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Applications in Flow Cytometry

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1 Applications in Flow Cytometry
some of the most common aplications of Flow Cytometry Vast subject won’t go into much detail main objective is to give you a brief overview exemplify what can be done with technique Cláudia Bispo IGC – April 4, 2013

2 Outline Potential Applications of Flow Cytometry Cell State
Immunophenotyping Cell cycle Apoptosis Cell proliferation Cell Function Cell activation Calcium flux Cytokine Secretion Activation of signalling pathways Levels of intracellular reactive oxygen species outline, during this presentation I will be talk about applications assess the state of the cellular populations further study functional state. these applications can be used most samples, some minor optimization steps can be necessary for bacterial and other microbiological samples few techniques that can be useful to people from microbiology Microbiology Dead/Live Discrimination Absolute counting Time points

3 Evaluate Cell State

4 CAUTION – Abs selection:
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 immunophenotyping, the most common of flow assays. uses labeled antibodies to identify cells of interest, with multiple detectors allow analysis of multiple parameters cell by cell simultaneously, EXAMPLE FIG identification of cellular subsets, labeled concurrently for different cellular markers it was possible to identify Just as a footnote, important to know your instrument & select fluorochromes that match the laser line and filter setup in the equipment to prepare experiment correctly and not waste unnecessary reagents 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 Excitation / Emission :
Cell Cycle DNA content analysis - Propidium Iodide (PI) G2 M G1 S G0 G0/G1 G2/M S-phase Evaluation of DNA content of cells - still one of the biggest applications in this field.  can provide a great deal of information about the state of cells on your sample  effect of certain stimuli, ex: transfected genes or drug treatment. This is achieved by labeling cell nuclei with Propidium Iodide (PI) & then analyzing the fluorescence properties of cellular pop. As resting and G1 cells will have one copy of DNA, 1x fluorescence intensity  1st peak to the left Cells in G2/M phase, two copies of DNA  2x the fluorescence intensity. Since the cells in S phase synthesizing DNA  fluorescence values between 1st - 2nd populations just like we can see in this histogram. EXAMPLE FIG 3 regions can subjectively selected  percentage of cells in each phase. Fluorescence (DNA content) Excitation / Emission : 488nm / max 617nm

6 Cell Cycle Analysis Cell Cycle Analysis Software Cell Number
G0/G1 Cell Number Examples: FlowJo ModFit LT  FCS Express  IDLYK …  G2/M S However, often … is not the most correct.  Useful if lot of debris or multiple DNA populations with different DNA content to determine accurate nr of cells in each phase best option use software that will mathematically analyse the DNA histogram & thus give a more accurate measurement of the percentage of cells in each phase. Fluorescence Intensity Accurate measurements allow for resolution of normal cells undergoing G1, S, G2 phases Also useful when multiple DNA populations present: measuring aneuploidy & polyploidy

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 104 S Phase 103 Anti-BrdU FITC 102 limitation of looking at a single fluorochrome: no kinetic information if S-phase cells are actually cycling / synthesizing DNA To assess: use BrdU plus PI on the sample BrdU is an analogue of thymidine  will be taken up into the DNA of cycling cells  After a specific amount of time, use an antibody against BrdU, find out if the cells took up BrdU during the process. EXAMPLE FIG plot, cells + for BrdU cells in S-phase region in the PI histogram, confirmed really cycling, excluding a few events that were not 101 G1 G2/M Excitation / Emission : PI 488nm / max 617nm BrdU varies by fluorophore Propidium Iodide

8 Pyronin Y plus Hoechst 33342/33258
Cell Cycle - G0/G1 discrimination Pyronin Y plus Hoechst 33342/33258 G0/G1 S G2/M G0 G1 Cell Count RNA Content RNA content Furthmore: to determine the level of senescence or resting state option to analyse cell cycle with dye combination Hoechst and Pyronin Y when cells are stained first with Hoechst and then with Pyronin possible to distinguish DNA from RNA. Pyronin binds preferentially to RNA & Hoechst to A-T DNA base pairs b/c resting cells arrested in G0 phase, have lower level of RNA and so are in red region of plot or bottom scheme  G0 and G1 phases can be separated looking at differences in RNA content DNA content (A-T base pairs) Excitation / Emission : PY 488nm / 575nm HO UV line / nm

