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Exploring the First Line of Defense: Research
Tools for the Innate Immune System
Sample & Assay Technologies
Welcome to our 4-part webinar series
"The Microscopic War: Microbes and Immunity”
Part 1: Host–Pathogen Interactions: Molecular Basis and Host
Defense Mechanisms
Part 2: Microbiome: from Identification to Characterization
Part 3: Exploring the First Line of Defense: Research Tools
for the Innate Immune System
Part 4: Toll-like Receptors in Inflammation
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Sample & Assay Technologies
Legal disclaimers
The products discussed in this presentation are intended for molecular biology
applications. These products are not intended for the diagnosis, prevention, or treatment
of a disease.
For up-to-date licensing information and product-specific disclaimers, please see the
respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user
manuals are available at www.QIAGEN.com, or can be requested from QIAGEN
Technical Services or your local distributor.
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Innate vs adaptive immunity
Innate
Adaptive
PAMPs/DAMPs
Rapid induction (hours)
Short-lived
Exploring the First Line of Defense: Research Tools for the Innate Immune System
Antigenic peptides
Slow induction (days)
Memory
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Sample & Assay Technologies
Phases of the innate immune response
Recognition
Recruitment/Activation
Recognize microbial
components
Macrophage recognition of
pathogens inflammation
Germline-encoded
receptors
Inflammation leads to PMN,
monocyte (M∅/DC), NK,
eosinophil recruitment via
chemokines
Non-clonal (ie, all cells of
the same lineage have
same receptors)
Effector
Phagocytosis and
microbial killing by PMN
and M∅
Cytotoxicity by NK cells
Induction of adaptive
immunity by DC and M∅
Non-cytokines or
chemokines (complement
fragments, fMLP) recruit
and activate innate immune
cells
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Epithelia, peptides, innate-like lymphocytes
Microbial invaders enter the body through skin and mucosa, and are met by several innate
defenses before encountering circulating cellular effectors.
.
Mucus/cilia (sweep out microbes before they can adhere)
Chemical defenses
β-defensins (epithelial cells and leukocytes, skin, tongue, respiratory tract)
α-defensins (Paneth cell granules in intestine, neutrophil granules)
Lysozyme, phospholipase A (saliva, tears)
pH and digestive enzymes (stomach)
Innate-like lymphocytes are lymphocytes with limited receptor diversity:
Intraepithelial γ/δ T lymphocytes – GI
B-1 B cells – peritoneal and pleural cavities
NK-T cells – thymus, peripheral lymphoid organs
.
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Induction of innate immunity by PRRs
Pathogen-associated molecular patterns (PAMPs)
Lipopolysaccharide
Flagellin
Single- and double-stranded nucleic acids
CpG
Lipoteichoic acid
Zymosan
etc…
Danger-associated molecular patterns (DAMPs)
HMGB1
Heat shock proteins
Pattern recognition receptors (PRRs)
Toll-like receptors (TLRs)
NOD-like receptors (NLRs)
Mannose receptor
Cytosolic DNA sensors
RIG1-like receptors (RLRs)
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Cellular and molecular effectors of innate immunity
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Phagocytes and natural killer (NK) cells
Neutrophils
Macrophages
Long-lived phagocytes
Short-lived phagocytes
Mannose receptor, scavenger
receptors
Granules (acid hydrolase,
myeloperoxidase, defensins,
cathepsin G, lysozyme,
lactoferrin, elastase, etc)
Complement receptors
ROS, RNI
Respiratory burst
Proinflammatory cytokine
production (IL-1β, IL-12, TNF-α,
IL-6,CXCL8)
Produce growth factors for tissue
remodeling
NETs (neutrophil extracellular
traps: chromatin + serine
proteases)
Proinflammatory cytokines
(IL-12, TNF-α)
NK cells
Cytotoxic cells (via perforin and
granzymes, or ADCC)
Expansion/activation in response
to IL-15, IL-12, Type I IFNs
Produce IFN-γ, IL-1, and IL-2
Recognize Class I MHC
(inhibitory & activating receptors)
Blood, spleen localization
Memory?
