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Day 1
Introduction
9:00 am to
10:30 am
FLOW CYTOMETRY
TRAINING
Robert Salomon
(Flow Manager and Senior Flow Cytomerty Scie...
Theory Session – 0900 till 1300
 Introductions to the Lab and Staff
 Self Introductions
 Basics of flow cytometry
 App...
Practical Session – 1330 till 1600
 Lab orientation
 Key Cytometry hardware Systems overview
 Review of Acquisition Sof...
Cell Sorting – 900 till 1230
 Recap, discussion, exam questions
 Cell Sorting
 Automacs Pro
 FACS (fluorescent Activat...
Theory Session – 0900 till 1300
 Introductions to the Lab and Staff
 Self Introductions
 Basics of flow cytometry
 App...
INTRODUCTION TO GARVAN FLOW
Robert Salomon
 Flow Cytometry Manager
 Senior Flow Cytometry Scientist
 ISAC Emerging Leader 2014-2019
r.salomon@garva...
Eric Lam
 Flow Cytometry Operator
e.lam@garvan.org.au
Lab 92958431
INTRODUCTION – FLOW STAFF
Vitri Dewi
 Flow Cytometry Technician
v.dewi@garvan.org.au
Lab 92958431
INTRODUCTION – FLOW STAFF
Hira Saeed
 Flow Cytometry Technician
h.saeed@garvan.org.au
Lab 92958431
INTRODUCTION – FLOW STAFF
1. Name and position ?
2. Group/lab affiliation ?
3. Flow Experience ?
4. Brief introduction to your project ?
INTRODUCTIO...
BASICS OF FLOW CYTOMETRY ?
Measurement
METRY
BASICS OF FLOW CYTOMETRY ?
Measurement
METRY
Cells
CYTO
BASICS OF FLOW CYTOMETRY ?
Measurement
METRY
Cells
CYTO
Flow
Flow
Flow Cytometry
Measurement
METRY
Cells
CYTO
Flow
Flow
FLOW CELL
Hydrodynamic
focusing of
sample to
laser
intercept -
(interrogation
point)
Legend
Laser intercept
Core Stream
INTRODUCTION TO FLOW:
ANIMATION
WHY USE FLOW ?
 Rapid analysis ( 3k- 200k events/second)
 Individual event analysis
 Quantifiable results
 Multiple pa...
WHY USE FLOW ?
 Rapid analysis ( 3k- 200k events/second)
 Individual event analysis
 Quantifiable results
 Multiple pa...
Imaging Flow Cytometry
Cells per field/sec) Approximately 100 20,000
Number of Parameters <6 <24
Quantifiable Maybe (using...
1. Cells in single cell suspension
2. Fluorescent probes
3. Cytometer
PREREQUISITES FOR FLOW CYTOMETRY
The key to good
res...
 Flow Cytometry can be split into two main categories
USING FLOW CYTOMETRY
Cell Analysis Cell Sorting
Cellular Characteri...
15mins
Coffee and Morning Tea
COFFEE
 Flow cytometry can be used to characterise, identify and
separate cells or events based on physical and functional
attri...
 Phenotyping
 Apoptosis and cell death
 Cell cycle, cell division and DNA synthesis
 Cell tracking
 Transduction/tran...
 Usually done with fluorescently tagged antibodies
 Have my Cancer Cells spread to the Lymph Nodes ?
 Do macrophages in...
1. Does treatment/Condition X lead to apoptosis and/or Cell
Death?
2. What is the Mechanism for Death and how quickly does...
Functional identification Reagent
Early Cell Membrane Changes • Phosphatidylserine flipping : ANX V
• Violet Asymmetry Pro...
 What stage of the Cell cycle are my cell in?
 What proportion of cells have synthesised new DNA ?
 How many times have...
http://flow.garvan.org.au/flowcytometryinformation/cell -cycle-protocol
CELL CYCLE STAGING
Stoichometrically binding DNA I...