9 Apoptosis Changes in light scatter DNA denaturation
apoptotic cells can be recognized by characteristic pattern of morphological, biochemical and molecular changes, summarized: Flow cytometry use some of these  detect and identify apoptotic cells majority based on the measurement of light scatter, sensitivity of DNA to denaturation, detection of changes in plasma memb or in signaling pathways. positive control give useful information about what to expect from analysis b/c the basal level of apoptosis and necrosis varies a lot in cellular population and might not give the necessary info cells in which apoptosis has been induced by (for example, by fas induction, treatment with the topoisomerase I inhibitor, etc…) 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:
Regarding changes in light scatter, as cells die/become apoptotic refractive index of internal cytoplasm becomes similar to that of the extracellular medium reduction FSC signal,  intracellular changes & alterations of cytoplasmic memb  increase SSC  EXAMPLE FIG cells cultured with rapamycin, an apoptosis inducer OR activation inhibitor % of cells in region decreases but other ex. changes is morphology might allow for an easy discrimination adding viability dye  proper differentiation live/dead with light scatter low level resolution of dead and apoptotic cells in some cases not enough Changes in light scatter: low level resolution of apoptotic cells

11 Propidium Iodide (fixed cells)
Apoptosis Propidium Iodide (fixed cells) DNA Degradation several viability dyes, stain dead cells, looking into DNA content in fixed samples during apoptosis, DNA is degraded  within the DNA there are nicks and fragmentation. looking at DNA content with PI cells that have lost DNA will take up less stain and so appear to the left of the G1 peak  amount of cells on SubG1 peak other viability dyes are available depend on the experiment: stain other fluorochromes, if you plan to fix and permeabilize your cells, etc. This method relies on the fact that after DNA fragmentation, there are small fragments of DNA that are able to be eluted following washing of the sample. This means that after staining with a quantitative DNA-binding dye, like PI, cells that have lost DNA will take up less stain and like we can see in this histogram, appear to the left of the G1 peak. Other viability dyes : 7-AAD Zombie Aqua To‐Pro3 ...

12 Annexin V-fluorochrome plus Propidium Iodide (non-fixed cells)
Apoptosis Annexin V-fluorochrome plus Propidium Iodide (non-fixed cells) To assess cell apoptosis in live cells, apoptotic/necrotic cells: Annexin V + PI. Annexin-V is a specific phosphatidylserine-binding protein that can be used to detect apoptotic cells. In normal cells  this residues found in inner part of cytoplasmic memb. during apoptosis  translocated & externalised  Annexin V is able to bind to them In general, this is an early event in apoptosis b/c cells are not fixed we can further exclude dead cells using PI --- Annexin-V is available conjugated to a number of different fluorochromes. 

13 Annexin V plus Propidium Iodide
Apoptosis Annexin V plus Propidium Iodide thus, distinguish four populations: • Viable cells, not undergoing detectable apoptosis: Annexin V (–) and PI (–) • Early apoptotic cells: Annexin V (+) and PI(–) • Late apoptotic cells: Annexin V (+) and PI (+) • Cells that have died through non-apoptotic pathway: Annexin V (–) and PI (+) commonly: PI in their samples but as you can see only these 2 pop will be identified Excitation / Emission : Annexin varies by fluorophore PI 488nm / 617nm

14 Analysis by Flow Cytometry
Apoptosis (intracellular staining) Fix and permeabilize Add Antibody Analysis by Flow Cytometry In addition to all these methods,   intracellular staining for specific signaling pathways involved in apoptosis have also become a widespread practice in flow cytometry general protocol for this type of analysis are…

15 Apoptosis – Bcl-2 family members
evaluate apoptotic states through Bcl-2 expression, protein blocks apoptotic death Just like as we can see in the EXAMPLES when cells are  stimulated & have higher cell viability the expression levels of this protein are higher when apoptosis is induced (rapamycin) mean expression levels decrease correspondely correlate effects Bcl-2 proteins block apoptotic death by controlling mitochondrial memb permeability & interfering with pro-apoptotic proteins. Excitation / Emission: Varies by fluorophore

16 Apoptosis – Activated forms of Caspases
Untreated Etoposide fundamental aspect  activation of caspases, many available conjugated Ab to bind to the active forms of caspases. EXAMPLE use cleaved Caspase-3 Antibody (Alexa Fluor 488 conjugate) clear difference between the expresssion during normal state  low expression levels when a cytotoxic agent is added  expression of active form of caspase is greatly incresased Flow cytometric analysis of Jurkat cells, untreated (blue) or etoposide-treated (green), using Cleaved Caspase-3 (Asp175) Antibody (Alexa Fluor® 488 Conjugate). Excitation / Emission: Varies by fluorophore Ex A488: 488nm / 520nm

