Table 1. Antibodies, staining indexes and usage of antibodies in the panels.
| Antibody* | Clone | Format | THACT† (μg/ml) | SI-1‡ | SI-2 | SI-3 | SI-4 | SI-5 | AFCPSV§ (μg/ml) |
|---|---|---|---|---|---|---|---|---|---|
| CD3 | SK7 | FITC | 6.67 | 156 | 158 | 99.2 | 69.5 | 33.6 | 0.75 |
| CD3 | UCHT1 | BV510 | 6.67 | 347 | 269 | 116 | 59.6 | 199 | 0.83 |
| CD3 | UCHT1 | BUV737 | 3.33 | 369 | 278 | 196 | 113 | 70.6 | 0.42 |
| CD4 | RPA-T4 | V450 | 6.67 | 183 | 173 | 144 | 95.3 | 53.4 | 0.83 |
| CD4 | RPA-T4 | V500 | 3.33 | 76.9 | 69.3 | 46.4 | 28.4 | 14.4 | 1.25 |
| CD4 | RPA-T4 | PE-Cy7 | 3.33 | 1285 | 1126 | 857 | 367 | 200 | 0.63 |
| CD8 | RPA-T8 | APC | 0.6 | 548 | 740 | 538 | 363 | 164 | 0.05 |
| CD8 | RPA-T8 | BB515 | 1.67 | 1609 | 1089 | 446 | 188 | 62.4 | 0.26 |
| CD11c | B-ly6 | PE | 6.67 | 167 | 127 | 104 | 78.3 | 51.9 | 0.83 |
| CD14 | M5E2 | PE-Cy7 | 3.33 | 55 | 87.8 | 58.3 | 80.2 | 63.5 | 0.38 |
| CD16 | 3G8 | V450 | 13.33 | 77.9 | 157 | 109 | 72.4 | 36.6 | 5 |
| CD16 | NKP15 | FITC | 10 | 122 | 134 | 90.4 | 55.4 | 24.6 | 3.33 |
| CD19 | HIB19 | BB515 | 3.33 | 211 | 192 | 130 | 97.8 | 45.1 | 0.75 |
| CD19 | HIB19 | PE-CF594 | 3.33 | 414 | 524 | 613 | 605 | 529 | 0.38 |
| CD21 | B-ly4 | PE | 5 | 304 | 369 | 420 | 457 | 511 | 0.38 |
| CD24 | ML5 | PE-Cy7 | 3.33 | 238 | 178 | 127 | 88.3 | 48.6 | 0.75 |
| CD25 | 2A3 | BB515 | 1.67 | 21 | 21.5 | 19.7 | 14.3 | 12.2 | 0.83 |
| CD27 | M-T271 | PE-CF594 | 3.33 | 603 | 603 | 375 | 314 | 158 | 0.75 |
| CD27 | L128 | BV711 | 6.67 | 147 | 166 | 184 | 155 | 132 | 1.5 |
| CD28 | CD28.2 | APC | 1.67 | 84.3 | 72.9 | 62 | 42.9 | 34.6 | 0.83 |
| CD38 | HIT2 | BV421 | 13.32 | 34.1 | 32 | 27.5 | 21.5 | 11.8 | 2.99 |
| CD39 | 7 TU66 | BUV737 | 6.67 | 24.4 | 25.2 | 19.9 | 13.7 | 8.98 | 3.33 |
| CD45 | HI30 | BUV395 | 6.67 | 59.9 | 75.6 | 57.7 | 53.3 | 52.1 | 0.63 |
| CD45RA | L48 | PE-Cy7 | 3.33 | 106 | 110 | 94.3 | 80 | 57.8 | 0.42 |
| CD45RO | UCHL1 | BV786 | 1.67 | 113 | 66.3 | 50.9 | 31.6 | 13.2 | 0.63 |
| CD56 | NCAM16.2 | PE | 1.67 | 96.1 | 59.4 | 144 | 100.5 | 78.9 | 0.31 |
| CD57 | NK-1 | PE-CF594 | 6.67 | 851 | 742 | 304 | 138 | 67.3 | 1.25 |
| CD62L | SK11 | BV711 | 13.33 | 77.1 | 64.2 | 42.4 | 43.6 | 47.5 | 1.06 |
| CD64 | MD22 | APC-R700 | 1.67 | 126 | 157 | 129 | 72.4 | 45.6 | 0.63 |
| CD88(C5aR)** | D53-1473 | PE | 13.32 | 19.9 | 12.3 | 4.35 | 2.61 | 2.31 | 14.2 |
