IQFCS

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• IQFCS
• International Qualex
• Flow Cytometry
• Systems

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Vision of IQFCS

• High quality products for quantitative assessment
  of living cells
• Apoptosis
• Apoptosis vs. Necrosis
• In vivo apoptosis detection
• Functional assay
• Proliferation
• Cytotoxicity
• Non-functional assays
• Non-bleaching antibodies

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What is Flow Cytometry?
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Flow CytometryThe use of focused light
(lasers) are used to interrogate cells delivered
by a hydrodynamically focused fluidics system.
Sheath fluid
Flow Chamber
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Optical Design

Analysis of the cells allow for separation of the
different populations based from size (Forward
Scatter) and granularity (side scatter)
PMT 5
PMT 4
Sample
PMT 3
Dichroic
Filters
Flow cell
PMT 2

PMT 1
Scatter
Laser
Sensor
Bandpass
Filters
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Light Scatter Gating
Side Scatter Projection
Neutrophils
Forward Scatter
Forward Scatter Projection
Monocytes
Lymphocytes
200
400
600
800
1000
0
90 Degree Scatter
Human white blood cells
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Optical Design
The Flow cytometer also detects various cell
properties based upon fluorescent tags through
the different filters

PMT 5
PMT 4
Sample
PMT 3
Dichroic
Filters
Flow cell
PMT 2

PMT 1
Scatter
Laser
Sensor
Bandpass
Filters
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US foreign patents pending
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Using different fluorescent tags allows for the
detection of multiple marker on a single cell
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Antibody Staining
The use of high quality antibodies and
fluorescent tags are required for proper
analysis.
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RBClyse

• Not only is it important to use high quality
  antibodies but technique is also important to
  obtain the best results
• IQFCS offers RBClyse
• allowing phenotypic analysis from whole blood

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RBClyse Buffer

• Add approximately 100 µl blood
• Lyse RBC with RBClyse
• Stain cells
• Fix (optional)
• Analyze

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RBClyse
RBClyse allows for phenotyping cells without
purifying lymphocytes using a lymphocyte
gradient.
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AQ488/647 for Flow Cytometry
Side Scatter
Isotype-AQ488
Isotype-AQ488
IQFCS offers a line of bright, non-photobleaching
antibodies allowing for a clear analysis of
cells. In this example, there is no non-specific
background
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AQ488/647 for Flow Cytometry
Side Scatter
Anti-CD4-AQ488
Anti-CD4-AQ488
Using the highest quality antibodies along with a
bright fluorescent tag allows for easy
quantification of the positive cells.
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AQ488/647 for Flow Cytometry

Anti-CD8-AQ647
Isotype-AQ488
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Photobleaching

• A major problem with fluorescent products is
  photobleaching. IQFCS offers a series on
  non-photobleaching fluorescent dyes.

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Non-Photobleaching

• Human PBMCs
• Stained with anti-CD4-AQ488 (FITC) or
  commercially available anti-CD4-FITC
• Incubated under fluorescent light

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Photobleaching
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Conclusion

• Bright clear separation
• Clear separation from high and low markers
• Tight CV
• No photobleaching

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AQ flourescent dye series

• AQ488 Flow Cytometry/Fluorescent microscopy
• AQ555 Fluorescent microscopy
• AQ647 Flow Cytometry/Fluorescent microscopy

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Functional assays
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Proliferation

IQFCS offers optimized kits to determine specific
cell population proliferative response following
antigen stimulation.
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AQ647 antibodies
The specialized antibodies allow for clear
proliferative response of high and low populations
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Apoptosis

• In the past, apoptosis was determined by
• Tunneling
• Fas/Fas legend
• Annexin staining
• Caspase antibodies
• DNA lettering
• Mitochondria gradient

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Apoptosis probes

• IQFCS optimized non-antibody based cell permeant
  apoptotic probes

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Detection Products

• FMK Inhibitor based caspase detection
• Fluorescence labeled inhibitors.
• All reagents are cell permeant and require no
  cell lysis, permeabilization steps or antibodies
• Analysis by fluorescence microscopy, fluorescence
  plate reader or flow cytometry
• Products for poly caspases and individual
  caspases 1, 2, 3/7, 4, 5, 6, 8, 9, and 10

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How they work
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Neuroblastoma Cell
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Normal Keratoconus corneal fibroblasts
Cristina Kenney, M.D., Ph.D.
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Drosophila eye
Rebecca Hays Ph.D
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Correlation between FLICA and other reagents

FAM-VAD is more sensitive that Annexin
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FLICA vs. TUNEL
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Apoptosis vs. Necrosis
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High throughput drug screen assay

• Drug screen assay is designed to detect cytolytic
  responses from drugs or proteins.
• In this example, protein A or B were incubated on
  a confluent epithelial layer

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Apoptosis vs. Necrosis
No protein
7-AAD
FAM-VAD
FAM-VAD
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Apoptosis vs. Necrosis
7-AAD
FAM-VAD
FAM-VAD
Protein A
Protein B 12 h
post inoculation
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Apoptosis vs. Necrosis
7-AAD
FAM-VAD
FAM-VAD
Protein B 24 h Post inoculation
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Total Cytotoxicity Assay

• Easy - run samples in a single tube
• Safe - no radioactivity
• Fast - finish in one day
• Patent Pending

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Assay Overview
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Purify target cells (K562, monocytes, etc.)
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Draw a gate around the target cells
Create a live/dead vs. caspase graph
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Apoptotic Cells
12.51 251
501
1001 E/T Ratios
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Examples

• Gamma delta lymphocytes activated with BCG
  vaccination. Olin et al., Journal of
  Immunological Methods 297, 1-11.

