Assays for Immunology ResearchIncuCyte® S3 Live-Cell Analysis System

Immune system characterization of health and disease states requires quantitative data from dynamic experimental models of the cells that comprise both the innate and adaptive responses and their targets. The IncuCyte® S3 Live-Cell Analysis System enables real-time, automated analysis of the dynamic changes in immune cells and their interactions inside your incubator, providing deeper functional insight.

Key Advantages:– Real-time, image-based measurements in

a physiologically-relevant environment.

– Optimized protocols in 96- and 384-well assay formats.

– Applications, reagents and consumables for non-invasive, non-disruptive analysis of cell health, phenotype and function.

– Minimal manipulation and cell loss with mix-and-read reagents.

Immune cell activation & proliferationMonitor immune cell activation in real time.

Live-cell Immuno-cytochemistryAnalysis of cell surface protein dynamics and distribution.

Chemotaxis and TEMMeasure immune cell migration toward chemokines.

Immune System Mobilization

NETosisQuantify neutrophil extracellular traps in real time.

PhagocytosisReal-time analysis of internalization and clearance using pH sensitive probes.

Immune ResponseProtein Expression

32

Gain insight into immune cell response to the external environment with continuous imaging and analysis of proteins in living cells. IncuCyte® Live-Cell Immunocytochemistry offers a powerful solution for long-term tracking and quantification of cell surface protein markers that can be linked to cell function and morphology, generating greater insight into cellular processes.

Associate changes in surface protein expression with cell function and morphologyCouple the dynamic changes in protein expression with cell function and morphology in real time.

Perform kinetic immunophenotyping to identify subpopulationsLabel cell surface markers in living cells using IncuCyte FabFluor-488 Antibody Labeling Reagents and then analyze using IncuCyte S3 Cell-by-Cell Analysis Software Module - temporally classify cells into subsets based on CD antigens without cell lifting or fix/wash protocols.

IncuCyte® Live-Cell Immunocytochemistry

Subset analysis of activated T-cells: CD71 upregulation. (A-C) Cell-by-cell density plots showing increase in CD71-fluorescence as a function of time and cell area (phase) in activated T-cells. (D) Dynamic changes in the proportion of CD71+, but not CD4+ cells, following activation. (E) Subset analysis showing preferential upregulation of CD71 expression in large (>110 μm2) vs small (<110 μm2) cells.

Multiplex cell surface marker, phagocytic activity, and proliferation measurements plus visualize morphology to study differentiation. THP-1 monocytes exposed to various treatments in the presence of IncuCyte FabFluor-488 Antibody Labeling Reagent 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. Kinetic graphs highlight differential and time-dependent surface protein expression in response to various treatments. 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.

Key Advantages:– Reveal protein dynamics in a

physiological context using non-perturbing IncuCyte® FabFluor-488 Antibody Labeling Reagents.

– NEW! Leverage IncuCyte® S3 Cell-by-Cell Analysis Software Module for kinetic analysis of heterogeneous population changes - eliminating the need for cell lifting or fix/wash protocols.

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

D

0 24 48 72 96 120 Time (h)

0 24 48 72 96 120 Time (h)

Area (μm2)30 100 300

Area (μm2)30 100 300

Area (μm2)30 100 300

Undifferentiated

Proliferation Efferocytosis

Vitamin D3 (50 nM) PMA (100 nM)

Mean intensity

0 0

20

25

40

100

10

1

0.1

50

75

60% 100% Marker+ CD71+E

CD71

Large

SmallCD4

IgG

HD phase

CD11b-FabFluor-488

0

20

40

60

80%

0 24 48 72Time (h)

ConfluenceRed area

0 0

25 2

50 4

75% 6 μm2/well

0 24 48 72Time (h)

72 96 120Time (h)

0

20

40

60

80%

0 24 48 72Time (h)

0

20

40

60

80%

0 24 48 72Time (h)

Normal green area (green phase)

CD40

PMA

PMA

Undiffer.

Undiffer.

Vitamin D3

Vitamin D3

CD40

CD14

CD11b

CD40

CD11bCD11b

CD14

CD14

BA C48 h0 h 120 h

54

Immune cell activation and proliferation are fundamental for regulating immune responses and the extent to which they occur. IncuCyte® Immune Cell Activation, Proliferation and Cell Health Assays are an integrated solution for real-time visualization and automated analysis of immune cell proliferation, activation and cell health.

