Table 1.
Antibodies and immunostaining panels used for flow cytometry. Detailed description of the fluorochrome-conjugated antibodies and immunostaining panels used to immunophenotype and flow sort the assessed CD4+ T cell populations.
| Immunostaining Panel | Antibody | Fluorochrome | Clone | Manufacturer |
|---|---|---|---|---|
| DILT1D Treg immunophenotyping (Whole blood) | CD4 | AF700 | RPA-T4 | BioLegend |
| CD25a | APC | M-A251+2A3 | BD Biosciences | |
| CD127 | PE-Cy7 | eBioRDR5 | eBioscience | |
| CD45RA | BV785 | HI100 | BioLegend | |
| CD14 | PB | M5E2 | BioLegend | |
| CCR6 | AF488 | G034E3 | BioLegend | |
| IL-6R | PE | UV4 | BioLegend | |
| CD8 | APC-Cy7 | RPA-T8 | BioLegend | |
| CXCR3 | PerCP Cy5.5 | G025H7 | BioLegend | |
| CD62L | BV605 | DREG-56 | BioLegend | |
| IL-6RhiTreg immunophenotyping (Freshly isolated PBMCs) | CD3 | BV510 | UCHT1 | BioLegend |
| CD4 | BUV398 | SK3 | BD Biosciences | |
| CD25a | APC | M-A251+2A3 | BD Biosciences | |
| CD127 | PE-Cy7 | eBioRDR5 | eBioscience | |
| CD45RA | BV785 | HI100 | BioLegend | |
| CTLA-4 | PE-CF594 | BNI3 | BD Biosciences | |
| CD8 | APC-Cy7 | RPA-T8 | BioLegend | |
| CD62L | BV605 | DREG-56 | BioLegend | |
| PD-1 | BV711 | EH12.2 | BD Biosciences | |
| IL-6R | BV421 | M5 | BD Biosciences | |
| HELIOS | FITC | 22F6 | BioLegend | |
| Ki-67 | PerCP Cy5.5 | B59 | BD Biosciences | |
| FOXP3 | PE | 259D | BioLegend | |
| IL-6RhiTIGIT- Treg immunophenotyping (Freshly isolated PBMCs) | CD3 | BV510 | UCHT1 | BioLegend |
| CD4 | BUV737 | SK3 | BD Biosciences | |
| CD25a | APC | M-A251+2A3 | BD Biosciences | |
| CD127 | PE-Cy7 | eBioRDR5 | eBioscience | |
| CD45RA | BV785 | HI100 | BioLegend | |
| HLA-DR | AF700 | L243 | BioLegend | |
| CD8 | APC-Cy7 | RPA-T8 | BioLegend | |
| CD62L | BV605 | DREG-56 | BioLegend | |
| CCR6 | BV711 | 11A9 | BD Biosciences | |
| TIGIT | PerCP eFluor710 | MBSA43 | eBioscience | |
| CD161 | PE | HP-3G10 | BioLegend | |
| IL-6R | BV421 | M5 | BD Biosciences | |
| HELIOS | FITC | 22F6 | BioLegend | |
| Ki-67 | BUV398 | B59 | BD Biosciences | |
| FOXP3 | PE-CF594 | 259D | BioLegend | |
| FACS Sorting (Freshly isolated PBMCs) | CD3 | FITC | UCHT1 | BioLegend |
| CD4 | AF700 | RPA-T4 | BioLegend | |
| CD25 | APC | M-A251 | BD Biosciences | |
| CD127 | PE-Cy7 | eBioRDR5 | eBioscience | |
| CD45RA | BV785 | HI100 | BioLegend | |
| IL-6R | PE | M5 | BD Biosciences | |
| TIGIT | PerCP eFluor710 | MBSA43 | eBioscience | |
| Proliferation dye | eFluor450 | – | eBioscience | |
| IL-17/IL-10 production (Freshly isolated CD4+T cells) | CD3 | BV510 | UCHT1 | BioLegend |
| CD4 | BUV737 | SK3 | BD Biosciences | |
| CD25a | APC | M-A251+2A3 | BD Biosciences | |
| CD127 | PE-Cy7 | eBioRDR5 | eBioscience | |
| CD45RA | BV785 | HI100 | BioLegend | |
| TIGIT | PerCP eFluor710 | MBSA43 | eBioscience | |
| HELIOS | FITC | 22F6 | BioLegend | |
| FOXP3 | PE-CF594 | 259D | BioLegend | |
| IL-17 | AF700 | BL168 | BioLegend | |
| IC | AF700 | MOPC-21 | BioLegend | |
| IL-10 | PE | JES-9D7 | BioLegend | |
| IC | PE | RTK2071 | BioLegend | |
| Viability Dye | eFluor780 | – | eBioscience | |
| Chemokine receptor profiling (Freshly isolated PBMCs) | CD4 | BUV737 | SK3 | BD Biosciences |
| CD25a | APC | M-A251+2A3 | BD Biosciences | |
| CD127 | PE-Cy7 | eBioRDR5 | eBioscience | |
| CD45RA | BV785 | HI100 | BioLegend | |
| TIGIT | PerCP eFluor710 | MBSA43 | eBioscience | |
| CD27 | BUV398 | L128 | BD Biosciences | |
| CD8 | APC-Cy7 | RPA-T8 | BioLegend | |
| IL-6R | BV421 | M5 | BD Biosciences | |
| CCR4 | BV510 | L291H4 | BioLegend | |
| CCR6 | AF488 | G034E3 | BioLegend | |
| CCR10 | PE | 6588-5 | BioLegend | |
| CXCR3 | AF700 | G025H7 | BioLegend | |
| CXCR5 | PE-CF594 | J252D4 | BioLegend | |
| CD15s | BV711 | CSLEX1 | BD Biosciences | |
| RORγt / gut-homing immunophenotyping (Freshly isolated CD4+T cells) | CD4 | BUV737 | SK3 | BD Biosciences |
| CD25a | APC | M-A251+2A3 | BD Biosciences | |
| CD127 | PE-Cy7 | eBioRDR5 | eBioscience | |
| CD45RA | BV785 | HI100 | BioLegend | |
| TIGIT | PerCP eFluor710 | MBSA43 | eBioscience | |
| CD3 | BV510 | UCHT1 | BioLegend | |
| CCR6 | BV711 | 11A9 | BD Biosciences | |
| CD8 | APC-Cy7 | RPA-T8 | BioLegend | |
| IL-6R | BV421 | M5 | BD Biosciences | |
| ITGB7 | FITC | FIB504 | BioLegend | |
| HELIOS | FITC | 22F6 | BioLegend | |
| ITGA4 | BV605 | 9F10 | BioLegend | |
| CD62L | BV605 | DREG-56 | BioLegend | |
| CCR9 | PE | L053E8 | BioLegend | |
| RORγ/γt | PE | AFKJS-9 | eBioscience | |
| FOXP3 | PE-CF594 | 259D | BioLegend | |
| Ki-67 | BUV398 | B59 | BD Biosciences |
a
Two clones of anti-CD25 that bind to different epitopes were used simultaneously to enhance CD25 staining. IC, Isotype control.