Cpd efluor 670

Splenic B cells were isolated from bone marrow chimeras (MojoSort Mouse Pan B Cell Isolation Kit; BioLegend). Control and WNK1-deficient B cells were labeled with CellTracker blue (CMAC, 20 µM) and CellTracker orange (CMTMR, 10 µM; both Invitrogen), respectively, for 20 min at 37°C. CD4⁺ T cells were isolated from WT C57BL/6J mice and from GFP⁺ OTII mice (EasySep Mouse CD4⁺ T cell Isolation Kit; Stemcell). WT polyclonal CD4⁺ T cells were labeled with CPD eFluor670 (10 µM; eBioscience) for 20 min at 37°C. Control and WNK1-deficient B cells (5 × 10⁶ each) were transferred i.v. in a 1:1 ratio together with GFP⁺ OT-II TCR transgenic CD4⁺ T cells and polyclonal CD4⁺ T cells (3 × 10⁶ each) into recipient mice that had received 15 µg HEL-OVA, 0.2 µg LPS in Alum (total volume of 20 µl) s.c. in the foot hock 18 h earlier. At 24-h after cell transfer, intravital imaging of reactive popliteal LNs was performed on a TrimScope (LaVision Biotec) equipped with a MaiTai NIR Laser (Spectraphysics) tuned to 780 nm and a Nikon 25× objective with NA 1.0 (Ficht et al., 2019 (link)). For analysis of cell migration and interactions, z-stacks of 11 images from 150 to 250 µm² areas were recorded every 30 s for 30 min at the B cell follicle border. Image sequences were analyzed by semi-automated tracking using Imaris (Bitplane) and by visual inspection for interaction time.

Splenocytes were stimulated with 100 U/ml of mIL-2, 10 µg/ml mIL-4, 1 µg/ml mIL-5 (all Peprotech) and 2 µg/ml F(ab′)2 fragments goat anti-mouse IgM (Jackson ImmunoREsearch), 2 µg/ml hamster anti-mouse CD40 mAb (3/23, BD), or 100 nM ODN74 5′–AAAAAAAAAAAAAACGTTAAAAAAAAAAA–3′ (Microsynth). Proliferation was assessed using the cell proliferation dye CPD eFluor 670 (eBioscience) and viability by 7-amino-actinomycin D (7AAD) or TO-PRO3 (Invitrogen) and Annexin-V exclusion and flow cytometric analysis.

Bead-isolated CD4⁺HLA-G⁺ T cells from patients with AD (AD-ACLF, n=3) were tested for their suppressive capacities in co-cultures with allogeneic PBMCs isolated from HCs. Prior to co-culture, allogeneic PBMCs were stained with 10 μM cell proliferation dye (CPD) eFluor 670 (eBioscience, Hatfield, UK) as per manufacturer’s protocol. Cells were cultured at different responder:HLA-G⁺ suppressor ratios (16:1, 8:1, 4:1 and 2:1) in TexMACS serum-free medium (Miltenyi Biotec) in the presence of anti-CD3 monoclonal antibody stimulation (α-CD3, 0.5 μg/mL) (eBioscience) for 5 days at 37°C in 5% CO₂. Proliferation was then measured on gated CD3⁺ T cells by dilution of the CPD-eFluor 670 dye using flow cytometry. Suppressive capacity was measured as percentage of suppression calculated as: [100−(% proliferation of responders:suppressors/% proliferation of responders only)×100].30 (link)