Table 1.
Details of the antibody panel used for peripheral blood staining. The conjugated metal, target antigen, clone, supplier, and final concentration of each antibody is reported. The inclusion or exclusion of each antigen in manual gating and FlowSOM analysis is indicated. For manual gating, a tick indicates use of the marker in the manual gating strategy to classify cell populations. For FlowSOM analysis, ‘type’ markers were included in clustering parameters, while ‘state’ markers did not contribute to the clustering algorithm but were compared for median expression within clusters. Two markers were conjugated to a fluorophore rather than a metal (pPLCγ2-PE and RORγt-Alexa Fluor 647) and a metal-conjugated anti-secondary antibody (175Lu-anti-PE and147Sm-anti-Cy5 respectively) was instead used to visualise these markers. Seven markers were excluded from analysis due to suboptimal staining quality. Transcription factors were stained for their phosphorylated form (denoted with a ‘p’ suffix).
| Staining Parameters |
Manual Gating | FlowSOM |
|||||
|---|---|---|---|---|---|---|---|
| CD3+ | CD19−CD3− | ||||||
| Metal | Antigen | Clone | Supplier | Conc. (μg/mL) | |||
| 104Pd | CD45 | HI30 | Biolegend | 4 | ✓ | ||
| 106Pd | CD45 | HI30 | BD | 6 | ✓ | ||
| 115In | CD8 | RPA-T8 | Biolegend | 4 | ✓ | Type | |
| 141Pr | CD235ab | HIR2 | Biolegend | 1.6 | ✓ | ||
| 142Nd | CD19 | HIB19 | Biolegend | 1 | ✓ | ||
| 143Nd | CD56 | REA196 | Miltenyi Biotec | 0.5 | ✓ | Type | Type |
| 144Nd | TCRγδ | B1 | Biolegend | 2 | ✓ | Type | |
| 145Nd | CD4 | RPA-T4 | Biolegend | 1 | ✓ | Type | |
| 146Nd | CD123 | 6H6 | Biolegend | 0.5 | ✓ | Type | |
| 147Sm | Anti-Cy5 | CY5-15 | Sigma | 4 | - | – | – |
| 148Nd | CD16 | B73.1 | BD | 1 | ✓ | Type | |
| 149Sm | CD25 | M-A251 | Biolegend | 4 | ✓ | Type | |
| 150Nd | pSTAT5 | 47 | BD | 6 | Excluded | ||
| 151Eu | CD39 | A1 | Biolegend | 4 | Type | ||
| 152Gd | CD66b | G10F5 | BD | 3 μL/mL | ✓ | ||
| 153Eu | pSTAT1 | 14/P-STAT1 | BD | 2 | Excluded | ||
| 154Gd | pAKT | J1.233.371 | BD | 4 | State | State | |
| 155Gd | CD27 | M-T271 | BD | 1 | State | ||
| 156Gd | pp38 MAPK | 30/p38 MAPK | BD | 4 | Excluded | ||
| 158Gd | pSTAT3 | 4/P-STAT3 | BD | 8 | Excluded | ||
| 159Tb | CD197 (CCR7) | 150503 | R&D Systems | 6 | Excluded | ||
| 160Gd | CD14 | M5E2 | BD | 4 | ✓ | Type | |
| 161Dy | CD141 | AD5-14H12 | Miltenyi Biotec | 8 | ✓ | Type | |
| 162Er | Foxp3 | PCH101 | eBioscience | 3 | Type | ||
| 163Dy | CD1c (BDCA-1) | L161 | Biolegend | 1 | ✓ | Type | |
| 164Er | CD45RO | UCHL1 | BD | 6 | Type | ||
| 165Ho | CD61 | VI-PL2 | Biolegend | 2 | ✓ | ||
| 166Er | pp65 NFκB | K10–895.12.50 | BD | 4 | State | State | |
| 167Er | CD11c | Bu15 | Biolegend | 2 | ✓ | Type | |
| 168Er | pERK1/2 | 4B11B69 | Biolegend | 2 | State | State | |
| 169Tm | CD45RA | HI100 | BD | 2 | ✓ | Type | |
| 170Er | CD3 | UCHT1 | Biolegend | 2 | ✓ | State | |
| 171Yb | CD62L | DREG-56 | Biolegend | 4 | ✓ | Type | |
| 172Yb | CD130 (gp130) | AM64 | BD | 8 | State | State | |
| 173Yb | pMK2 | P24-694 | BD | 4 | State | State | |
| 174Yb | HLA-DR | L243 | Biolegend | 2 | ✓ | Type | |
| 175Lu | Anti-PE | PE001 | Biolegend | 4 | - | – | – |
| 176Lu | CD127 (IL-7Rα) | A019D5 | Biolegend | 2 | ✓ | Type | State |
| 209Bi | T-bet | 4B10 | BD | 2 | Excluded | ||
| PE | pPLCγ2 | K86–689.37 | BD | 200 μL/mL | State | State | |
| AF647 | RORγt | Q21-559 | BD | 4 | Excluded | ||