Igg1 apc

For analysis of B‐cell populations and T regulatory cells, live single‐cell suspensions were prepared from spleens and stained with propidium iodide (PI; catalog number P4864; Sigma/Merck), CD45R/B220‐PE‐Cy7 (1/400; catalog number RA3‐6B2; BioLegend, San Diego, CA, USA), CD138‐PE (1/400; catalog number 281–2; BioLegend), GL7‐Pacific Blue (1/200; BioLegend), peanut agglutinin (PNA)–fluorescein isothiocyanate (1/400; catalog number L7381; Sigma/Merck), IgG1‐APC (1/400; catalog number RMG1‐1; BioLegend), CD4‐Pacific Blue (1/400; catalog number RM4‐5; BD Biosciences), CD25‐APC (1/100; catalog number PC61; BD Biosciences) and FoxP3‐PE (catalog number MF23; BD Biosciences). Antibodies were diluted in FACS buffer (Hanks' Balanced Salt Solution with 2% fetal calf serum). For staining with anti‐FoxP3, cells were fixed and permeabilized with the Mouse FoxP3 buffer set (BD Biosciences). B‐cell populations were defined as follows: plasma cells, B220^lowCD138^hi; conventional B cells, B220^hi CD138^low; GC B cells, B220^hiGL7^hiPNA^hi; non‐GC B cells, B220^hiGL7^lowPNA^low; isotype switched, B220^hiGL7^hiPNA^hiIgG1^hi; nonisotype switched, B220^hiGL7^hiPNA^hiIgG1^low. T regulatory cells were identified as CD4⁺CD25⁺FoxP3⁺. Cells were analyzed on a BD FACSCanto II (BD Biosciences) after exclusion of PI⁺ dead cells. Data were analyzed using FlowJo version 10.6.2 cell cycle analysis software (BD Biosciences).

Immune capture and bead-based flow cytometry were carried out as described previously (8 (link),24 (link),25 (link)). In short, 10 µg exosomes in 100 µl PBS were incubated with 1 µg biotin-labeled anti-CD63 (Cat: 353018, Biolegend, San Diego, CA, USA) for 2 h at room temperature on a shaker. Next, 10 µl ExoCap Streptavidin magnetic beads (MBL Life Science, Woburn, MA, USA) were added and incubated for another 2 h at room temperature on a shaker. Samples were washed using a magnetic rack and subsequently stained with the following antibodies/isotype controls for 1 h at room temperature on a shaker: CD14-PE (0.5 µg, Cat: 12-0149-42) and IgG1-PE (0.5 µg, Cat: 12-4714-42) (both from eBioscience/Thermofisher Scientific), CD16-APC (0.8 µg, Cat: 36076) and IgG1-APC (0.8 µg, Cat: 400122) (both from Biolegend), CD44v3-APC (10 µl, FAB5088A) and IgG2b-APC (10 µl, IC0041A) (both from R&D, Minneapolis, MN, USA). The stained complexes were washed twice using a magnetic rack and finally resuspended in 300 µl PBS for flow cytometry. Detection was performed using a Gallios flow cytometer with Kaluza 1.0 software (Beckman Coulter, Brea, CA, USA) and 10000 events were acquired. Data are presented as relative fluorescent intensity (RFI) which is the mean fluorescence intensity of the stained sample divided by the mean fluorescence intensity of the corresponding isotype control.

The chondrocytes were fixed using a fixation/permeabilization solution kit (cat. no. 554715; BD Biosciences) and stained with CD34 (cat. no. 343607; BioLegend, Inc.; 1:100), CD44 (cat. no. 338807; BioLegend, Inc.; 1:100), CD59 (cat. no. 304711; BioLegend, Inc.; 1:100), CD74 (cat. no. 326811; BioLegend, Inc.; 1:100), CD90 (cat. no. 328109; BioLegend, Inc.; 1:100), CD105 (cat. no. 323205; BioLegend, Inc.; 1:100), CD146 (cat. no. 361015; BioLegend, Inc.; 1:100), CD164 (cat. no. 324805; BioLegend, Inc.; 1:100), SOX9-Alexa-647 (cat. no. 565493; BD Pharmingen; BD Biosciences; 1:200), ACAN-PE (cat. no. sc-33695; Santa Cruz Biotechnology, Inc.; 1:100), IgG1-Alexa-647 (cat. no. 557732; BD Pharmingen; BD Biosciences; 1:100), IgG1-PE (cat. no. 400112; BioLegend, Inc.; 1:100), IgG1-APC (cat. no. 400122; BioLegend, Inc.; 1:100), IgG1-FITC (cat. no. 400109; BioLegend, Inc.; 1:100) or IgG2a-APC (cat. no. 400221; BioLegend, Inc.; 1:100) for 30 min at 4˚C. The dilution of the antibody for FACS was ranged from 1:100-1:200. After staining, cells were washed with Perm/Wash Buffer (cat. no. 554715; BD Biosciences) and analyzed by flow cytometry (FACS Canto II; BD Biosciences). Data were analyzed using FlowJo version 10.7 software (FlowJo LLC).