ. The fluorescent intensity and area of every fluorescent cell was integrated for an image to determine the fluorescent intensity per unit area. Eight or nine images were analyzed for every condition and the results of F10-EVs and Nanog⁺F10-EVs were expressed as relative values to that of PBS.