Arise from circulating monocytes
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Mast cells
Basophils
Helminths, viruses and
bacteria (also allergy)
Least abundant
granulocyte
Respond to
complement, IgE, TLR
stimulation
Histamine,
proteoglycans, lipid
mediators, IL-4, IL17E
Release histamine,
heparin, proteases,
TNF and other
proinflammatory
cytokines, eicosanoids,
etc.
Macroparasite and
helminth defense,
allergy
IgE-activated,
matures via IL-3
Eosinophils
Nuocytes
Produce major basic
protein, eicosanoids,
histamine, peroxidases,
acid phosphatase,
growth factors, and
cytokines
Important in Type 2
innate response
against helminth
infection (Neill et al,
Nature 2010)
Activated by IL-5, IL-3,
and GM-CSF
lung inflammation
Respond to IL-25 and
Helminths, viral infection, IL-33, produce IL-13
allergy
Involved in allergic
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Dendritic cells
“Professional antigen presenting cells (APCs)”, linking innate and adaptive immunity (macrophages and B
cells are also considered professional APCs)
Present as immature DCs (highly phagocytic) in tissues; upon antigen capture, migrate to lymph nodes
and spleen and mature, providing Ag presentation and costimulation to T cells
Multiple subsets and sub-subsets based on surface markers, localization, and function
Produce IL-12, IL-15, TNF-α, and Type I IFNs, and respond to chemokines through CCR2, CCR5, CCR6,
and CCR7
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Complement cascade
Three types:
Classical
Alternative
Lectin-induced
Initiation:
C1q, mannose-binding lectin, or C3b bind
microbe surfaces (or C1q binds C-reactive
protein)
3 pathways all lead to cleavage of C3
Outcomes:
Opsonization
Chemotaxis/activation of leukocytes (C3a,
C5a)
Membrane-attack complex
Results from C3b generation
Consists of C5b-9
Effective against Neisseria species
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Cytokines and chemokines
Cytokines produced by innate immune cells
Type I IFNs, IFN-γ
TNF-α
IL-1, 6, 8, 10, 12, 15, 18
Cytokines that enhance/inhibit innate immune activity
Enhance: IFN-γ, TNF-a, IL-15, IL-12
Inhibit: IL-10, TGF-beta
Important chemokine receptors
CXCR1, 2 (neutrophils)
CCR1, 2, 3 (leukocytes)
CCR5 (DCs, NK cells, monocytes)
CCR6, 7 (DCs)
CXCR3 (ligand: CXCL10, monocytes and NK
cells)
Important chemokines
CXCL8 (produced by M∅, recruits PMN)
CCL2 (produced by monocytes, fibroblasts,
recruits monocytes, DCs, NK cells)
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Important signaling networks
Cytokines
JAK/STAT
MAPK
IRFs
PI3K
NFκB
Chemokines
PI3K/AKT
MAPK
NFκB
TLRs
NFκB
JNK/MAPK
IRFs
PI3K
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Outcomes of an innate immune response
Microbial killing
Complement
Neutrophil and macrophage microbicidal mechanisms
NK cell cytotoxicity against infected cells
Type I IFNs induce antiviral state in infected cells
Induction of adaptive immunity
Dendritic cells and macrophages present antigen to T cells and provide costimulation
Chemokine and cytokine production lymphocyte expansion, activation
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Technologies for innate immune research
Pathway-focused gene expression analysis
Featured publications: Derbigny, W.A. et al.
2012, Infect. Immun.
RT2 Profiler PCR Array, Mouse Inflammatory
Cytokines
Signaling pathway reporter arrays
Featured publication: Zughaier, S.M. 2011, J.
Leukocyte Biol.
Cignal Finder 10-Pathway Reporter Array
RT2 Profiler PCR Array, Human TLR and
Human Apoptosis
Cytokine analysis
Featured publication: Rahman, S. et al. 2011 J.
Immunol.
Multi-Analyte ELISArray
RT2 Profiler PCR Array, Mouse IFN-α and IFNβ Response, Mouse Dendritic and Antigen
Presenting Cells
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
RT2 Profiler PCR Arrays
84 of the most relevant genes in biological and disease pathways
Gene lists identified through state-of-the-art bioinformatics and text-mining tools
Integrated controls for genomic DNA contamination, normalization, and PCR processes
Web-based data analysis software at no additional cost
Compatible with most real-time PCR instruments
Immunity-related pathways:
Innate & Adaptive Immune Response
Inflammatory Cytokines & Receptors
Dendritic & Antigen-Presenting Cells
Inflammasomes
IFN-α/β Response
NFkB Signaling
MAPK Signaling
PI3K/AKT Signaling
(and more… 140+ pathways,
including custom arrays)
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Application data
Which cytokines alter expression after PMA-ionomycin treatment?