CELL CYCLE STAGING
Dye Notes
DAPI UV/ 405 nm excited, Membrane impermanent,
Hoechstt 33342 UV/ 405 nm excited, Membrane pe...
 DNA base analogues BrDU and EdU (thymadine analogue) are
incorporated into newly synthesised DNA if synthesis occurs in
...
Cells are first loaded with Cytoplasmic dye and at every division
the dye is equally split between the daughter cells
CELL...
CELL TRACKING
 Dyes and regents are available for long and short term
tracking
 Where did my cells go ?
 Are these the ...
Length of tracking Dye/ Reagent
Short • Calcein Dyes (live cells only) - hours
• Cell Tracker ™ (thiol-reactive) - 15
mins...
By Linking a Gene of
interest to the sequence
for a fluorescent
protein the fluorescent
protein is produced
when ever your...
FUNCTIONAL ANALYSIS
Calcium Flux Gene Expression
Transporter activity FRET Systems
https://www.bcm.edu/research/labs/goode...
EXAMPLE EXPERIMENT :
UNDERSTANDING CANCER
Question 1: Can I give the mouse cancer ?
EXAMPLE EXPERIMENT :
UNDERSTANDING CANCER
Question 2: Where does the Cancer spread too ?
1) Lymph Nodes
2) Lungs
3) Blood
...
EXAMPLE EXPERIMENT :
UNDERSTANDING CANCER
Question 3: What Genes do the Cancer cells Express?
1) RT PCR
2) Microarray
3) R...
EXAMPLE EXPERIMENT :
UNDERSTANDING CANCER
Question 4: What changes occur when the Cancer
Spreads ?
1) Lymph Nodes
2) Lungs...
EXAMPLE EXPERIMENT :
UNDERSTANDING CANCER
Question 5: How much heterogeneity is in my cancer ?
1) PCR
2) RT PCR
3) RNA Seq...
EXAMPLE EXPERIMENT :
UNDERSTANDING CANCER
Question 6: Does the Cancer recruit host cells ?
EXAMPLE EXPERIMENT :
UNDERSTANDING CANCER
Question 7: Are the host cells functionally active ?
www.icms.qmul.ac.uk
EXAMPLE EXPERIMENT :
UNDERSTANDING CANCER
Question 8: Can I treat the cancer ?
EXAMPLE EXPERIMENT :
UNDERSTANDING CANCER
Question 9: Can I find the Cancer Initiating cell?
EXAMPLE EXPERIMENT :
UNDERSTANDING CANCER
Question 8: Can I create a new cancer cell line ?
Immunology
 What is the nature of the immune response?
 What cytokines are the immune cells making ?
 CBA kits
 Is the...
How can flow help you ?
Do you have an application where you think
flow cant help ?
OTHER APPLICATIONS
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Flow Cytometry Training : Introduction day 1 session 1

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Flow Cytometry Training talks - part 1
This forms the first session of the Garvan Flow , Flow Cytometry Training course. this is a 1 1/2 day training course aimed at giving new and experienced researchers a better understanding of cytometry in medical and biological research.

