S A L K I N S T I T U T E F O R B I O L O G I C A L S T U D I E S

Carolyn O’Connor Director, Flow Cytometry Core Facility

Salk.edu/fccf coconnor@salk.edu

Flow Cytometry Core Facility … cell analysis & cell sorting

bacteria monitoring

Brewing & Winemaking

Food & Pharmaceutical

Manufacture

yeast analysis

Complete Blood Count

DNA abnormality

Cancer Diagnosis Immunodeficiency

disorders

Immunology Developmental

biology

Neuroscience

Stem cells Aging

Cancer biology

Transplantation

Metabolism

Microbiology

Aquatic Ecology phytoplankton, algae, bacteria

Plant cell biology

… cell analysis & cell sorting

Biomedical Research

Plant & Marine Biology Industrial

Flow Cytometry

Clinical & Diagnostics

1947 Spore detector *US Army sponsored

Cytometry … the science of measuring cells

1953 Cell Counter *Counting and sizing

1969-1972 Fluorescence based cytometers

1965 Droplet based

cell sorter Ink-jet printing: droplet technology

Flow Cytometry … cell analysis & cell sorting

Multi-cellular organisms are complex

what cells are present?

what are their roles?

how many of each cell?

what is the significance

of each cell?

are they

interacting?

health

disease aging

Identifying cells by antibody labeling

we can color code cells using fluorescent labelled antibodies antibodies recognize and bind unique shapes

specific proteins on cells

e.g. tumor biopsy liquid suspension.. no solids!!

CD4 T cells CD8 T cells

CD4 T cell

CD8 T cell

Two types of immune cells

CD8 protein

fluorescent proteins engineered fluorescent proteins

genes inserted into cell genomes

expressed in stem cells

form fluorescent tissues

DNA dyes can inform cell cycle phase

Probes can be combined in sophisticated experiments

Other examples of labels

pH Mitochondrial function

Reactive Oxygen Species

phagocytosis cell health oxidative stress

fluorescent indicators

Forward Scattered Light detector

Side scattered light and

fluorescence detectors

“Hydrodynamic focusing“ region

OUT To vibrating nozzle and/or waste tank

Sample profiling, cell by cell Fluorescent

sample IN

saline

Photons emitted

Blocker bar

Detectors convert photons to voltage for software interface

*Magnitude proportional to #

photons

Forward Scattered Light ~ size

Side Scattered Light ~ cell “complexity”

Series of dichroic mirrors

Scatter profile of blood

Scatter profile of blood

Fluorescent probe derived photons of

different wavelengths are quantified

Cells of interest can be purified from a

mixture

Isolating cells by “FACS”

…fluorescence is measured

… the drops get charged -

+ -

CD4 T cells CD8 T cells

CD8 T cells

an electric field guides the charged drops

CD4 T cells

+

+

–

–

of cells are created

Starting mixture

CD8

T ce

lls

CD4 T cells

Sorting technology in the core

InfluxTM sorter

10 sorted beads ~size of blood cell

Holder for 6 population (“6-way”) sorting

5 lasers 11 colors

How can we use this technology for

cancer research?

Pancreatic cancer

Lowest survival rate

3rd leading cause of cancer related deaths in the US

Innovative approaches still are failing.

using patient’s own immune system

to attack cancer

Better treatments are needed

what cells are present?

what are their roles? how many of each cell?

what is the significance

of each cell?

are they

interacting?

Tumor Micro-environment “Immuno-suppressive”

Cancer Supporting

What’s unique about pancreatic cancer?

“… coalition formed to accelerate next

generation immunotherapy ”

US Cancer Moonshot Initiative..

Pancreatic Tumor Microenvironment

Pancreatic stellate cell

Tumor cell

Pericyte

Lymphatic vessel

Blood vessel

Immune cells

DCs MØ

T cell

adapted from Junttila & Sauvage, Nature, 2013

HUGE cast of characters!

what are they doing?

Cells of the tumor micro-environment

IDENTIFY and ISOLATE: Complex 10+ antibody combinations

Multiple cell types (6 way sorting)

Determine activity: functional gene expression

who are they?

Informing novel therapy design

Micro-environment “Cancer supporting” state

Novel therapies for pancreatic cancer

Morgan Truitt, PhD – Laboratory of Dr. Ronald Evans

Novel therapeutic target: Tumor micro-environment

“Anti-tumor” state Micro-environment

00.10.20.30.40.50.60.7

Control Immunotherapy Drug A +Immunotherapy

Tumor Size (g)

Unraveling complex biology Disease model e.g. pancreatic cancer

Dissected complexity e.g. why can’t the immune system attack?

Develop novel strategies e.g. target the tumor micro-environment

Multi-color flow cytometry

Technology & Expertise

High content data

Accelerated discovery

A technology paradigm

Fluorescence Technology

Instrumentation

20 channels: cell sorter

50 channels: analytical cytometer

Novel Fluorescent

Polymers: Nobel Prize winning

technology (2000)

Fluorescent protein

engineering

Flow Cytometry Core Facility Conor Fitzpatrick

Pancreatic Cancer Project Morgan Truitt, Evans Laboratory

We are supported by:

J. Belcovson Sort Chamber

Salk Cores Administration Travis Berggren Angelee Ferber

Acknowledgements

Central Australia

Carolyn O’ConnorManaging Director, Flow Cytometry Core