In this experiment, human PBMCs were treated with PMA and ionomycin, and then analyzed
using the Common Cytokines RT2 Profiler PCR Array. This volcano plot shows both foldchange and the statistical significance, and demonstrates that 23 genes, including IL-10, IFNgamma, IL-2, and TNF were upregulated, while IL-1beta and 5 other genes were
downregulated in response to treatment.
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
How does gene expression change during infection?
Case study #1
Characterizing overall responses of gene networks, as opposed to just a few genes, can
give a more comprehensive picture of the changes that are occurring. How can you profile
the most relevant genes to your system of interest all at once?
Identifying a role for TLR3 in the innate immune response to Chlamydia muridarum
infection in murine oviduct epithelial cells
Derbigny, W.A. et al. 2012, Infect. Immun.
Context:
Research aim: to determine how oviduct epithelial cells participate in host defense
against Chlamydia.
Technique: used Mouse Inflammatory Cytokines RT2 Profiler PCR Array to compare
gene expression in infected wild-type or TLR3-deficient OE cells.
Significance: demonstrated a role for TLR3 and IFN-β in initiating inflammatory
responses against an intracellular bacterial pathogen.
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Experimental design and results
Case study #1, Derbigny et al.
Compared gene expression changes under the following conditions using the Mouse
Inflammatory Cytokines PCR Array:
Wild-type cells plus C. muridarum
TLR3-deficient cells plus C. muridarum alone
TLR3-deficient cells plus IFN-β and C. muridarum
TLR3-deficient cells plus IFN-β alone
Results
Wild-type infected cells: most chemokines and interleukins, CXCL15, CCR10.
Deleting TLR3: diminished response to infection, except CXCL15 effects. CCR9 and Lta
showed modest compared to WT cells.
IFN-β + infection of TLR3-/-: partially rescued chemokine and Casp1 responses
IFN-β sans C. muridarum: still stimulated some chemokine response, but not as much
as with infection
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Conclusions
Case study #1, Derbigny et al.
The team concluded that TLR3 and IFN-β are major mediators of the inflammatory
response to C. muridarum in OE cells, possibly in a cell-type-specific manner (as other
studies had showed no involvement of TLR3 in the macrophage response).
The ability to easily compare expression of many pathway-related genes in several
different conditions permitted the team to dissect the specific roles of the receptor, the
cytokine, and the pathogen in stimulating responses.
The CXCL10 findings from the PCR Array, as well as ELISA for CXCL10 and IL-6,
prompted the team to examine whether IFN-beta was exerting its effects through
enhancement of TLR2 signaling. Indeed, IFN-beta caused upregulation of TLR2
expression in OE cells.
RT2 Profiler PCR Arrays provide an excellent starting place for identifying which genes
in a specific biological pathway are affected by infection, cytokine stimulation, and gene
knockdown.
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Technologies for innate immune research
Pathway-focused gene expression analysis
Featured publications: Derbigny, W.A. et al.
2012, Infect. Immun.
RT2 Profiler PCR Array, Mouse Inflammatory
Cytokines
Signaling pathway reporter arrays
Featured publication: Zughaier, S.M. 2011, J.
Leukocyte Biol.
Cignal Finder 10-Pathway Reporter Array
RT2 Profiler PCR Array, Human TLR and
Human Apoptosis
Cytokine analysis
Featured publication: Rahman, S. et al. 2011 J.
Immunol.