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Flow Cytometry Training : Introduction day 1 session 1

  1. 1. Day 1 Introduction 9:00 am to 10:30 am FLOW CYTOMETRY TRAINING Robert Salomon (Flow Manager and Senior Flow Cytomerty Scientist)
  2. 2. Theory Session – 0900 till 1300  Introductions to the Lab and Staff  Self Introductions  Basics of flow cytometry  Applications for Flow Cytometry Morning tea - Provided  Getting Started  Panel Design  Controls and compensation  Data Analysis and Interpretation  Data Acquisition Overview  Instrumentation Lunch – provided Practical Session SESSION 1 Outline – 5 mins Day 1
  3. 3. Practical Session – 1330 till 1600  Lab orientation  Key Cytometry hardware Systems overview  Review of Acquisition Software  Mock Experiment Cytometry Exam to be completed after practical before tomorrows session TRAINING SCHEDULE Outline – 5 mins Day 1
  4. 4. Cell Sorting – 900 till 1230  Recap, discussion, exam questions  Cell Sorting  Automacs Pro  FACS (fluorescent Activated Cell Sorting)  Troubleshooting  Certificates TRAINING SCHEDULE Outline – 5 mins Day 2
  5. 5. Theory Session – 0900 till 1300  Introductions to the Lab and Staff  Self Introductions  Basics of flow cytometry  Applications for Flow Cytometry Morning tea - Provided  Getting Started  Panel Design  Controls and compensation  Data Analysis and Interpretation  Data Acquisition Overview  Instrumentation Lunch – provided Practical Session SESSION 1 Outline – 5 mins Day 1
  6. 6. INTRODUCTION TO GARVAN FLOW
  7. 7. Robert Salomon  Flow Cytometry Manager  Senior Flow Cytometry Scientist  ISAC Emerging Leader 2014-2019 r.salomon@garvan.org.au Lab 92958431 Office 92958432 INTRODUCTION – FLOW STAFF
  8. 8. Eric Lam  Flow Cytometry Operator e.lam@garvan.org.au Lab 92958431 INTRODUCTION – FLOW STAFF
  9. 9. Vitri Dewi  Flow Cytometry Technician v.dewi@garvan.org.au Lab 92958431 INTRODUCTION – FLOW STAFF
  10. 10. Hira Saeed  Flow Cytometry Technician h.saeed@garvan.org.au Lab 92958431 INTRODUCTION – FLOW STAFF
  11. 11. 1. Name and position ? 2. Group/lab affiliation ? 3. Flow Experience ? 4. Brief introduction to your project ? INTRODUCTION – ATTENDEES
  12. 12. BASICS OF FLOW CYTOMETRY ? Measurement METRY
  13. 13. BASICS OF FLOW CYTOMETRY ? Measurement METRY Cells CYTO
  14. 14. BASICS OF FLOW CYTOMETRY ? Measurement METRY Cells CYTO Flow Flow
  15. 15. Flow Cytometry Measurement METRY Cells CYTO Flow Flow
  16. 16. FLOW CELL Hydrodynamic focusing of sample to laser intercept - (interrogation point) Legend Laser intercept Core Stream
  17. 17. INTRODUCTION TO FLOW: ANIMATION
  18. 18. WHY USE FLOW ?  Rapid analysis ( 3k- 200k events/second)  Individual event analysis  Quantifiable results  Multiple parameter analysis  Statistical relevance
  19. 19. WHY USE FLOW ?  Rapid analysis ( 3k- 200k events/second)  Individual event analysis  Quantifiable results  Multiple parameter analysis  Statistical relevance
  20. 20. Imaging Flow Cytometry Cells per field/sec) Approximately 100 20,000 Number of Parameters <6 <24 Quantifiable Maybe (using complex analysis tools) 12-16 bit (< 65,356 channels) Yes* 18 bit resolution (262,144 channels) Ave. No. of Analysed Cells 1000- 10 x 100 cell fields >10,000 Anatomical localisation Yes N0 WHY USE FLOW ?
