Proliferation Spot T-Cell Kit for Imaging

Product Information

Introduction

The ClickTech Proliferation Spot T-Cell Kit (ProliSpot) is an innovative assay platform that combines two complementary principles in one workflow:

• EdU-based DNA synthesis detection for precise identification of proliferating T cells.
• ELISpot-based cytokine capturing for antigen-specific functional profiling of immune cells at a single cell level.

ProliSpot is an integrated approach that enables simultaneous measurement of cell division and immune activation at single-cell resolution without radioactive labeling, harsh DNA denaturation, or the need for flow cytometry analysis.

Kit Variants

1. ClickTech Proliferation Spot T-Cell Kit 488 (480 Wells) for Imaging

FITC channel (~488 nm) for fluorescence detection

2. ClickTech Proliferation Spot T-Cell Kit 647 (480 Wells) for Imaging

Cy5 channel (~647 nm) for far-red detection

Both kits are optimized for automated imaging, high-throughput analysis, and single-cell resolution, making them suitable for:

• Immunological research
• Vaccine development
• CAR-T potency testing
• Autoimmunity and cancer immunology studies

Scientific Background

EdU Assays: Detect DNA replication by incorporating 5-ethynyl-2′-deoxyuridine (EdU) into newly synthesized DNA, visualized via Click Chemistry. Advantages include high sensitivity and no need for harsh DNA denaturation (unlike necessary in BrdU based assays).

ELISpot Assays: Quantify cytokine secretion by capturing proteins on antibody-coated membranes, visualized as discrete spots. ELISpot is highly sensitive for immune function but does not measure proliferation.

ProliSpot combines both, enabling researchers to correlate proliferation and antigen-specific cytokine secretion in the same well.

Materials and Methods

1. Cell Preparation: Peripheral blood lymphocytes (PBLs) were stimulated for 48 hours with S. aureus Toxin E at 250,000 cells per well.
2. Plate Setup: Cells were coated onto ProliSpot-prepared glass-bottom plates with PVDF membranes and PEI activation for optimal protein binding.
3. EdU Detection: Incorporation of EdU was visualized via copper(I)-catalyzed azide-alkyne cycloaddition (Click Chemistry) using 6-FAM Picoly azide and Eterneon Red Picoly azide.
4. Imaging: Automated imaging performed with a 10× air objective, 6×6 mosaic acquisition, and stitching.
5. Analysis: Image segmentation, cell counting, and fluorescence intensity measurement (EdU-488 per cell) were conducted using automated software.

Results

ClickTech Proliferation Spot T-Cell Kit (ProliSpot) for ELISpot analysis was developed using baseclicks Sensitive EdU Cell Proliferation Kit for Imaging (BCK-EdUPro488IM100). The protocol and solutions were in particular adapted on plate coating efficiency and subsequent fluorescent detection quality. After establishing the procedure for the ELISpot detection and manufacturing process we compared the performance.

Coating Efficiency: Comparison between the manufactured ProliSpot kit and the adapted procedure for ELISpot detection basis of BCK-EdUPro488IM100 on adhesion efficiency (Figure 1) and detection capacity (Figure 2) showed almost identical performance of the kit to the established lab procedure.

Figure 1: Images of cell adhesion on coated plates. Generally 100.000 cells per well were analyzed and constant adhesion performance was observed from different blood donors for the ClickTech Proliferation Spot T-Cell Kit (ProliSpot) and the laboratory procedure. A. Image of a well DAPI emission (blue cell nucleus). B. Close up DAPI emission (blue cell nucleus) C. Close up FAM emission (green cells).

A.

B.

C.

Figure 2: baseclick’s newly developed ClickTech Proliferation Spot T-Cell Kit (ProliSpot) exhibited similar performance in A. adhesion of cells to the plate surface, B. fluorescence intensity per well and C. toxin stimulation evaluation testing.

Discussion

ProliSpot demonstrated high performance in cell coating, EdU incorporation and fluorescence detection allowing automated imaging capturing in ELISpot machines.

Conclusion

The ClickTech Proliferation Spot T-Cell Kit offers a sensitive, reliable, and scalable method for T-cell proliferation analysis. Its dual-readout capability, DNA synthesis and antigen-specific cytokine detection, provides a comprehensive view of immune responses, making it a powerful tool for advanced immunological research, diagnostic and drug development.

Workflow Overview

1. Cell stimulation
2. EdU incorporation
3. ProliSpot plate preparation
4. Cell transfer & fixation
5. Click Chemistry detection (FITC or Cy5)
6. Automated imaging & analysis

Technical Summary

Comparison of Methods

Kit Components

• EdU reagent
• Fluorescent azide dye (488 or 647)
• DMSO
• Reaction Buffer
• Reactor system
• Buffer Additive
• Saponin
• BSA-c
• ProliSpot PVDF plates
• Polyethylenimine (PEI) coating solution
• DAPI nuclear stain
• Glutaraldehyde
• Detailed protocol for human T-cell workflows

Key Advantages

• Dual Readout: Proliferation + immune function in one assay
• High Sensitivity: Detects rare antigen-specific T cells
• Automation Ready: Compatible with high-throughput imaging systems
• Safe & Efficient: No radioactive labeling or harsh DNA denaturation