IncuCyte® Activation, Proliferation & Cell Health Assays

Key Advantages:– NEW! Automatically generate

label-free total cell counts using the IncuCyte S3 Cell-by-Cell Analysis Software Module – from over 2,000 assay wells in parallel!

– Automatically detect and quantify dynamic changes in cell morphology upon activation.

– Multiplex with IncuCyte cell health

reagents for real-time analysis of apoptosis and cell death.

Uncover cell-specific cytotoxic treatment effects on immune cell health Label a population of interest and multiplex with readouts of cell health using non-perturbing reagents and the IncuCyte® S3 Cell-By-Cell Analysis Software Module.

Observe and quantify dynamic changes in cell morphology Use the power of live-cell analysis to quantify morphology changes associated with immune-cell activation using the IncuCyte® S3 Cell-By-Cell Analysis Software Module.

Quantify immune cell proliferation in real-time Automated label-free analysis of cell proliferation all within a physiologically relevant environment!

Enlargement and morphological change in activated T-cells.PBMCs were treated with anti-CD3 and IL-2, or vehicle control, and monitored over time with the IncuCyte® S3 system. (A-D) Activation induced a time-dependent increase in average area and eccentricity (all cells). Note the change in eccentricity preceded the increase in area. The cell-by-cell area distribution (E) and density plots (F-H) highlight the increased heterogeneity over time following activation, and the appearance of a population of large cells with high eccentricity. Values shown are the mean ± SD of 4 wells.

Measure cell health in sub populations of cells. Human CD8+ T-lymphocytes are susceptible to vincristine-induced apoptotic cell death. (A) Frequency histograms of IncuCyte® FabFluor-488 conjugated to either -CD8, -CD45 and -IgG-labeled hPBMCs. (B and C) IncuCyte images showing color-coded subsets of healthy and apoptotic (IncuCyte® Annexin V+) CD8+ or CD8- cells (4 groups) following treatment with vincristine (300 nM) or vehicle (48 h). Vincristine induced a concentration- and time-dependent reduction in the proliferation of CD8+ cells (D) and a concomitant increase in apoptosis (E). Concentration-response curves yielded IC50 or EC50 values of 4 nM for anti-proliferation and 8 nM for induction of apoptosis (F).

Label-free cell counting. IncuCyte S3 Live-cell Analysis System (20x) images of NucLight Red-labeled Jurkat cells: (A) HD Phase + Phase Mask (B) Phase Mask only (C) Phase + Red Fluorescence (D) Phase Mask + Red Fluorescence. Scale bar = 50μM. (E) Time-course analysis of label free cell count (open symbols) and nuclear count (closed symbols) at different initial cell plating densities (5-40K, 96-well plate). Note the close similarity of values obtained by the phase object and nuclear label counting methods.

Count

Frequency

Cell count

Cell count

Apoptosis ApoptosisEccentricity

Area (μm2)

Mean intensity

Time (h)

Average eccentricity

A. Phase + Mask

C. Phase + Red

C. Non-activated

B. Vehicle C. Vincristine, 300 nM

D. a-CD3/IL2 activated

B. Mask

D. Red + Mask

0 0 24 2448 4872 7296 96120 120 Time (h) Time (h)

70 0.55

0.60

0.65

100

130 μm2

Average areaA

E

A

D FE

E

F G H0 h 48 h 120 h

B0.70

Activated Activated

Non-activated

Non-activated

0

0.1

0

100

1

24 48 72

300

10

Time (h)0 24 48 72 96 120

Time (h)0 24 48 72 96 120

Time (h)0 24 48 72 96 120

96

Area (μm2)30 100 300

Area (μm2)30 100 300

Area (μm2)30 100 300

0

0

0

230

50

1 1

50 50

25 25

75

2 2

75 75

25

0 00 0

0.5

460

100%

3 x 104 3 x 104

100% 100%

Cell count

0

5

15

10

1.0 0 h

IgG

120 h

CD45

Apoptosis

Cell count

Vehicle

Vehicle

1.2

1.2

3.7

3.711

11

33

33

100

100

300

300

48 h

CD8

5K

10K

20K

40K

76

Chemotaxis is the directional movement of cells in response to a chemical stimulus and is an essential component of immune responses, tumor metastasis, wound healing, and blood vessel formation. IncuCyte® Chemotaxis Migration and TEM Assays are an integrated solution for real-time visualization and automated analysis of chemotactic migration.