Multi-Analyte ELISArray
RT2 Profiler PCR Array, Mouse IFN-α and IFNβ Response, Mouse Dendritic and Antigen
Presenting Cells
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Cignal Reporter Assays & Arrays
Functionally verified assays for 45 pathways:
Type I IFN
IFN-γ
NFκB
MAPK
PI3K/AKT
STAT3
TGF-β
And more…
Cignal Finder 10-Pathway Arrays:
Immune Signaling
Cancer
Development
Stem Cell & Differentiation
Nuclear Receptors
Stress & Toxicity
Cignal 45-Pathway Array
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Application data
Determining the signaling pathways activated in response to
cytokine stimulation
HeLa cells were reverse transfected with the Immune Response 10-Pathway Cignal
Finder Reporter Array. 16 hours after transfection, medium was changed to assay
medium. 32 hours after transfection, cells were treated with 5 ng/ml TNFα or left
untreated. After 6 hours treatment, dual-luciferase assays were performed. Results are
expressed as fold change.
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Which signaling pathways are being triggered?
Several signaling pathways can produce innate immune responses. How do you know
which are at work in your system?
Neisseria meningitidis capsular polysaccharides induce inflammatory responses via
TLR2 and TLR4-MD-2
Zughaier, S. M., J. Leukoc. Biol., March 2011
Context:
Research aim: determining how CPS-triggered signaling proceeds
Human TLR Signaling and Human Apoptosis RT2 Profiler PCR Arrays
Cignal Finder 10-Pathway Reporter Array to identify active signaling pathways
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Experimental context
Case study #2, Zughaier
Pathways interrogated:
NFκB
PKC/Ca++ (NFAT)
Type I IFNs (ISRE)
IFN-γ (GAS)
MAPK/ERK (SRE)
MAPK/JNK (AP-1)
TGF-β (SMAD)
cAMP-PKA (CRE)
C-EBP
Glucocorticoid receptor (GRE)
Context:
Meningococcal endotoxin (LOS) produces a strong innate immune response through
TLR4, but the innate response to another crucial virulence factor, capsular
polysaccharide (CPS), was uncharacterized
TLR2 and TLR4-MD2 were identified as the CPS receptors on macrophages.
TLRs 2 and 4 can signal through NFκB or MAPK pathways. Which ones for CPS?
Purified CPS from LOS-deficient strain of N. meningitidis (IpxA mutant)
Dosed HEK/TLR2/6 or HEK/TLR4-MD2-CD14 cells with CPS or LOS after transient
reverse transfection with reporters
Stimulated THP-1 cells with CPS-lpxA, LOS, or Rhizobium LPS (TLR4 ligands) to
compare gene induction profiles
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Findings and conclusions
Case study #2, Zughaier
Findings:
Strong NFκB reporter activity in TLR4-MD2 and TLR2-6 stably transfected cells
stimulated with serogroup B CPS
Milder activity from Type I IFN/ISRE (TLR2-6), MAPK-JNK/AP-1 (both), and MAPERK/SRE (TLR4-MD2).
Also observed variation in gene expression profiles between cells stimulated with CPSlpxA vs LOS (including TNF, NOD1, CD40LG, LTA, and CARD6).
Conclusions:
Cignal Finder 10-Pathway Reporter Array identified 4 inflammatory signal transduction
pathways potentially involved in TLR-mediated recognition of CPS
The RT2 Profiler PCR Array determined that CPS induces qualitatively distinct gene
expression responses compared to LOS
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Technologies for innate immune research
Pathway-focused gene expression analysis
Featured publications: Derbigny, W.A. et al.
2012, Infect. Immun.
RT2 Profiler PCR Array, Mouse Inflammatory
Cytokines
Signaling pathway reporter arrays
Featured publication: Zughaier, S.M. 2011, J.
Leukocyte Biol.
Cignal Finder 10-Pathway Reporter Array
RT2 Profiler PCR Array, Human TLR and
Human Apoptosis
Cytokine analysis
Featured publication: Rahman, S. et al. 2011 J.
Immunol.
Multi-Analyte ELISArray
RT2 Profiler PCR Array, Mouse IFN-α and IFNβ Response, Mouse Dendritic and Antigen
Presenting Cells
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Multi-Analyte ELISArrays
Common Cytokines
Inflammatory Cytokines
TLR-induced Cytokines I: Viral
TLR-induced Cytokines II: Microbial
Autoimmunity
Th1 / Th2 / Th17 Cytokines
Common Chemokines
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Application data
Are Th1 or Th2 cytokines being produced?
Time-dependent (0, 6, 18, 24, and 48 h) patterns of Th1/Th2 cytokine induction by human
peripheral blood mononuclear cells (PBMC) in response to PMA (50 µg/ml) and
ionomycin (1 µg/ml) were monitored. The relative amount of each cytokine was profiled at
the same time using the ELISArray Kit.
