Broad Immune Profiling Assay Kit
16-Plex for Human PBMC Samples and ZellScannerONE™
AbKT-1002-10RXN
VistaPlex™ Assay Kits for ChipCytometry offer a ready to use, reliable and customizable solution to fast-track precise spatial multiplexing.
Designed to provide a convenient, flexible, and modular analysis of immune cells, the Broad Immune Profiling Assay kit enables quantification of 16 phenotypic biomarkers that define T Cell, B Cell, Leukocyte, NK Cell, NKT Cell, Monocyte, Dendritic and Exhaustion Cell populations and sub-populations in human PMBC samples. Validation was performed on the ChipCytometry™ platform on 4 blood donors in duplicate, in addition to individual antibody validation. The Broad Immune Profiling Assay Kit contains primary antibodies and antibody diluent and is optimized for ease of use and throughput.
| Biomarkers identified and quantified: | |||
|---|---|---|---|
| CD3 | CD25 | CD39 | CD8 |
| CD4 | CD27 | CD45RA | CD45 |
| CD56 | CD16 | CD14 | CD19 |
| CD279 | CD11c | HLA-DR | CD123 |
| Cell population characterized and quantified: | |||
|---|---|---|---|
| Leukocytes | T Cells | T-Cytotoxic | T-Helper |
| T-Regulatory | Naive CD4+ | Central Memory CD4+ | Effector CD4+ |
| Effector Memory CD4+ | Naive CD8+ | Central Memory CD8+ | Effector CD8+ |
| Effector CD8+ | NK Cells | NKT Cells | B Cells |
| Memory B Cells | Naive B Cells | Classical Monocytes | Non-classical Monocytes |
| Dedritic Cells | Myeloid Dendritic Cells | Plasmacytoid Dendritic Cells | Exhausted CD4 T-Cells |
| Exhausted CD8 T-Cells | |||
Details & Data
Gating Strategy
Hierarchical gating strategies define cell phenotypes. From image data, each cell is captured by automated segmentation, and the biomarker expression is quantified using HDR imaging to measure fluorescence intensity for every cell. Standard FCS files are generated to enable the identification of cell phenotypes via classic flow cytometry-like hierarchical gating.
| Cell Population | Parent Gate | Gating Strategy |
|---|---|---|
| Leukocytes | All | CD45+ |
| T cells | CD45+ Leukocytes | CD45+ CD3+ |
| T Cytotoxic | T Cells | CD45+ CD3+ CD4- CD8+ |
| T Helper | T Cells | CD45+ CD3+ CD4+ CD8- |
| T Regulatory | T Helper | CD45+ CD3+ CD4+ CD8- CD27+ CD45RA+ |
| Naive CD4+ | T Helper | CD45+ CD3+ CD4+ CD8- CD27+ CD45RA+ |
| Central Memory CD4+ | T Helper | CD45+ CD3+ CD4+ CD8- CD27+ CD45RA- |
| Effector CD4+ | T Helper | CD45+ CD3+ CD4+ CD8- CD27- CD45RA+ |
| Effector Memory CD4+ | T Helper | CD45+ CD3+ CD4+ CD8- CD27- CD45RA- |
| Naive CD8+ | T Cytotoxic | CD45+ CD3+ CD4- CD8+ CD27+ CD45RA+ |
| Central Memory CD8+ | T Cytotoxic | CD45+ CD3+ CD4- CD8+ CD27+ CD45RA- |
| Effector CD8+ | T Cytotoxic | CD45+ CD3+ CD4- CD8+ CD27- CD45RA+ |
| Effector Memory CD8+ | T Cytotoxic | CD45+ CD3+ CD4- CD8+ CD27- CD45RA- |
| NK Cells | CD45+ Leukocytes | CD45+ CD3- CD56+ |
| NKT Cells | CD45+ Leukocytes | CD45+ CD3+ CD56+ |
| B Cells | CD 45+ Leukocytes | CD45+ CD3- CD19+ |
| Memory B Cells | B Cells | CD45+ CD3- CD19+ CD27+ |
| Naive B Cells | B Cells | CD45+ CD3- CD19+ CD37- |
| Classical Monocytes | CD3- Leukocytes | CD45+CD3-CD14+CD16- |
| Non-Classical Monocytes | CD3- Leukocytes | CD45+CD3-CD14+CD16+ |
| Dendritic Cells | CD45+CD3-CD14-CD19- | CD45+CD3-CD14-CD19-HLA-DR+CD56- |
| Myeloid Dendritic Cells | Dendritic Cells | CD45+CD3-CD14-CD19-HLA-DR+CD56-CD11c+CD123- |
| Plasmacytoid Dendritic Cells | Dendritic Cells | CD45+CD3-CD14-CD19-HLA-DR+CD56-CD11c-CD123+ |
| Exhausted CD4 T Cells | T Helper | CD45+CD3+CD56-CD4+CD8-CD279+ |
| Exhausted CD8 T Cells | T Cytotoxic | CD45+CD3+CD56-CD4-CD8+CD279+ |
Reliable
The ChipCytometry™ platform captures biological variability, while maintaining technical reproducibility. For the Broad Immune Profiling Assay Kit, technical replicates were tested on 4 different donors. Fresh PMBC samples were used to confirm intra-assay and inter-assay reproducibility. To ensure high quality data can be obtained on a variety of sample types, the assay kit was successfully tested on cryopreserved samples and samples collected with Cyto-Chex BCT tubes.
Ready to Use and Customizable
The Broad Immune Profiling Assay Kit has been optimized to maximize throughput by minimizing the number of cycles required for analysis. One open imaging channel for filter set FS395 in each cycle allows for easy customization. Additional cycles may be added for further customization of your high-plex assay.
| Cycle | Antibody | Corresponding Filter Set | Vol. Antibody | Diluent Volume | Incubation Time |
|---|---|---|---|---|---|
| 1 | CD3 | FSPerCP | 10μL | 260μL | 5 min |
| CD25 | FS560 | 10μL | |||
| CD39 | FS488 | 10μL | |||
| CD8 | FS421 | 10μL | |||
| 2 | CD4 | FSPerCP | 10μL | 260μL | 5 min |
| CD27 | FS560 | 10μL | |||
| CD45RA | FS488 | 10μL | |||
| CD45 | FS421 | 10μL | |||
| 3 | CD56 | FSPerCP | 10μL | 260μL | 5 min |
| CD16 | FS560 | 10μL | |||
| CD14 | FS488 | 10μL | |||
| CD19 | FS421 | 10μL | |||
| 4 | CD279 | FSPerCP | 10μL | 260μL | min |
| CD11c | FS560 | 10μL | |||
| HLA-DR | FS488 | 10μL | |||
| CD123 | FS421 | 10μL |
Broad Immune Profiling Assay Kit Documentation
Download the Protocol for easy-to-use instructions for the Broad Immune Profiling Assay kit. The Assay Template file can be imported onto the ZKW App to pre-load the assay instructions for quick and easy assay set up.
Store all kit contents at 4°C, protected from light.
Additional materials required: ZellSafe™ Chips (28050606/01-010) and Wash Buffer (28050606/07-004).