Neutrophils are the first line of defense at the site of an infection and play an essential role in the innate immune system, employing multiple strategies to degrade and kill microbes, including the release of neutrophil extracellular traps (NETs). Automatically quantify neutrophils undergoing NETosis in real time with the IncuCyte® S3 Live-Cell Analysis System and IncuCyte® Cytotox Green Reagent.

Scavenging and engulfing micro-organisms, apoptotic cells and tumor cells is a central role in host defense. The IncuCyte® pHrodo® Reagents and IncuCyte Live-Cell Analysis System enable real-time, automated analysis of phagocytosis inside your cell culture incubator.

IncuCyte® Chemotaxis Assays IncuCyte® NETosis Assay

IncuCyte® Phagocytosis Assays

Visualize every cell throughout your experiment in real time Gain deep phenotypic insight into cell movement and morphology with proprietary IncuCyte® ClearView Chemotaxis plates.

SDF-1a mediated chemotaxis of human T cells. (A) CD3/CD28 activated T-cells were seeded on an ICAM coated ClearView plate and migration toward SDF-1a was quantified by measuring the loss of cell area on the top of the membrane. (B) Activated T-cells were also monitored for their ability to migrate through a HUVEC monolayer in response to SDF-1a chemoatrractant gradient. Optically clear IncuCyte ClearView plates allowed for the visualization of endothelial monolayer integrity and leukocyte diapedesis throughout the time course of the assay.

IncuCyte® ClearView 96-well Chemotaxis Plates. Combine the optical clarity of microfluidics devices with the throughput of transmembrane assays. Each well of an IncuCyte ClearView plate provides an optically clear surface for label-free imaging and analysis of chemotactic cell migration. Cells are added to the upper chamber and chemoattractant to the lower reservoir plate. Chemotactic transmembrane migration is automatically quantified as the cells migrate through laser etched pores (yellow circles) toward chemoattractant.

Real-time analysis and visualization of phagocytosis. J774A.1 mouse macro- phages visualized engulfing IncuCyte® pHrodo® Green E. coli Bioparticles® which, on entering the acidic environment of the phagosome, increase in fluorescence. Images and movies enable confirmation of signal and provide insight into the dynamic morphological changes (e.g. formation of phagocytic cups) associated with phagocytosis. The signal was ablated by inhibitors of phagocytosis including cytochalasin D and nocodazole. 96-well plate view illustrates the time courses of phagocytosis and assay consistency.

Validate NETosis with IncuCyte® HD Phase images. IncuCyte images of differentiated HL-60 cells stimulated using PMA in the presence of IncuCyte® CytoTox Green reagent. As nuclear contents are moved to the plasma membrane and released, the network of extracellular fibers composed of DNA is bound by the Cytotox Green reagent, as seen by fluorescent enhancement overtime.

Key Advantages:– Visually monitor every cell in your

experiment, assess morphology and gain phenotypic insight from images and movies.

– Measure label-free or labeled cell migration without fixing, staining or cell scraping steps.

– Investigate cell migration on biologically relevant surfaces or across an endothelial monolayer.

Key Advantages:– Simple mix-and-read protocols suitable for

pharmacological screening – no washing, no fixing, no lifting.

– Automated analysis over the entire assay time course without removing sensitive cells from the incubator.

– Visualize the distinct morphological changes associated with NETosis.

Key Advantages:– Visualize and validate dynamic phagocytic

clearance by immune cells with images and movies.

– Generate quantitative, reproducible and specific measurements of engulfed cells.

– Efficiently study the model of your choice in

either 96- or 384-well plate format.

Analyze sensitive cells using a 96-well approach with high reproducibility Capture cell migration or transendothelial migration in real time all from within your incubator.

