7 aad dye

Uptake of the TLR9 agonist, ODN 2216 FITC (Invivogen, San Diego, CA, USA) was assessed in magnetically sorted CD4+ T cells using flow cytometry. Briefly, 10⁵ cells were exposed to 500 ng/mL of the ligand overnight (14 hours) and the uptake of ODN 2216 was evaluated on the basis of a shift in fluorescence per cell as median fluorescence intensity (MFI) (Figure S1D). GpC ODN 2243 was used as a control to assess whether the effects are specific to CpG ODN 2216. For experiments involving assessment of uptake, ODN 2243 was labelled using Ulysis™ Alexa Fluor 488 nucleic acid labelling kit as per manufacturer instructions (Thermofisher Scientific Inc., Waltham, MA). Cells without labelled ODN were used as negative control (NC) to see baseline fluorescence in the FITC channel in each setup. Dead cells can non‐specifically bind to ODNs including ODN 2216 (Figure S1E). In view of this, all further analysis was performed on live cells by tight gating on scatter gate, followed by gating of 7‐AAD dye (e‐bioscience) negative events, for all experiments which did not require permeabilization (Figure S1). On an average, 98‐99% of cells after forward and side scatter tight gating were live cells. Further analysis was done on live cells only.

Analysis of spleen cells was carried out using a multi-color FACS analysis, following a standard procedure (39 (link), 40 (link)). Cells were stained with a combination of directly conjugated mAbs, washed, and analyzed using FACSCanto II (BD Biosciences, San Jose, CA, USA). The antibodies used were CD3-FITC, CD4-APC, and CD8-APC-Cy7 (Biolegend, San Diego, CA, USA), CD19-PE-Cy7 and CD11c-PE (eBioscience), and CD11b-PE-Cy7 (BD Biosciences). Non-viable cells staining positive with 7AAD dye (eBioscience) were excluded from the analysis. Data collected on 50,000 cells were analyzed using FACSDiva software (BD Biosciences).