The following fluorochrome-conjugated antibodies (mAb) were used. For the in vitro assay, Dead Cell Marker-FarRed (Life Technologies), CD3-DAPI (Biolegend, San Diego, CA, USA), CD45-BV510 (Biolegend), CD4-FITC (eBiosciences, Thermo Fisher Scientific, Waltham, MA, USA), CD8-PE (eBiosciences), CD11c-PE-Cy7 (eBiosciences), and CellTrace Far Red-APC (Invitrogen, Waltham, MA, USA) were used. For the in vivo studies, Dead Cell Marker-Aqua (Life Technologies), CD3-DAPI (Biolegend), CD45.2-PE (eBiosciences), CD45.1-eFluor780 (eBiosciences), CD4-FITC (eBiosciences), CD8-BV421 (BD), CD11c-PE-Cy7 (eBiosciences), CD25-PE Cy7 (eBiosciences), CD19-BV711 (BD) and CellTrace Far Red-APC (Invitrogen) were used. For cell surface analysis of proliferation, CD4+ T cells were collected after the co-culture, washed two times with FACS Stain Buffer (BD), and stained with the indicated antibody mastermix for 20 min at 4 °C. Then, the cells were harvested and fixed with Fix/Perm (eBioscience) and finally analyzed for Celltrace dilution with flow cytometry. Samples were acquired on a BD LSRFortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and data were analyzed using FlowJo v10 (Tree Star Inc., San Carlos, CA, USA). Calculations of percent proliferated T cells were essentially performed according to [30 (link)].
Lapazio L., Braun M, & Grandien K. (2021). H2-M and H2-O as Targeting Vehicles for the MHC Class II Processing Compartment Promote Antigen-Specific CD4+ T Cell Activation. Vaccines, 9(10), 1053.
+ Open protocol