17 Cell Proliferation Tracking Cell Proliferation with CFSE (Carboxyfluorescein Succinimidyl ester) Dilution of CFSE Cell Divisions STAIN WITH CFSE CELL 1 2 3 4 Evaluate cell proliferation, use of CFSE allows to track cell proliferation through - diffusion of this fluorescein molecule - freely into cells - Retained in their cytoplasm, - not affecting its cellular function cells labelled with this dye  proliferate, after mitosis each daughter cell: contain half as much dye as the parent  half as bright. After each division the level of fluorescence will halve  by measurement of the green fluorescence  monitor the nr of divisions. EXAMPLE, cells have been labelled & stimulated - follow divisions up to 5 generations. Original + daughter cells Also, combining surface markers: specific subpopulations, like in this case with B cells. Using a modelling program such as FlowJo or ModFit, further determine how many cells belong to each generation. Excitation / Emission: 488nm / 521 nm

18 Tracking Cell Proliferation with CFSE
IL-7 IL-7+ DMSO IL-7+ PI3K Inhibitor IL-7+ Erk Inhibitor few other examples when the cells are stimulated or inhibited to proliferate

19 Evaluate Cell Function

20 Cell Activation FSC x SSC – Cell size similar to apoptotic cells,
also possible to evaluate the changes in morphology of activated cells When cells are activated  bigger  cellular population with higher FSC and SSC distribution. through light scatter with a low level resolution Sofia Marques (IMM) Filipa Lopes (IMM)

21 Cell Activation Activation markers: CD69, CD71, etc
more detailed and specific way to assess cellular activation specific expression of certain activation markers, just like CD69 or CD71 … to assess effect of stimuli large range of antibodies to choose from and combined with others  det. activated state specific subpop Excitation / Emission: Varies by fluorophore

22 Calcium Flux Excitation / Emission: Indo1 UV line / 405nm
Effects of T cell receptor stimulation by B-Cells on ionized calcium concentration ([Ca2+]i). Fluorescence-imaging of human erythrocytes treated with PGE2 using the calcium fluorophor Fluo-4 Calcium  very important cell signal changes in intracellular levels are a good indicator if the cell is responding to a stimulus. mobilization observed  using fluorescent dyes The most common UV-excited dye Indo-1  fluoresces at a different wavelength when bound to calcium & when it is not. By measuring fluorescence at both wavelengths  ratioing the two signals, a flux can be seen THROUGH course of 20min Fluo-4 is a excited by a 488 laser  when binds to Ca2+ inside the cell  fluorescent --- Calcium ions have an important role in cell signalling and the intracellular concentration of calcium ions may show transient changes in response to external stimuli. There are several fluorescent dyes whose properties depend on the amount of bound Ca++. For flow cytometry, the most useful of these is indo-1 whose emission wavelength changes on binding calcium (see Figure 3.4, Chapter 3). It is loaded into the cell in the form of the acetoxymethyl ester. Indo-1 is excited in the UV. A mixture of fluo-3 and Fura red can be used for work with a flow cytometer limited to excitation at 488 nm. Fluo-3 has a maximal fluorescence at 530 nm; its fluorescence increases with increasing Ca++ concentration. Fura Red has a maximal fluorescence at 650 nm which decreases with increasing Ca++ concentration. Excitation / Emission: Indo1 UV line / 405nm Fluo nm / 516nm Sofia Marques (IMM) Filipa Lopes (IMM)

23 Activation of signalling pathways
Evaluation of the activation signaling pathways another way to assess cellular responses to environmental stimuli critical decision processes within the cell combining phospho-specific antibodies & flow cytometry  tool for detection of protein phosphorylation at the single cell level. General phospho-protein staining technique: after certain stimuli cells are then fixed, permeabilized & stained with phospho-specific antibodies. b/c the Abs bind only to the phosphorylated form of the proteins  increase in fluorescence correlates with an increase in phosphorylation. stimulus A  increase in red fluorescence because the red protein is phosphorylated. B  green FL The combination of stimuli A and B induces phosphorylation of both proteins making the cells both green and red fluorescent, that is double positive for both dyes. Krutzik et al. Clin Immunology (2004)

24 Phospho-protein detection
Activation of signalling pathways Phospho-protein detection pStat1 Multiple signaling cascades can be monitored simultaneously: different Abs determine the specificity of stimulus analyze rare subsets of cells within complex populations Better discrimination of High vs. Low responders Better evaluate population heterogeneity improvement comparing with Western Blot: clearly 2 different kinds of samples appear identical even reliably distinguish different levels of expression Ability to analyze rare subsets of cells within complex populations Better evaluate population heterogeneity Discrimination of High vs. Low responders Krutzik et al. Clin Immunology (2004)

25 Combining Surface Markers with Phospho-staining
Another advantage of using flow cytometry incorporating surface markers into staining protocols different sub pops analyzed concurrently for protein phosphorylation maximize results from precious samples…  Evaluate protein phosphorylation in different subpopulations concurrently

26 Levels of intracellular reactive oxygen species
Excitation / Emission: 488nm / 605nm other type of evaluation is analyze the oxidative burst after stimulus several compounds, in their reduced form are non-fluorescent  become fluorescent upon oxidation. ex: dihydroethidium, when oxidized to ethidium bind to DNA of cells and fluoresce red. incubation with 10 mM muramyl dipeptide for 15 min. (small window of time – time points useful to not miss) 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.

