Pe cy7 il10

Dulbecco’s Modified Eagle Medium (DMEM/HEPES, Cat #12430-054) and Penicillin/Streptomycin (Cat #15240062) were from Gibco (Carlsbad, CA). Fetal bovine serum (FBS) and G418 sulfate were obtained from Invitrogen (Carlsbad, CA). Rabbit antibody against β-actin (Cat #4970S) was from Cell Signaling (Beverly, MA). Rabbit antibody against NHE1 (Cat #ab67314) and rat antibody against CD8 (Cat #ab22378) were from Abcam Ltd. (Cambridge, MA). Rat anti-mouse CD31 (Cat #550274) was from BD Pharmingen (San Jose, CA). Rabbit anti-ionized calcium-binding adapter molecule 1 (iba1) was from Wako (Richmond, VA). PerCP/Cy5.5-CD45, PE-P2RY12, BV421-TGFβ, BV605-TNFα, APC/780-IL-1β, PE/Cy7-IL10, PerCP/Cy5.5-CD8a, APC/Cy7-CD4, Alexa Fluor 700-CD25, PE-FoxP3, BV605-PD-1, PE-Gr-1, APC-NK1.1, and Pacific Blue Granzyme B were obtained from Biolegend (San Diego, CA). eFluor 450-CD16/32, PE/Cy7-CD206, APC-IFNγ was purchased from eBioscience (San Diego, CA). APC-IL-6 were obtained from thermos fisher (Waltham, MA). BUV737-CD11b, Alexa Flour 700-CD86 were obtained from BD Biosciences (San Jose, CA). PE-Ym1were from Abcam Ltd. (Cambridge, MA). Rabbit anti-Laminin (Cat #L9393) and cariporide (HOE642) was purchased from Sigma Chemicals (St. Louis, MO). Anti-mouse PD-1 (RMP1-14) and IgG2a isotype control were from BioXcell (West Lebanon, NH).

For surface marker staining, mononuclear cells were previously blocked with anti-CD16/32 antibody (Thermo Fisher Scientific) for 15 min and then incubated with the following anti-mouse antibodies: PE-CD19, PE-B220 and Pacific blue-CD45 (all purchased from Thermo Fisher Scientific).
For intracellular marker staining, cells were previously stimulated with 50 ng/mL PMA and 750 ng/mL ionomycin in the presence of 20 µg/mL brefeldin A for 5 h, then surface-stained for 30 min. After surface marker staining, cells were resuspended in Fixation/Permeabilization Buffer Set (Thermo Fisher Scientific) and stained with PE-Cy7-IL-10 (BioLegend, San Diego, CA, USA); APC-IL-12A (Thermo Fisher Scientific) and FITC-EBI3 (Novus Biologicals, Littleton, CO, USA) for 45 min according to the manufacturer’s protocol.
For intranuclear (p-STAT3) marker staining, cells were previously surface stained and then fixed and permeabilized using a transcription factor staining buffer set (Thermo Fisher Scientific) and stained with PE-p-STAT3 (Thermo Fisher Scientific) for 45 min according to the manufacturer’s protocol.
Isotype antibodies were used as negative controls. Flow cytometry analysis was performed using BD Celesta Analyzer.