Flow Cytometry Core Facility

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1 Flow Cytometry Core Facility cell analysis & cell sorting Carolyn O Connor Director, Flow Cytometry Core Facility Salk.edu/fccf coconnor@salk.edu 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

Transplantation Immunodeficiency disorders Complete Blood Count Clinical &

Stem cells Neuroscience Immunology Biomedical Research Metabolism Aging Cancer

Ecology phytoplankton, algae, bacteria cell analysis & cell sorting Food &

2 Transplantation Immunodeficiency disorders Complete Blood Count Clinical & Diagnostics DNA abnormality Cancer Diagnosis Developmental biology Microbiology Stem cells Neuroscience Immunology Biomedical Research Metabolism Aging Cancer biology Flow Cytometry Plant cell biology Plant & Marine Biology Aquatic Ecology phytoplankton, algae, bacteria cell analysis & cell sorting Food & Pharmaceutical Manufacture bacteria monitoring Brewing & Winemaking yeast analysis Industrial

3 Cytometry the science of measuring cells 1947 Spore detector *US Army sponsored 1953 Cell Counter *Counting and sizing 1965 Droplet based cell sorter Ink-jet printing: droplet technology Fluorescence based cytometers

4 Flow Cytometry Multi-cellular organisms are complex cell analysis & cell sorting what cells are present? what are their roles? health disease aging what is the significance of each cell? how many of each cell? are they interacting?

Identifying cells by antibody labeling specific

! CD4 T cell CD8 protein CD4 T cells CD8 T cells

5 Identifying cells by antibody labeling specific proteins on cells Two types of immune cells CD8 T cell e.g. tumor biopsy liquid suspension.. no solids!! CD4 T cell CD8 protein CD4 T cells CD8 T cells antibodies recognize and bind unique shapes we can color code cells using fluorescent labelled antibodies

Other examples of labels fluorescent proteins

inform cell cycle phase genes inserted into

fluorescent tissues fluorescent indicators ph

Species phagocytosis cell health oxidative

6 Other examples of labels fluorescent proteins engineered fluorescent proteins DNA dyes can inform cell cycle phase genes inserted into cell genomes expressed in stem cells form fluorescent tissues fluorescent indicators ph Mitochondrial function Reactive Oxygen Species phagocytosis cell health oxidative stress Probes can be combined in sophisticated experiments

Sample profiling, cell by cell saline Hydrodynamic focusing region

detectors *Magnitude proportional to # photons Forward Scattered

7 Sample profiling, cell by cell saline Hydrodynamic focusing region Series of dichroic mirrors Fluorescent Side Scattered Light ~ cell sample complexity IN Photons emitted Scatter Fluorescent profile probe of derived blood photons of different Scatter wavelengths profile of are quantified blood Side scattered Detectors convert photons to light and voltage for software interface fluorescence detectors *Magnitude proportional to # photons Forward Scattered Light ~ size OUT Blocker To vibrating bar nozzle and/or waste tank Forward Scattered Light detector

8 Isolating cells by FACS Starting mixture CD4 T cells CD8 T cells of cells are created CD8 T cells CD4 T cells fluorescence is measured the drops get charged an electric field guides the charged drops Cells of interest can be purified from a mixture CD4 T cells CD8 T cells

9 Sorting technology in the core Influx TM sorter 5 lasers 11 colors 10 sorted beads ~size of blood cell Holder for 6 population ( 6-way ) sorting

10 How can we use this technology for cancer research?

11 Pancreatic cancer 3rd leading cause of cancer related deaths in the US Lowest survival rate

using patient s own immune what cells system are present? to attack cancer what are their roles?

12 Better treatments are needed What s unique about pancreatic cancer? how many of each cell? US Cancer Moonshot Initiative.. Innovative approaches still are failing. using patient s own immune what cells system are present? to attack cancer what are their roles? Tumor Micro-environment coalition formed to accelerate next generation immunotherapy Immuno-suppressive Cancer Supporting what is the significance of each cell? are they interacting?

cell types (6 way sorting) who are they? HUGE cast of characters! what are they doing?

13 Cells of the tumor micro-environment Pancreatic Tumor Microenvironment T cell MØ Immune cells DCs IDENTIFY Pancreatic and ISOLATE: stellate cell Tumor cell Complex 10+ antibody combinations Multiple cell types (6 way sorting) who are they? HUGE cast of characters! what are they doing? adapted from Junttila & Sauvage, Nature, 2013 Blood vessel Pericyte Lymphatic vessel Determine activity: functional gene expression

14 Informing novel therapy design Novel therapies for pancreatic cancer Morgan Truitt, PhD Laboratory of Dr. Ronald Evans Micro-environment Cancer supporting state Novel therapeutic target: Tumor micro-environment Tumor Size (g) Control Immunotherapy Drug A + Immunotherapy Micro-environment Anti-tumor state

15 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

16 A technology paradigm Fluorescent protein engineering 20 channels: cell sorter Accelerated Fluorescence Technology discovery Technology & Expertise Novel Fluorescent Polymers: Nobel Prize winning technology (2000) Instrumentation High content data 50 channels: analytical cytometer

17 Acknowledgements Sort Chamber J. Belcovson Flow Cytometry Core Facility Conor Fitzpatrick Salk Cores Administration Travis Berggren Angelee Ferber Pancreatic Cancer Project Morgan Truitt, Evans Laboratory We are supported by:

19 Central Australia Carolyn O Connor Managing Director, Flow Cytometry Core

Select the Sex of Your Next Calf Prior to Mating: Using Sexed Semen 1

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