Spatial Immune Profiling Assay Kit
14-Plex for Human FFPE Samples and ZellScannerONE™
AbKT-3001-10RXN
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 key immune cells, the Spatial Immune Profiling Assay Kit enables quantification of 13 phenotypic biomarkers that define key immune cell populations and subpopulations in human FFPE samples. Validation was performed on the ChipCytometry™ platform on 3 tissue types in triplicate, in addition to individual anti body validation. The Spatial Immune Profiling Assay Kit contains 13 primary antibodies, antibody diluent, secondary antibodies, fluorescence quencher, and DNA stains; and is optimized for ease of use and throughput.
| Biomarkers identified and quantified: | ||||
|---|---|---|---|---|
| CD3 | CD27 | Ki-67 | CD45 | CD45RA |
| FoxP3 | CD68 | EpCAM | PD-1 | CD8 |
| PD-L1 | Granzyme B | CD4 | Nuclear Stains | |
| Cell population characterized and quantified: | |||
|---|---|---|---|
| Leukocytes | Epithelial cells | T-cells | Macrophages |
| T helper cells | T regulatory cells | Proliferating T-helper cells | Exhausted T-helper cells |
| Central Memory T-helper cells | Naive T-helper cells | Effector T-helper cells | Effector Memory T-helper cells |
| T cytotoxic cells | Proliferating T cytotoxic cells | Exhausted T cytotoxic cells | Naive T cytotoxic cells |
| Effector T cytotoxic cells | Effector memory 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.
Representative dot plots demonstrating a gating strategy to identify immune cell subtypes.
Example Gating Strategy
| Population | Marker Definition |
|---|---|
| Nucleated cells | DNA+ |
| Leukocytes | DNA+ CD45+ CD326- |
| Epithelial cells | DNA+ CD45- CD326+ |
| T-cells | DNA+ CD45+ CD326- CD3+ CD68- |
| CD4+ T cells | DNA+ CD45+ CD326- CD3+ CD68- CD4+ |
| Treg | DNA+ CD45+ CD326- CD3+ CD68- CD4+ FOXP3+ |
| Proliferating T-helper | DNA+ CD45+ CD326- CD3+ CD68- CD4+ FOXP3- Ki-67+ CD279- |
| Exhausted T-helper | DNA+ CD45+ CD326- CD3+ CD68- CD4+ FOXP3- Ki-67- CD279+ |
| Central Memory T-helper | DNA+ CD45+ CD326- CD3+ CD68- CD4+ FOXP3- CD27+ CD45RA- |
| Naive T-helper | DNA+ CD45+ CD326-CD3+ CD68- CD4+ FOXP3- CD27+ CD45RA+ |
| Effector T-helper | DNA+ CD45+ CD326- CD3+ CD68- CD4+ FOXP3- CD27- CD45RA+ |
| Effector Memory T-helper | DNA+ CD45+ CD326- CD3+ CD68- CD4+ FOXP3- CD27- CD45RA- |
| CD8+ Tcells | DNA+ CD45+ CD326- CD3+ CD68- CD4- CD8+ |
| GranB+ CD8 | DNA+ CD45+ CD326- CD3+ CD68- CD4- CD8+ GranB+ |
| Proliferating CD8 | DNA+ CD45+ CD326- CD3+ CD68- CD4- CD8+ Ki-67+ CD279- |
| Exhausted CD8 | DNA+ CD45+ CD326- CD3+ CD68- CD4- CD8+ Ki-67- CD279+ |
| Central Memory CD8 | DNA+ CD45+ CD326- CD3+ CD68- CD4- CD8+ CD27+ CD45RA- |
| Naive CD8 | DNA+ CD45+ CD326- CD3+ CD68- CD4- CD8+ CD27+ CD45RA+ |
| Effector CD8 | DNA+ CD45+ CD326- CD3+ CD68- CD4- CD8+ CD27- CD45RA+ |
| Effector Memory CD8 | DNA+ CD45+ CD326- CD3+ CD68- CD4- CD8+ CD27- CD45RA- |
Reliable
The ChipCytometry™ platform captures biological variability, while maintaining technical reproducibility. For the Spatial Immune Profiling Assay Kit, technical replicates were tested on 3 different FFPE tissue types. FFPE tissue samples were used to confirm intra-assay and inter-assay re producibility. To ensure high quality data can be obtained on a variety of sample types, the assay kit was successfully tested in triplicate on a variety of FFPE samples.
Ready to Use and Customizable
The Spatial Immune Profiling Assay Kit has been optimized to maximize throughput by minimizing the number of cycles required for analysis. Open imaging channels in cycles 2 and 5 allows for easy customization. Additional cycles may be a dded for further customization of your high-plex assay.
Spatial Immune Profiling Assay Kit Stain Plan
| Cycle | Target | Filter Set | Antibody Volume | Diluent Volume | Incubation Time |
|---|---|---|---|---|---|
| 1 | Ki67 | FS488 | 50 μL | 450 μL | 1 hr |
| CD27 | FS560 | 50 μL | |||
| CD3 | FSPerCP | 50 μL | |||
| 2 | CD4 | FS488 | 50 μL | 500 μL | 1 hr |
| CD45 | FSPerCP | 50 μL | |||
| 3 | CD68 | FS488 | 50 μL | 450 μL | 1 hr |
| FoxP3 | FS560 | 50 μL | |||
| CD45RA | FSPerCP | 50 μL | |||
| 4 | CD8 | FS488 | 50 μL | 450 μL | 1 hr |
| PD-1 | FS560 | 50 μL | |||
| EpCAM | FSPerCP | 50 μL | |||
| 5 | Granzyme B | FS488 | 50 μL | 500 μL | 1 hr |
| PD-L1 | FS560 | 50 μL |
Documentation
Download the Protocol for easy-to-use instructions for the Spatial 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).