1h 45m 1h 55m 2h 05m 2h 15m 3h 15m

Cytochalasin D Latrunculin A VehicleNocodazole

Migration (6 h) Total T-cell area normalized to initial value (top)

Total T-cell area normalized to initial value

Chemotaxis of Primary T-Cells to SDF-1a

Primary T-Cell, TEM toward SDF-1a

HD Phase + Fluorescence

Total T-cell area normalized to initial value (30 h)

Time (h) Log [SDF-1a] (M)

Log [SDF-1a] (M)Time (h)

A

B

0 -10

-9 0

3 -96 -8

-810

9 -7

-720

12 -6

-630

0.0

0.0

0.0

1.0

0.5

0.4

0.5

0.5

1.5

0.8

1.5

1.0 1.0

2.0

1.2

2.0

0.0

Negative control0.69 nM2.06 nM6.17 nM18.52 nM55.56 nM166.77 nM500 nM

Control3.125 nM6.25 nM12.5 nM

25 nM50 nM200 nM100 nM

EC50=17.5 nM

EC50=23 nM

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© 2018 Essen BioScience. All rights reserved. IncuCyte®, Essen BioScience® and all names of Essen BioScience products are registered trademarks and the property of Essen BioScience unless otherwise specified. Essen Bioscience is a Sartorius Company. Specifications subject to change without notice. Doc Number: 8000-0538-E00.

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Ordering informationProduct Description Cat. No.

ImmunocytochemistryIncuCyte® S3 Cell-By-Cell Analysis Software Module 1 each 9600-0031

IncuCyte® Mouse IgG2a FabFluor-488 Antibody Labeling Reagent 1 vial, 50 μg 4743

IncuCyte® Mouse IgG2b FabFluor-488 Antibody Labeling Reagent 1 vial, 50 μg 4744

IncuCyte® Mouse IgG1 FabFluor-488 Antibody Labeling Reagent 1 vial, 50 μg 4745

ChemotaxisIncuCyte® ClearView 96-Well Chemotaxis Plate 1 plate 4582

IncuCyte® ClearView 96-well Chemotaxis Plates Case of 10 Plates 4648

IncuCyte® Chemotaxis Analysis Software Module 1 each 9600-0015

Immune Cell Activation, Proliferation, and Cell HealthIncuCyte® S3 Cell-By-Cell Analysis Software Module 1 each 9600-0031

IncuCyte® Caspase-3/7 Green Apoptosis Assay Reagent 20 μL 4440

IncuCyte® Caspase-3/7 Red Apoptosis Assay Reagent 20 μL 4704

IncuCyte® Annexin V Red Reagent for Apoptosis 1 vial, 100 tests 4641

IncuCyte® Annexin V Green Reagent for Apoptosis 1 vial, 100 tests 4642

IncuCyte® Cytotox Red Reagent for Counting Dead Cells 5 μL x 5 4632

IncuCyte® Cytotox Green Reagent for Counting Dead Cells 5 μL x 5 4633

IncuCyte® Mouse IgG2a FabFluor-488 Antibody Labeling Reagent 1 vial, 50 μg 4744

IncuCyte® Mouse IgG2b FabFluor-488 Antibody Labeling Reagent 1 vial, 50 μg 4745

IncuCyte® Mouse IgG1 FabFluor-488 Antibody Labeling Reagent 1 vial, 50 μg 4745

NETosisIncuCyte® Cytotox Green Reagent for Counting Dead Cells 5 μL x 5 4633

PhagocytosisIncuCyte® pHrodo® Red E. coli Bioparticles® for Phagocytosis 1 vial, 2 mg 4615

IncuCyte® pHrodo® Green E. coli Bioparticles® for Phagocytosis 1 vial, 2 mg 4616

IncuCyte® pHrodo® Red Zymosan Bioparticles for phagocytosis 1 vial, 1 mg 4617

IncuCyte® pHrodo® Green Zymosan Bioparticles® for Phagocytosis 1 vial, 1 mg 4618

IncuCyte® pHrodo® Red S. aureus Bioparticles® for Phagocytosis 1 vial, 2 mg 4619

IncuCyte® pHrodo® Green S. aureus Bioparticles® for Phagocytosis 1 vial, 2 mg 4620

IncuCyte® pHrodo® Red Cell Labeling Kit 1 kit 4649

To place an order or request additional information

E-mail:sales@essenbio.com

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For Japan, Australia and other countries around the world:+81-3-5826-4795

Harness the power of live-cell analysis with a full range of IncuCyte reagents and consumables to revolutionize the way you quantify cell behavior. To view a complete listing of our reagents and consumables visit: essenbioscience.com/reagents

Further ReadingSee more exciting data, movies, application notes and scientific posters by visiting www.essenbioscience.com/immunology