  21. 21. 1. Cells in single cell suspension 2. Fluorescent probes 3. Cytometer PREREQUISITES FOR FLOW CYTOMETRY The key to good result is good sample Preparation
  22. 22.  Flow Cytometry can be split into two main categories USING FLOW CYTOMETRY Cell Analysis Cell Sorting Cellular Characterisation Cellular Characterisation & Separation (FACS) B cell = 15% T cell = 23% GFP pos cell = 15% RBC = 47 % 1 2 3 4
  23. 23. 15mins Coffee and Morning Tea COFFEE
  24. 24.  Flow cytometry can be used to characterise, identify and separate cells or events based on physical and functional attributes as long as the detection system is based on a light readout. APPLICATIONS OF FLOW CYTOMETRY CELL + Marker + Cytometer(s) = Experimental Outcome
  25. 25.  Phenotyping  Apoptosis and cell death  Cell cycle, cell division and DNA synthesis  Cell tracking  Transduction/transfection confirmation  Functional analysis – calcium flux, gene expression, dye efflux, mitochondrial activity EXAMPLE EXPERIMENTS Analysis Cell Sorting
  26. 26.  Usually done with fluorescently tagged antibodies  Have my Cancer Cells spread to the Lymph Nodes ?  Do macrophages increase when gene X is switched on ?  What immune cells are recruited to the cancer site ?  What are the Differences between the Primary and Secondary cancer lessions ? PHENOTYPING Naïve B cells • CD20 pos • CD27 neg • CD10 neg • igG neg
  27. 27. 1. Does treatment/Condition X lead to apoptosis and/or Cell Death? 2. What is the Mechanism for Death and how quickly does it occur ? APOPTOSIS AND CELL DEATH Membrane alterations Caspases TUNEL Assay Membrane Integrity Membrane alterations Mitochondrial Changes Caspase Activation DNA Changes Membrane Integrity DNA condensation & FragmentationCaspase activity DAPI, PI or 7-AAD Membrane changes membrane potential and integrity FLICA detection
  28. 28. Functional identification Reagent Early Cell Membrane Changes • Phosphatidylserine flipping : ANX V • Violet Asymmetry Probe • Monomeric Cyanine Dyes : PO-PRO™-1, YO-PRO®-1, and TO-PRO®-3 Mitochondrial Changes • DiOC2(3) • JC-1 • DilC1(5) • MitoTracker® • MitoProbe® Caspase Activation • Caspase 3/7 activity: CellEvent®, Vybrant ® FAM™ (FLICA® regaents), PARP cleavage DNA Changes • DNA Fragmentation – TUNEL • Chromatin Condensation – Hoechstt 33342, Vybrant® DyeCycle™ Violet Membrane integrity PI, DAPI,7AAD, Fixable Live/Dead, ……….. APOPTOSIS AND CELL DEATH REAGENTS
  29. 29.  What stage of the Cell cycle are my cell in?  What proportion of cells have synthesised new DNA ?  How many times have my cells divided ? CELL CYCLE, CELL DIVISION AND DNA SYNTHESIS http://www.personal.psu.edu/staff/d/r/drs18/bisciImages/cycle.jpg
  30. 30. http://flow.garvan.org.au/flowcytometryinformation/cell -cycle-protocol CELL CYCLE STAGING Stoichometrically binding DNA Intercalating Dyes can be used to Quantitate changes in DNA content during the cell cycle. DNAContent G0/G1 | S | G2/M | G0/G1 Cell Cycle Phase
  31. 31. CELL CYCLE STAGING Dye Notes DAPI UV/ 405 nm excited, Membrane impermanent, Hoechstt 33342 UV/ 405 nm excited, Membrane permanent PI Blue/green excited, Membrane impermanent, Requires RNAse treatment Vybrant DyeCycle™ Variable excitation, Membrane permanent, no RNAse treatment required Draq 5 Red excitation, Membrane permanent, no RNAse treatment required (water soluble) 7-AAD Similar to PI but does not require RNAse treatement
  32. 32.  DNA base analogues BrDU and EdU (thymadine analogue) are incorporated into newly synthesised DNA if synthesis occurs in the presence of the Analogue DNA SYNTHESIS G2/M cells https://www.lifetechnologies.com/order/catalog/product/B23151 Undivided cells S phase cells