| CD123 | 7G3 | PE-CF594 | 1.67 | 279 | 304 | 263 | 230 | 94.7 | 0.2 |
| CD127 | HIL-7R-M21 | PE | 26.68 | 83.1 | 87.8 | 93.2 | 94 | 76.5 | 2.5 |
| CD127 | HIL-7R-M21 | PE-CF594 | 1.67 | 26.1 | 19.9 | 14.6 | 7.39 | 3.32 | 1.25 |
| CD137(4-1BB)** | 4B4-1 | BV421 | 6.67 | 15.6 | 15.9 | 13.4 | 10.5 | 9.66 | 0.8 |
| CD141(BDCA3) | A1A4 | BV711 | 1.67 | 10.4 | 8.96 | 9.02 | 7.56 | 7.4 | 0.83 |
| CD154** | TRAP1 | APC | 0.6 | 7.55 | 7.05 | 6.43 | 6.11 | 5.5 | 0.3 |
| CD183(CXCR3) | 1C6 | PE-CF594 | 6.67 | 37.9 | 28.3 | 19.8 | 12.6 | 11.2 | 6.0 |
| CD197(CCR7)* | 150503 | PE | 1.67 | 20.6 | 15 | 11.8 | 9.77 | 6.01 | 1.67 |
| CD303(BDCA2)* | AC144 | APC | 6.67 | 96.2 | 63.4 | 48.1 | 27.6 | 18 | 3.33 |
| FOXP3 | 259D/C7 | PE-CF594 | - | - | - | - | - | - | 10 |
| HLA-DR | G46-6 | BV510 | 3.33 | 177 | 110 | 63.9 | 43.8 | 27.8 | 0.83(P3) 1.5(P5) |
| IgD | IA6-2 | BV510 | 3.33 | 126 | 128 | 122 | 108 | 70.4 | 0.75 |
| IgM** | G20-127 | APC | 0.6 | 69.9 | 50.8 | 38.9 | 32.7 | 25.8 | 0.4 |
| TCRαβ | T10B9.1A-31 | BUV737 | 3.33 | 50 | 29 | 14.2 | 6.43 | 3.63 | 3.33 |
| TCRγδ | 11F2 | PE-Cy7 | - | - | - | - | - | - | 2.0 |
| Lineage (CD3, CD14, CD19,CD20, CD56) |
SK7, SJ25C1, L27, MφP9, NCAM16.2 | FITC | - | - | - | - | - | - | 5.28 |
*Used antibodies were supplied by BD Biosciences, except CD303 and CCR7 were obtained from Miltenyi Biotec and R&D Systems respectively.
†THACT: The highest antibody concentration for titration.
‡SI-1: Staining index-1 (SI-1) under the highest antibody concentration. 100μl of anticoagulated-WPB and 160μl total staining volume were used for antibody titration under 5–6 serial dilutions of antibodies (1, 1/2, 1/4, 1/8, 1/16 or 1/32) at room temperature (22°C to 30°C) for 15 minutes. The majority of these antibodies were assessed for their SI on lymphocytes or combined monocytes based on FCS vs SSC. The SI of CD25, CD127, CD154 and 4-1BB on CD3+CD4+ T cells, CD28, CD62L and CCR7 on CD3+ T cells, CD8 on CD4- negative lymphocytes, and CD56 on CD19 negative lymphocytes were assessed.
**The SI of C5aR were assessed on granulocytes. Anticoagulated-WPB was washed with cold PBS twice before IgM staining. The antibody titrations for 4-1BB and CD154 were performed on 1×106 PBMCs that were stimulated with CD3/CD28 beads overnight.
§AFCPSV: antibody final concentration in the panel staining volume.
P3: Panel 3
P5: Panel 5