Easysep mouse cd4 t cell isolation kit

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.

CD4⁺ T cells isolated from Foxp3^Cre and Foxp3^CremiR-142^fl/fl spleens by EasySep Mouse CD4⁺ T Cell Isolation Kit (STEMCELL Technologies, Vancouver, British Columbia, Canada) were sorted for YFP expressing cells using BD FACSAria II instrument. CD4⁺YFP⁺ T_reg cells were stimulated with anti-CD3 (1 μg/mL) specific antibodies for 48 hours, stained with Annexin V-PE (BioLegend) and analyzed by flow cytometry. For the IFNγ-induced apoptosis of T_reg cells assay, splenocytes were cultured for 24 hours in the presence or absence of IFNγ (10 ng/mL) and then harvested and stained with Annexin V-PE for fluorescence activated cell sorting (FACS) analysis.

In order to sort Tregs (CD4⁺, GFP⁺) from spleens of B6 Foxp3^EGFP or DEREG mice, CD4⁺ cell subpopulation was enriched prior to sorting. To this end, isolated splenocytes were subjected to immunomagnetic positive selection for CD19⁺ using EasySep™ Mouse CD19 Positive Selection Kit II (STEMCELL Technologies) and then the negative fraction was subsequently subjected to negative selection using EasySep™ Mouse CD4⁺ T cell Isolation Kit (STEMCELL Technologies). When needed, CD4⁺ cells were additionally stained with anti-CD69-PE and anti-CD44-PE-Cy7 monoclonal antibodies as described above. To sort CD8⁺ cells, the fraction of splenocytes devoid of CD19⁺ cells was stained with anti-CD8a-PerCP-Cy5.5 monoclonal antibody. Then the cells were sorted using BD FACS Aria™ III Cell Sorter (BD Biosciences).