For flow cytometric analysis, single-cell suspensions were stained as described (25 (link)). Intracellular staining was conducted using a Fixation/Permeabilization kit (eBioscience, San Diego, CA, USA) following incubation of cells with Leukocyte Activation Cocktail (BD, San Diego, CA, USA) with Brefeldin A or Monensin (eBioscience). Fragment crystallizable (Fc) receptors were blocked using purified CD16/32 monoclonal antibody (mAb) (BD). Viable lymphocytes were assessed using Live/Dead Fixable Dead Cell Stain Kit (Invitrogen, Carlsbad, CA, USA). Matching isotype mAbs were used to control for background staining.
Anti-mouse CD5 (53-7.3), B220 (RA3-6B2), CD3 (17A2) and CD39 (24DMS1) were purchased from eBioscience. Anti-mouse CD19 (6D5) was from Biolegend (San Diego, CA, USA). Anti-mouse CD4 (RM4-5), CD25 (PC61) and Foxp3 (MF23) were purchased from BD.
Human cells were stained with CD20 APC-Cy7 (L27) and CD5 PE-Cy7 (L17F12), (BD, San Diego, CA, USA). TLR9 PE (26C593.2) was purchased from Enzo Life Sciences (Farmingdale, NY, USA). CD19 PE (HIB19) was purchased from eBioscience. A BD LSRII flow cytometer was used for assessment of cell suspensions (San Jose, CA, USA). Analysis was conducted using FlowJo software (Treestar, Ashland, OR, USA).
Anti-mouse CD5 (53-7.3), B220 (RA3-6B2), CD3 (17A2) and CD39 (24DMS1) were purchased from eBioscience. Anti-mouse CD19 (6D5) was from Biolegend (San Diego, CA, USA). Anti-mouse CD4 (RM4-5), CD25 (PC61) and Foxp3 (MF23) were purchased from BD.
Human cells were stained with CD20 APC-Cy7 (L27) and CD5 PE-Cy7 (L17F12), (BD, San Diego, CA, USA). TLR9 PE (26C593.2) was purchased from Enzo Life Sciences (Farmingdale, NY, USA). CD19 PE (HIB19) was purchased from eBioscience. A BD LSRII flow cytometer was used for assessment of cell suspensions (San Jose, CA, USA). Analysis was conducted using FlowJo software (Treestar, Ashland, OR, USA).