Presentation is loading. Please wait.

Presentation is loading. Please wait.

Applications in Flow Cytometry IGC – April 4, 2013.

Similar presentations


Presentation on theme: "Applications in Flow Cytometry IGC – April 4, 2013."— Presentation transcript:

1 Applications in Flow Cytometry IGC – April 4, 2013

2 Outline Immunophenotyping Cell cycle Apoptosis Cell proliferation Cell activation Calcium flux Cytokine Secretion Activation of signalling pathways Levels of intracellular reactive oxygen species Cell State Cell Function Dead/Live Discrimination Absolute counting Time points Microbiology Potential Applications of Flow Cytometry

3 Evaluate Cell State

4 Immunophenotyping Uses labeled antibodies (Abs) to identify cells of interest Determination of cell surface antigens Allows for detailed identification of cellular subsets (simultaneously measure multiple parameters cell by cell) Targets on both surface and intracellularly CAUTION – Abs selection: Fluorophore’s excitation spectrum must match the laser line used, and its emission must fall within detection filter sets in the cytometer CAUTION – Abs selection: Fluorophore’s excitation spectrum must match the laser line used, and its emission must fall within detection filter sets in the cytometer

5 Cell Cycle DNA content analysis - Propidium Iodide (PI) G2 M G1 S G0 G0/G1 S-phase G2/M Fluorescence (DNA content) Excitation / Emission : 488nm / max 617nm Excitation / Emission : 488nm / max 617nm

6 Cell Cycle Analysis Cell Cycle Analysis Software G 0 /G 1 S G 2 /M Fluorescence Intensity Cell Number Accurate measurements allow for resolution of normal cells undergoing G1, S, G2 phases Also useful when multiple DNA populations present: measuring aneuploidy & polyploidy Examples: FlowJo ModFit LT FCS Express IDLYK … Examples: FlowJo ModFit LT FCS Express IDLYK …

7 Cell Cycle - Bromodeoxyuridine (BrdU) method Propidium Iodide plus BrdU staining BrdU is thymidine analog Taken up by cells in S-phase Usually in combination with PI Anti-BrdU FITC G1 G2/M S Phase Propidium Iodide Excitation / Emission : PI 488nm / max 617nm BrdU varies by fluorophore Excitation / Emission : PI 488nm / max 617nm BrdU varies by fluorophore

8 Cell Cycle - G0/G1 discrimination Pyronin Y plus Hoechst 33342/33258 G0/G1 S G2/M G0 S G2/M G1 Cell Count RNA Content Excitation / Emission : PY 488nm / 575nm HO UV line / nm Excitation / Emission : PY 488nm / 575nm HO UV line / nm DNA content (A-T base pairs) RNA content

9 Apoptosis Changes in light scatter DNA denaturation Changes in plasma membrane Changes in cell organelles / signaling pathways * positive control is useful

10 Apoptosis CELL DEATH – FSC x SSC Changes in light scatter: low level resolution of apoptotic cells Changes in light scatter: low level resolution of apoptotic cells

11 Apoptosis Propidium Iodide (fixed cells) DNA Degradation Other viability dyes : 7-AAD Zombie Aqua To‐Pro3...

12 Apoptosis Annexin V-fluorochrome plus Propidium Iodide (non-fixed cells)

13 Apoptosis Annexin V plus Propidium Iodide Excitation / Emission : Annexin varies by fluorophore PI 488nm / 617nm Excitation / Emission : Annexin varies by fluorophore PI 488nm / 617nm

14 Apoptosis (intracellular staining) Fix and permeabilize Add Antibody Analysis by Flow Cytometry

15 Apoptosis – Bcl-2 family members Excitation / Emission: Varies by fluorophore Excitation / Emission: Varies by fluorophore

16 Apoptosis – Activated forms of Caspases Flow cytometric analysis of Jurkat cells, untreated (blue) or etoposide-treated (green), using Cleaved Caspase-3 (Asp175) Antibody (Alexa Fluor® 488 Conjugate). Untreated Etoposide Excitation / Emission: Varies by fluorophore Ex A488: 488nm / 520nm Excitation / Emission: Varies by fluorophore Ex A488: 488nm / 520nm

17 Tracking Cell Proliferation with CFSE (Carboxyfluorescein Succinimidyl ester) STAIN WITH CFSE Dilution of CFSE Cell Divisions CELL Cell Proliferation Excitation / Emission: 488nm / 521 nm Excitation / Emission: 488nm / 521 nm

18 Tracking Cell Proliferation with CFSE IL-7 IL-7+ DMSO IL-7+ PI3K Inhibitor IL-7+ Erk Inhibitor

19 Evaluate Cell Function

20 Cell Activation FSC x SSC – Cell size Sofia Marques (IMM) Filipa Lopes (IMM)

21 Cell Activation Activation markers: CD69, CD71, etc Excitation / Emission: Varies by fluorophore Excitation / Emission: Varies by fluorophore

