Live-Cell Immunocytochemistry

Reveal the Dynamics of Surface Protein Expression with a New Approach to Immunocytochemistry

Immunocytochemistry (ICC) is an important cell imaging technique for visualizing protein localization, trafficking pathways and protein-protein interactions. With specialized microscopes, researchers can resolve labeled cell structures with nanometer resolution.

However, studying changes in protein distribution and expression over time, which may occur as cells differentiate, interact or respond to external stimuli, is more challenging.

With the new Incucyte® Live-Cell Immunocytochemistry protein detection technique utilizing non-perturbing Incucyte® Fabfluor-488 and Incucyte® Fabfluor-594 Antibody Labeling Dyes, you can now complement your existing immunocytochemistry protocols and start gaining new insights into the dynamics of surface protein expression.

White Paper: Live-Cell Immunocytochemistry

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Introducing Incucyte® Live-Cell Immunocytochemistry

Incucyte® Live-Cell Immunocytochemistry Assay combines the power of automated imaging and analysis with a simple-to-use, antibody labeling approach to generate kinetic, image-based measurements of surface protein expression – inside your tissue culture incubator.  

Live-Cell Immunocytochemistry offers a powerful solution for long-term tracking and quantification of cell surface protein markers which can then be linked to cell function and morphology, allowing for greater insight into cellular processes.

Key Advantages

The Incucyte® Live-Cell Analysis System, Incucyte® Fabfluor-488 and Incucyte® Fabfluor-594 Antibody Labeling Dyes provide a turnkey solution for quantifying the long-term dynamic changes in cell surface protein expression and distribution in 96- and 384-well formats, so you can derive meaningful results faster.

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Quantify Surface Protein Expression Dynamics

Measure the dynamic changes in cell surface proteins in response to inflammatory stimuli to provide additional insight into regulation of the immune-cell signaling pathways in tumors.

Figure 1. Automated analysis of Incucyte® Fabfluor-488 Dye fluorescence reveals cell type-, time- and concentration-dependent increase in PD-L1 expression (right). Incucyte® Nuclight Red MDA-MB-231 (breast) or SKOV-3 (ovarian) cancer cells were incubated with IFNγ in the presence of Incucyte® Fabfluor-488-α-PD-L1 antibody complex and Incucyte® Opti-Green background suppressor. Quantification of the green fluorescent area shows that IFNγ induces a time- and concentration- dependent increase in PD-L1 expression in MDA-MB-231 cells. Time-course profiles show differential PD-L1 expression in MDA-MB-231 (high expresser) and SKOV-3 (medium expresser) cells.

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Rapid, single step labeling plus automated acquisition and analysis of images from up to six 96-well plates in parallel to get answers faster.

Coupling Protein Expression Dynamics to Morphology & Function

Easily link long-term changes in protein abundance and distribution to changes in morphology and function in real time.

Figure 2. Multiplex cell surface marker, phagocytic activity, and proliferation measurements plus visualize morphology to study differentiation (right). THP-1 monocytes were exposed to media (undifferentiated), vitamin D3 or PMA in the presence of Incucyte® Fabfluor-488 antibody complexed to CD11b, CD14 or CD40. PMA showed a marked change in cell morphology (HD-phase contrast images) compared to media alone or vitamin D3 treated cells. The kinetic graphs highlight differential and time-dependent surface protein expression in response to the various treatments. Interestingly, PMA, but not media or vitamin D3, yields a decrease in cell proliferation (confluence) and concordant increase in phagocytic potential as measured by efferocytosis of apoptotic Jurkat cells labeled with pHrodo® Red Cell Labeling Kit for Incucyte®.

Derive Deeper Insight With Cell-Cell Interaction Studies

Observe and quantify cell-cell interactions via cell surface protein expression markers, enabling insight into complex co-culture models.

Figure 3. Visualize and quantify immune interactions within a mixed culture using Incucyte® Fabfluor-488 Antibody Labeling Dye. Incucyte® Cytolight Red A549 tumor cells were mixed with either pre-activated or non-activated PBMCs in the presence of Incucyte® Fabfluor-488-α-CD45 and Incucyte® Opti-Green to label the total lymphocyte population. Images at 2 hours post PBMC addition, show interactions between CD45+ cells (green) and A549 cells (red). Quantification of the interaction (overlay, yellow mask in images) reveals a marked increase in the interaction of activated effector cells with the target cells indicating cell engagement for immune killing of tumor cells (as shown in the bar graphs).

(Video)  Confirm cell-cell interactions in an immune cell killing model over time. Incucyte® Nuclight Red A549 lung cancer cells were cultured with human PBMCs in the presence of Incucyte® Fabfluor-488 tagged CD8 antibody. Activation of PBMCs with CD3/IL-2 increased PBMC interaction with tumor cells. Labeling of immune-cells with Incucyte® Fabfluor-488-CD8 confirmed polarity, where the CD8+ region of the PBMC appeared to contact the A549 tumor cell.

Validation Data

Validation of Non-Perturbing, Specific Long-Term Labeling Reagents

The Incucyte® Fabfluor-488 Dyes are non-perturbing and produce specific long term labeling for powerful analysis of surface protein dynamics on live cells over hours or days.

Figure 4. Visualize and quantify expression of surface markers overtime using Incucyte® Fabfluor-594 Dye in combination with Incucyte® Surface Fit background subtraction and Incucyte® Cell-by-Cell Analysis. HT-1080 cells cultured in the presence of Incucyte® Fabfluor-594labeled α-CD71 display red fluorescence while those cultured with Incucyte® Fabfluor-594 IgG isotype control display no cellular fluorescence. Addition of Incucyte® Opti-Green or Incucyte® Fabfluor-594 labeled antibody had no effect on cell growth over 2 days (A). Only cells treated with labeled CD71 display an increase in Red Object Area over time (B) which when normalized for cell area, shows a constant expression of CD71 (C).

Specific Antigen Detection to Identify and Quantify Mixed Cell Populations

Evaluate complex co-culture models and characterize their cellular interactions with specific surface markers that identify changes in protein expression over time.

Figure 5. Specificity and longevity of surface marker expression over time using Incucyte® Fabfluor-488 Dye. Ramos (B cell like) cells were grown in the presence of various Incucyte® Fabfluor-488 labeled antibodies in the presence of Incucyte® Opti-Green. Long term labeling with non-specific (CD45) and specific (CD20) B cell markers was observed. No fluorescent signal was seen for a T cell marker (CD3) or isotype control antibodies.  When Ramos and Jurkat (T cell like) cells were mixed in fixed ratios, the expected level of CD20 staining was measured, demonstrating the utility in mixed cultures.

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Literature and Documentation


Incucyte® Reagents, Consumables and Software

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Product Guide

Incucyte® Fabfluor-488 Antibody Labeling Dyes

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Product Guide

Incucyte® Fabfluor-594 Antibody Labeling Dyes

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Poster: AAI 2018

Fluorescent Fab/Ab to track live cell surface markers and cell populations

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