7 aad viability staining

For DC isolation, spleens or thymuses were cut and digested with collagenase III and Dnase I, followed by light-density separation and then immune-magnetic bead depletion using the procedure described elsewhere (23 (link)). For generation of Fms-like tyrosine kinase 3 (Flt3) ligand (FL)-cultured bone marrow-derived pDCs (24 (link), 25 (link)) in vitro, total bone marrow cells were cultured for 7–9 days in culture medium consisting of RPMI1640 medium supplemented with essential and nonessential amino acids, 5.5 × 10⁻⁵mol/L 2-mercaptoethanol, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% FBS (Gibco) with 100 ng/mL recombinant murine FL (Peprotech). Dead cells were excluded by 7-AAD viability staining (eBioscience). DCs were stained with combinations of mAbs to CD11c (N418), SiglecH (eBio440C), B220 (RA3-6B2), CD172a (P84), CD8a (53-6.7), and CD24 (M1/69). CD11c^intSiglecH⁺B220⁺ were used to gate pDCs population. The antibodies were purchased from eBioscience (San Diego, CA, USA) or Biolegend (San Diego, CA, USA). Cells were analyzed with a LSRII flow cytometer (BD) or sorted with a FACSAria III machine (BD). FACS data were analyzed and displayed by FlowJo software (Tree Star).

Cell viability was assessed using 7-AAD viability staining (eBioscience, San Diego, CA) or Fixable Viability Dye eFluor™ 506 (eBioscience, San Diego, CA) according to the manufacturer’s protocols. In short, adherent cells were harvested by trypsinization and pooled with any detached cells recovered from the supernatant following centrifugation at 200×g for 10 min. For 7-AAD staining cells were washed with PBS, and stained with 5 μL dye per 1 × 10⁶ cells for 5 min at RT. Cells were analyzed using a BD FACS Canto II (BD Biosciences, San Jose, CA) with BD FACS Diva software (version 6.1.3). For the Fixable Viability Dye eFluor™ 506 labeling, cells were washed twice in ice-cold PBS followed by staining on ice for 30 min using a 1:1000 working dilution. Unbound dye was removed by washing with PBS and the cells were fixed in 1% PFA. Cells were analyzed using a CytoFLEX LX (Beckman Coulter). Data analysis was performed using FlowJo v10.07 software (FlowJo LLC, Ashland, OR). Heat shocked cells were taken along in all experiments as a positive control. To this end, cells were incubated for 3 min at 65 °C followed by immediate placement on ice for 1 min.