22 Calcium Flux Fluorescence-imaging of human erythrocytes treated with PGE 2 using the calcium fluorophor Fluo-4 Effects of T cell receptor stimulation by B-Cells on ionized calcium concentration ([Ca 2+ ] i ). Sofia Marques (IMM) Filipa Lopes (IMM) Excitation / Emission: Indo1 UV line / 405nm Fluo-4 488nm / 516nm Excitation / Emission: Indo1 UV line / 405nm Fluo-4 488nm / 516nm

23 Activation of signalling pathways Krutzik et al. Clin Immunology (2004)

24 Activation of signalling pathways Phospho-protein detection Ability to analyze rare subsets of cells within complex populations Better evaluate population heterogeneity Krutzik et al. Clin Immunology (2004) Discrimination of High vs. Low responders pStat1

25 Combining Surface Markers with Phospho-staining  Evaluate protein phosphorylation in different subpopulations concurrently

26 Levels of intracellular reactive oxygen species Measurement of oxidative burst in human peripheral blood granulocytes using dihydroethidium. On oxidation, ethidium is produced which binds to DNA & fluoresces red. The histogram shows the red fluorescence before and after incubation with 10 mM muramyl dipeptide for 15 min. Excitation / Emission: 488nm / 605nm Excitation / Emission: 488nm / 605nm

27 Cytokine Secretion - Multiplex Bead Arrays Quantitation & detection of cytokine and signal transduction proteins, DNA & RNA - Magnetic bead-based technology - Up to 50 analytes /well of a 96 well plate - Up to results in 1 hour - Uses small sample volumes UIC UIC bead coated with capture antibody mixture

28 Multiplex Bead Arrays UIC UIC Or design custom and personalized panels Merck Millipore MAGPix

29 Microbiology Applications

30 Microbiology - Dead/Live Staining Various established methologies can be optimized o SYTO 9 /PI - Live/Dead BacLight viability kit o SYBR Green-I /PI - Barbesti (2000) o DiBAC 4 (3)/EB/PI - Flow Cytometry Protocols 2nd ed. o etc... Several kits available CAUTION: Make sure excitation/emission of kit dyes do not coincide with the ones on your sample CAUTION: Make sure excitation/emission of kit dyes do not coincide with the ones on your sample LIVE/DEAD kit for Bacteria LIVE/DEAD kit for Yeast & Fungi Excitation / Emission: 488nm / Varies by method Excitation / Emission: 488nm / Varies by method

31 Microbiology Monitoring Cell Cycle – DNA content Minor protocol adjustments/optimization might be needed comparatively with established protocols for eukaryotes E.coli: DRAQ5 - Silva et al. (2010) Yeast : PI or SYTOX Green - Knutsen (2011) Excitation / Emission: 647nm / 670nm Excitation / Emission: 647nm / 670nm Excitation / Emission: 488nm / 520nm Excitation / Emission: 488nm / 520nm

32 Time Points Assess treatment effect on population survival, protein expression, etc at indicated time points - effect on protein expression - counting: population proportions Microbiology CAUTION: Make sure you use a fluorescence control when protocol requires measuring & comparing protein expression CAUTION: Make sure you use a fluorescence control when protocol requires measuring & comparing protein expression

33 Cell counting Employs the use of reference beads on a regular cytometer Allows the examination of large number of cells per sample Combining Surface Markers will allow to count specific subpopulations Handheld Automated Cell Counter Allows cell differentiation by its volume (size) according to the Coulter principle Uses a disposable sensor Scepter UIC

34 Cell counting (2) Countess™ Automated Cell Counter (Invitrogen) Tali™ Image-Based Cytometer (Invitrogen) TC20 Automated Cell Counter (BioRad) etc... Image based UIC UIC UIC

35 Today’s Future Applications

36 Amnis Image Stream Merck Millipore

37 Amnis Image Stream

38 CyTOF – Mass Cytometer Omatsky et al. JAAS (2008), Bendall et al. (2011) Can measure 34 parameters simultaneously in a single cell Cells are labeled with Abs containing rare elements metal tags Cells are atomized & ionized in plasma (high temperatures) Tags are separated & identified by mass (time-of-flight)

39 Future Advances Heading further into the path of Single Cell Analysis – microfluidics & lab-on-a-chip systems Reduced laser size and capillary flow techniques mean smaller instruments Instruments can now image cell at point of laser interrogation More colours for immunofluorescence

40 Summary Flow Lab UIC Available Laser Lines at IGC’s Flow Cytometry Lab Bench Top AnalyzersCell Sorters FACScanFACScalibur CyAn ADP LSR Fortessa MoFloFACSAria Multiline UV ( nm) Violet (407 nm) Blue Violet (442 nm) Cyan (457 nm) Blue (488 nm) Green (514 nm) Yellow(561 nm) Red (640 nm)

41 Summary of UIC Flow Lab Cytometer Immuno- phenotyping / activation state Cell Cycle / Cell proliferation Apoptosis Calcium flux (with Fluo-4) Multiplex cytokine secretion Activation Signaling Pathways / ROS Microbiology applications FACSCalibur FACSScan CyAn ADP BD Fortessa Luminex MAGPix


Download ppt "Applications in Flow Cytometry IGC – April 4, 2013."

Similar presentations


Ads by Google