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.

1. Day 1
Introduction
9:00 am to
10:30 am
FLOW CYTOMETRY
TRAINING
Robert Salomon
(Flow Manager and Senior Flow Cytomerty Scientist)

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. 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. 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. 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. INTRODUCTION TO GARVAN FLOW

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. Eric Lam
 Flow Cytometry Operator
e.lam@garvan.org.au
Lab 92958431
INTRODUCTION – FLOW STAFF

9. Vitri Dewi
 Flow Cytometry Technician
v.dewi@garvan.org.au
Lab 92958431
INTRODUCTION – FLOW STAFF

10. Hira Saeed
 Flow Cytometry Technician
h.saeed@garvan.org.au
Lab 92958431
INTRODUCTION – FLOW STAFF

11. 1. Name and position ?
2. Group/lab affiliation ?
3. Flow Experience ?
4. Brief introduction to your project ?
INTRODUCTION – ATTENDEES

12. BASICS OF FLOW CYTOMETRY ?
Measurement
METRY

13. BASICS OF FLOW CYTOMETRY ?
Measurement
METRY
Cells
CYTO

14. BASICS OF FLOW CYTOMETRY ?
Measurement
METRY
Cells
CYTO
Flow
Flow

15. Flow Cytometry
Measurement
METRY
Cells
CYTO
Flow
Flow

16. FLOW CELL
Hydrodynamic
focusing of
sample to
laser
intercept -
(interrogation
point)
Legend
Laser intercept
Core Stream

17. INTRODUCTION TO FLOW:
ANIMATION

18. WHY USE FLOW ?
 Rapid analysis ( 3k- 200k events/second)
 Individual event analysis
 Quantifiable results
 Multiple parameter analysis
 Statistical relevance

19. WHY USE FLOW ?
 Rapid analysis ( 3k- 200k events/second)
 Individual event analysis
 Quantifiable results
 Multiple parameter analysis
 Statistical relevance

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. 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.  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. 15mins
Coffee and Morning Tea
COFFEE

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.  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.  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. 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. 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.  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. 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. 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.  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. 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. 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. 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. 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. 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. EXAMPLE EXPERIMENT :
UNDERSTANDING CANCER
Question 1: Can I give the mouse cancer ?

39. EXAMPLE EXPERIMENT :
UNDERSTANDING CANCER
Question 2: Where does the Cancer spread too ?
1) Lymph Nodes
2) Lungs
3) Blood
4) Bone

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. 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. 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. EXAMPLE EXPERIMENT :
UNDERSTANDING CANCER
Question 6: Does the Cancer recruit host cells ?

44. EXAMPLE EXPERIMENT :
UNDERSTANDING CANCER
Question 7: Are the host cells functionally active ?
www.icms.qmul.ac.uk

45. EXAMPLE EXPERIMENT :
UNDERSTANDING CANCER
Question 8: Can I treat the cancer ?

46. EXAMPLE EXPERIMENT :
UNDERSTANDING CANCER
Question 9: Can I find the Cancer Initiating cell?

47. EXAMPLE EXPERIMENT :
UNDERSTANDING CANCER
Question 8: Can I create a new cancer cell line ?

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. How can flow help you ?
Do you have an application where you think
flow cant help ?
OTHER APPLICATIONS