Flow Cytometry Training : Introduction day 1 session 1
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The document outlines a two-day flow cytometry training program led by Robert Salomon, covering theory and practical sessions on cell sorting and various applications of flow cytometry, including data analysis, instrumentation, and troubleshooting. It includes introductory segments, hands-on lab orientation, and a detailed discussion on cell analysis and sorting techniques. Key subjects addressed span from fundamental principles to advanced applications like cancer research and immune response analysis.
Flow Cytometry Training : Introduction day 1 session 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. 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
- 16. FLOW CELL Hydrodynamic focusing of sample to laser intercept - (interrogation point) Legend Laser intercept Core Stream
- 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