Spatial Immune Profiling Assay Kit

14-Plex for Human FFPE Samples and ZellScannerONE™
AbKT-3001-10RXN

ChipCytometry™ Assay Kits 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:
CD3CD27Ki-67CD45CD45RA
FoxP3CD68EpCAMPD-1CD8
PD-L1Granzyme BCD4Nuclear Stains
Cell population characterized and quantified:
LeukocytesEpithelial cellsT-cellsMacrophages
T helper cellsT regulatory cellsProliferating T-helper cellsExhausted T-helper cells
Central Memory T-helper cellsNaive T-helper cellsEffector T-helper cellsEffector Memory T-helper cells
T cytotoxic cellsProliferating T cytotoxic cellsExhausted T cytotoxic cellsNaive T cytotoxic cells
Effector T cytotoxic cellsEffector 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.

Example Gating Strategy

PopulationMarker Definition
Nucleated cellsDNA+
LeukocytesDNA+ CD45+ CD326-
Epithelial cellsDNA+ CD45- CD326+
T-cellsDNA+ CD45+ CD326- CD3+ CD68-
CD4+ T cellsDNA+ CD45+ CD326- CD3+ CD68- CD4+
TregDNA+ CD45+ CD326- CD3+ CD68- CD4+ FOXP3+
Proliferating T-helperDNA+ CD45+ CD326- CD3+ CD68- CD4+ FOXP3- Ki-67+ CD279-
Exhausted T-helperDNA+ CD45+ CD326- CD3+ CD68- CD4+ FOXP3- Ki-67- CD279+
Central Memory T-helperDNA+ CD45+ CD326- CD3+ CD68- CD4+ FOXP3- CD27+ CD45RA-
Naive T-helperDNA+ CD45+ CD326-CD3+ CD68- CD4+ FOXP3- CD27+ CD45RA+
Effector T-helperDNA+ CD45+ CD326- CD3+ CD68- CD4+ FOXP3- CD27- CD45RA+
Effector Memory T-helperDNA+ CD45+ CD326- CD3+ CD68- CD4+ FOXP3- CD27- CD45RA-
CD8+ TcellsDNA+ CD45+ CD326- CD3+ CD68- CD4- CD8+
GranB+ CD8DNA+ CD45+ CD326- CD3+ CD68- CD4- CD8+ GranB+
Proliferating CD8DNA+ CD45+ CD326- CD3+ CD68- CD4- CD8+ Ki-67+ CD279-
Exhausted CD8DNA+ CD45+ CD326- CD3+ CD68- CD4- CD8+ Ki-67- CD279+
Central Memory CD8DNA+ CD45+ CD326- CD3+ CD68- CD4- CD8+ CD27+ CD45RA-
Naive CD8DNA+ CD45+ CD326- CD3+ CD68- CD4- CD8+ CD27+ CD45RA+
Effector CD8DNA+ CD45+ CD326- CD3+ CD68- CD4- CD8+ CD27- CD45RA+
Effector Memory CD8DNA+ 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

CycleTargetFilter SetAntibody VolumeDiluent VolumeIncubation Time
1Ki67FS48850 μL450 μL1 hr
CD27FS56050 μL
CD3FSPerCP50 μL
2CD4FS48850 μL500 μL1 hr
CD45FSPerCP50 μL
3CD68FS48850 μL450 μL1 hr
FoxP3FS56050 μL
CD45RAFSPerCP50 μL
4CD8FS48850 μL450 μL1 hr
PD-1FS56050 μL
EpCAMFSPerCP50 μL
5Granzyme BFS48850 μL500 μL1 hr
PD-L1FS56050 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).

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