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INCUCYTE® APPLICATIONS

IncuCyte® Live-Cell Immunocytochemistry

Overview

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.

Conventional ICC is ill-suited in this regard, due to: 

  • Time consuming ‘end-point’ fix and stain protocols that yield a single image per time-point
  • Requirement to concatenate data from different samples (assay wells) to build time-courses, potentially introducing artefacts
  • Loss of environmental control, resulting in cellular changes that may lead to unwanted and misleading experimental results

Continuous imaging and analysis of proteins on living cells over time enables the tracking of spatio-temporal changes and provides further insight into how cells respond to their environment. With a live-cell approach to Immunocytochemistry, researchers can:

  • Reveal protein dynamics in a physiological context
  • Link protein dynamics to cell function and morphology
  • Evaluate complex co-culture models and characterize cell-cell interactions


With IncuCyte®’s new Live-Cell Immunocytochemistry protein detection technique utilizing non-perturbing IncuCyte® FabFluor-488 reagents, you can now complement your existing immunocytochemistry protocols and start gaining new insights into the dynamics of surface protein expression.

White Paper Cover Live-cell Immunocytochemistry

WHITE PAPER: Live-cell Immunocytochemistry

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LIVE-CELL IMMUNOCYTOCHEMISTRY

Introducing IncuCyte® Live-cell Immunocytochemistry

IncuCyte Live-cell Immunocytochemistry 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.  IncuCyte 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.

Video ICC

Key Advantages

Key Advantages

The IncuCyte live-cell analysis system and IncuCyte FabFluor-488 Antibody Labeling Reagents 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 protein expression and distribution over time using non-perturbing antibody labeling reagents — inside your incubator.

Read more below

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Unlock your productivity

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

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Couple protein expression dynamics to morphology & function

Associate changes in surface protein expression with cell function and morphology to reveal informative, temporal changes in cell behavior.

Read more below

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Derive deeper insight with cell-cell interaction studies

Visualize and quantify cell-cell interactions overtime in complex co-culture models, revealing insight into the interplay of cells.

Read more below


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 reagent fluorescence reveals cell type-, time- and concentration-dependent increase in PD-L1 expression (right). MDA-MB-231 NucLight Red (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.


Unlock your productivity

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

Immunocytochemistry ICC Quick Guide


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 IncuCyte pHrodo Red cell labeling Kit.


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.

Immunocytochemistry Figure 5 and 6

Figure 3. Visualize and quantify immune interactions within a mixed culture using IncuCyte FabFluor-488. A549 CytoLight Red 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).

Immunocytochemostry ICC video

Video.  Confirm cell-cell interactions in an immune cell killing model over time. Red fluorescent-expressing 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 mouse FabFluor-488 reagents are non-perturbing and produce specific long term labeling for powerful analysis of surface protein dynamics on live cells over hours or days.

Immunocytochemistry ICC Figure 7

Click image to enlarge

Figure 4. Visualize and quantify expression of surface markers overtime using IncuCyte FabFluor-488 reagent in combination with IncuCyte Opti-Green.  HT1080 cells cultured in the presence of IncuCyte FabFluor-488 labeled α-CD71 display green fluorescence while those cultured with IncuCyte FabFluor-488 IgG isotype control display no cellular fluorescence.  Addition of IncuCyte Opti-Green or IncuCyte FabFluor-488 labeled antibody had no effect on cell growth over 2 days (A).  Only cells treated with labeled CD71 display an increase in green 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 reagent. Ramos (B cell like) cells were grown in the presence of various 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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