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Natural Killer activity against K562 cells
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Antigen directed cytolytic activity against M.
bovis infected monocytes
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P0.04
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Cytolytic Activity
10
5
0
Non-vaccinated BCG-vaccinated

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Morphines effect on Gamma Delta lymphocytes

• Morphine was administered in-vivo prior to BCG
  vaccination Brain, Behavior, and Immunity article
  in press, 2006.

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Natural Killer activity
BCG
BCG-Morphine
Control
To-pro-3
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Conclusion

• FLICA probes are easy to use
• Increase sensitivity over other reagents
• Decreases assay time
• Allows to functional studies

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In vivo apoptosis detection
Patent Pending
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Morphine enhanced apoptosis in splenocytes
Morphine LPS LPS Morphine Control
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Morphine enhanced apoptosis in bone marrow
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Tissue
Control mice LPS only
mice Morphine LPS
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Chemotherapeutic efficacy
Mice receive 8 mg/kg of Arsenic Trioxide (ATO)
Robert Griffen Ph.D
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SR-VAD-FMK
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FAM-VAD-FMK
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Confirmation by Flow Cytometry
ATO 8.0 mg/kg 39 Apoptotic
Control 18 Apoptotic
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Bird brain in vivo
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Neurodegeneration
. P.E. Paulson, Ph. D
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chemotherapyefficacy
Eyedisease
Neuro-degeneration
Cardiac damage
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MCS Molecular Cytometry SystemsSingle nucleotide
polymorphisms (SNPs)/Nucleic Acid Detection

• MCS Based upon License from Los Alamos National
  Lab
• US Patents 6,287,766, 7,153,656 Other patent
  pending technologies
• CRADA with Los Alamos National Laboratory

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MCS Flow Cytometry Platform

• High sensitivity
• Low background
• Multi-color fluorescent detection
• Rapid analysis
• Quick turnaround and data analysis
• Very flexible in format and scale

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MCS Genomic Analysis using Multiplexing
Microsphere ArraysGAMMArrays

• Sensitive multicolor detection
• flow cytometry
• Free/bound resolution
• homogeneous assays
• Flexible substrate
• microspheres

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Tools for Genomic Analysis

• Assay Chemistries
• hybridization
• single base extension
• oligo ligation
• 5 nuclease (TaqMan, Invader)
• Assay Platforms
• gel electrophoresis
• microwell plates
• flat microarrays
• flow cytometry

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Single nucleotide polymorphisms

• Alterations in a single nucleotide
• Occur in 1 in every 1000 nucleotides
• 1.4 X 106 SNPs identified

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Single nucleotide polymorphisms

• Altered protein development
• Altered protein functions
• Loss of protein function
• Disease
• Increased virulence

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Use of MCS SNP Platform

• Key role in medical diagnostic
• Determination of risk factors
• Detection of pathogens
• Characterization of virulence

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SNP Lifecycle
Stage One High throughput SNP discovery Stage
Two SNP validation in diverse populations Sta
ge Three Large association studies to
correlate SNPs with phenotype Stage
Four Clinical trials Stage Five Clinical
diagnostics
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SNP Scoring Comparison
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MCS Future Technology Applications

• Personalized Medicine
• Human Predisposition to Disease
• Identification Characterization of Human
  Pathogens
• Human Forensics
• Nucleic Acid Assays in Animal, Environmental,
  Human Plant Diagnostics

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SNP Lifecycle
Stage One High throughput SNP discovery Stage
Two SNP validation in diverse populations Sta
ge Three Large association studies to
correlate SNPs with phenotype Stage
Four Clinical trials Stage Five Clinical
diagnostics
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How does it work?
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Addressed Capture Oligos
AAAA
TTTT(oligo 1 sequence)
AAAC
TTTG(oligo 2 sequence)
AAAG
TTTC(oligo 3 sequence)
AAAT
TTTA(oligo 4 sequence)
AACA
TTGT(oligo 5 sequence)
AACC
TTGG(oligo 6 sequence)
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Multiplexed Primer Capture
1 2 3 4 5 6 7 8 9 10 11 12
1 2 3 4 5 6 7 8 9 10 11 12 M
Address
Capture
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Bead-Based Minisequencing
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Bead-Based Minisequencing
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Multiplexed Minisequencing
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Multiplexed Minisequencing
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Multiplexed HLA Typing
G
A
C
C
G
G
C
C
C
C
A
C
T
C
C
C
T
C
T
T
T
T
A
A
A
A
A
A
G
A
G
A
Cai et al., Genomics, 2000
G
G
G
G
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Disease Susceptibility Chronic Beryllium Disease

• Be metal light and strong
• many industrial applications
• CBD immune hypersensitivity with delayed onset
  of 10-40 years
• 1-5 of exposed individuals develop CBD
• Genetic factor HLA DPB1 exon2 glu69

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Chronic Beryllium Disease
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Thank YouPlease let us know how we can help!

• Michael Olin Ph.D.
• Dan Moothart

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Watch this presentation at www.iqfcs.com