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
What comprises the cytokine milieu?
Case study #3, Rahman et al.
Innate immune responses produce a multitude of cytokines, which play distinct roles. Is it
possible to efficiently detect several related cytokines simultaneously?
Murine FLT3 Ligand-Derived Dendritic Cell-Mediated Early Immune Responses Are
Critical to Controlling Cell-Free Human T Cell Leukemia Virus Type 1 Infection
Rahman, S. et al. J. Immunol. 2011
Context:
Research aim: to clarify how DCs respond to HTLV-1 infection
RT2 Profiler PCR Arrays for IFNα/β Response, Dendritic & Antigen-Presenting Cells
Multi-Analyte ELISArray Kit to identify production of multiple cytokines
at once in the context of infection
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Background and experimental design
Case study #3, Rahman et al.
Cytokines detected:
IL-2
IL-4
IL-5
IL-6
IL-10
IL-12
IL-13
IL-17A
IL-23
IFN-γ
TNF-α
TGF-β1
Study background
Innate immunity was not thought to have a major part in HTLV-1 control; however, the
authors demonstrate a significant role for Flt3L-generated DC and Type I IFN in control
of cell-free virus (the type that would be encountered early in infection)
Which cytokines are produced in early HTLV-1 infection?
Cultured Flt3L-generated BMDC with cell-free virus to analyze cytokines
Compared cytokine induction between UV-irradiated and competent virus
Profiled gene expression upon DC infection with competent virus.
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Findings and conclusions
Case study #3, Rahman et al.
Findings
Multi-Analyte ELISArray – proinflammatory cytokines were secreted by DCs after viral
challenge (IL-6, TNF-α, IL-12). Th2 cytokines (IL-4, IL-5, IL-13) were not induced, but
IL-10 and TGF-β were secreted as well.
RT2 Profiler PCR Arrays
IFNα, IFNβ Response: Several IFN-responsive genes were upregulated after viral
challenge, as well as signaling molecules
DC & Antigen Presenting Cells: Some cytokines and chemokines were upregulated,
confirming the ELISArray results, and signaling and antigen uptake/presentation
genes were also enhanced. Many key chemokines (CCL2, 7, 8) and receptors
(CCR5, CCR3) were downregulated.
Conclusions
Multi-Analyte ELISArray Kits allowed detection of multiple cytokines at once, profiling
the cytokine response of innate immune cells (in this case, DC) to virus.
RT2 Profiler PCR Arrays confirmed the ELISA results and shed light on which
chemokines and IFN-responsive genes were being activated or deactivated in response
to virus.
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Summary
The innate immune system comprises a complex network of cellular and molecular
components
Research tools that allow simultaneous analysis of many immunity-related players at
once are an effective way to characterize innate responses to microbes
RT2 Profiler PCR Arrays profile expression of 84 genes simultaneously, and are
available for over 140 pathways. Many of these are related to innate immunity and host
defense, and custom arrays are also available.
Cignal Finder Reporter Arrays (10-Pathway and 45-Pathway) permit simultaneous
cell-based reporter analysis of several signaling pathways through DNA-based or
lentiviral vectors, using either GFP or luciferase.
ELISArrays are multiplex cytokine analysis assays using a traditional ELISA format.
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Contact us!
If you are interested in trying out these technologies in your research, please visit:
http://www.qiagen.com/search/rt2-profiler-pcr-arrays
http://www.qiagen.com/search/cignal-finder-reporter-arrays
http://www.qiagen.com/search/multi-analyte-elisarray-kits
When you’re ready, contact us at [email protected] to ask about our special RT2 Profiler PCR
Array starter pack offer!
Thank you for your time and attention!
Exploring the First Line of Defense: Research Tools for the Innate Immune System
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Sample & Assay Technologies
Welcome to our 4-part webinar series
"The Microscopic War: Microbes and Immunity”
Part 1: Host–Pathogen Interactions: Molecular Basis and Host
Defense Mechanisms
Part 2: Microbiome: from Identification to Characterization
Part 3: Exploring the First Line of Defense: Research Tools for
the Innate Immune System
Part 4: Toll-like Receptors in Inflammation
36