  33. 33. Cells are first loaded with Cytoplasmic dye and at every division the dye is equally split between the daughter cells CELL DIVISION http://www.vsh.com/Documentation/modfitlt/html/enhanced_cell_tracking_analysis.htm https://www.lifetechnologies.com/au/en/home/life-science/cell-analysis/flow-cytometry/cell-health-and-viability-assays-for-flow-cytometry/cell- proliferation-assays-for-flow-cytometry/celltrace-reagents-for-cell-proliferation.html
  34. 34. CELL TRACKING  Dyes and regents are available for long and short term tracking  Where did my cells go ?  Are these the cells that injected ?  What did my cells turn into ? Donor Vs Recipient LN Vs Spleen Vs Brain Vs Lungs
  35. 35. Length of tracking Dye/ Reagent Short • Calcein Dyes (live cells only) - hours • Cell Tracker ™ (thiol-reactive) - 15 mins – 1 hr • CellTrace (amine-reactive) Mid • Qtracker ®(Qdots nano crystals) – 6 to 10 Generations • CFSE and its derivatives – 6 to 8 generations • Lipid intercalators : CellVue® - days to weeks, DIL/DID Long • Fluorescent Proteins CELL TRACKING
  36. 36. By Linking a Gene of interest to the sequence for a fluorescent protein the fluorescent protein is produced when ever your gene of interest is transcribed & translated. TRANSDUCTION/TRANSFECTION CONFIRMATION Transduction = virus Transfection = no virus
  37. 37. FUNCTIONAL ANALYSIS Calcium Flux Gene Expression Transporter activity FRET Systems https://www.bcm.edu/research/labs/goodell/index.cfm?pmid=20017 The “Side Population” is a result of active transporter pumps Gene X Promoter GFP Gene Dyes such as Indo-1 change properties as they bind to Calcium during cell activation Donor fluorochrome Acceptor fluorochrome FRET Occurs within 10nm
  38. 38. EXAMPLE EXPERIMENT : UNDERSTANDING CANCER Question 1: Can I give the mouse cancer ?
  39. 39. EXAMPLE EXPERIMENT : UNDERSTANDING CANCER Question 2: Where does the Cancer spread too ? 1) Lymph Nodes 2) Lungs 3) Blood 4) Bone
  40. 40. EXAMPLE EXPERIMENT : UNDERSTANDING CANCER Question 3: What Genes do the Cancer cells Express? 1) RT PCR 2) Microarray 3) RNA Seq 4) Western blot 5) Proteomics
  41. 41. EXAMPLE EXPERIMENT : UNDERSTANDING CANCER Question 4: What changes occur when the Cancer Spreads ? 1) Lymph Nodes 2) Lungs 3) Blood 1) PCR 2) RT PCR 3) Microarray 4) RNA Seq 5) DNA Seq 6) Western blot 7) Microscopy 8) Cytokine assays 9) Epigenetics 10) Flow Cytometry
  42. 42. EXAMPLE EXPERIMENT : UNDERSTANDING CANCER Question 5: How much heterogeneity is in my cancer ? 1) PCR 2) RT PCR 3) RNA Seq 4) DNA Seq 5) Microscopy 6) Cytokine assays 7) Epigenetics A1 H12 Single cell Sorting Into 96/384 well plates
  43. 43. EXAMPLE EXPERIMENT : UNDERSTANDING CANCER Question 6: Does the Cancer recruit host cells ?
  44. 44. EXAMPLE EXPERIMENT : UNDERSTANDING CANCER Question 7: Are the host cells functionally active ? www.icms.qmul.ac.uk
  45. 45. EXAMPLE EXPERIMENT : UNDERSTANDING CANCER Question 8: Can I treat the cancer ?
  46. 46. EXAMPLE EXPERIMENT : UNDERSTANDING CANCER Question 9: Can I find the Cancer Initiating cell?
  47. 47. EXAMPLE EXPERIMENT : UNDERSTANDING CANCER Question 8: Can I create a new cancer cell line ?
  48. 48. Immunology  What is the nature of the immune response?  What cytokines are the immune cells making ?  CBA kits  Is there an antigen specific response  Tetramers EXAMPLE EXPERIMENT : UNDERSTANDING CANCER
  49. 49. How can flow help you ? Do you have an application where you think flow cant help ? OTHER APPLICATIONS

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