27 Cytokine Secretion - Multiplex Bead Arrays
SOON @ UIC Luminex MAGPix System bead coated with capture antibody mixture 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 Luminex System  analyzer based on the principles of flow cytometry magnetic bead-based technology  multiplex up to 50 analytes in a single microplate well (very small sample volumes) system uses differentially dyed beads to achieve multi-analyte profiling for proteins & nucleic acids. Each type of bead coated with specific antibody mixture specific for analytes measured, These beads then pass through the equipment flow cell, b/c they are magnetic they’re stop and optics able to measure amount of fluorescence being proportional to amount of cytokine bound to each bead (equivalent to ELISA reading) but more cost and time efficient

28 Multiplex Bead Arrays SOON @ UIC
Or design custom and personalized panels Merck Millipore MAGPix A wide range of molecules, such as cytokines, hormones, so on… can be analyzed and Many panels are available, examples Merk Millipore possibility to design your own panels for the molecules you’re interested in The procedure is really straight forward and you’ll be able to get up to results in 1 hour graph;:

29 Microbiology Applications
Like I said Most these applications can also be applied to bacterial and other microbiological samples Few techniques that can be useful specifically to people from microbiology

30 Microbiology - Dead/Live Staining
Various established methologies can be optimized SYTO 9 /PI  - Live/Dead BacLight viability kit SYBR Green-I /PI Barbesti (2000) DiBAC4(3)/EB/PI Flow Cytometry Protocols 2nd ed. etc... Several kits available Excitation / Emission: 488nm / Varies by method LIVE/DEAD kit for Bacteria LIVE/DEAD kit for Yeast & Fungi live dead staining various established methologies can be optimized for bacterial and other microbiological samples. encoutered several already published optimizations and several kits are also commercially available. One thing to account for is the excitation/emission spectra of these do not coincide with fluorescent tags of your sample LIVE/DEAD BacLight Bacterial Viability Kit SYTO 9 stain + PI These differ in their ability to penetrate healthy bacterial cells. alone, SYTO9  labels live and dead bacteria. PI  only bacteria with damaged membranes, SYBR-I + PI to detect viable bacteria in different samples SYBR-I (+) PI (-): viable bacteria; SYBR-I (-) PI (+): dead or damaged cells, PI (+), while SYBR-I (+): partially damaged membranes, the PI does not reach a concentration that is sufficient for an efficient energy transfer from SYBR-I to the PI bound to DNA. DiBAC4/EB/PI DiBAC4 which is used as an indicator of memb potential ΔΨ, with EB, which is retained by cells with intact membranes PI is used to demonstrate membrane permeability; once PI enters cells, it displaces EB from nucleic acids CAUTION: Make sure excitation/emission of kit dyes do not coincide with the ones on your sample

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) There are also few papers published on how to evaluate DNA content for microbiological samples DRAQ5 with E.coli PI or SYTOX Green with yeast Excitation / Emission: 647nm / 670nm Excitation / Emission: 488nm / 520nm

32 Microbiology Time Points
Assess treatment effect on population survival, protein expression, etc at indicated time points - effect on protein expression - counting: population proportions CAUTION: Make sure you use a fluorescence control when protocol requires measuring & comparing protein expression Time points: assess treatment effect, great importance no matter what is your research background. population survival, cell cycle, etc during determinated time points, Or the effect on protein expression In this last case it is of maximum importance  use a fluorescence control, such as fluorescent beads at least at the beggining of each analysis. eliminates de effect of several factors such as: PMT ou laser differences from day to day, facilitating the comparison of results  counting cells and assess population proportions

33 Cell counting Beads @ UIC Scepter
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 @ UIC Scepter Handheld Automated Cell Counter Allows cell differentiation by its volume (size) according to the Coulter principle Uses a disposable sensor to count cells on a regular cytometer you must use reference beads b/c most flow cytometers have no way of exactly controlling the flux and amount of sample, examine of a large number of cells reduce the time employed with each determination: for large-scale studies this may be ideal. possibility of combining surface markers during the process allow for determinations of specific subpop. The easiest way to accomplish this is  reference beads with known number of particles per mL can be added to the sample. There are other alternatives: Scepter: handheld cell counter differentiates cells by its volume Uses disposable sensor Coulter principle The Coulter method of sizing and counting particles is based on measurable changes in electrical impedance produced by nonconductive particles suspended in an electrolyte. A small opening (aperture) between electrodes is the sensing zone through which suspended particles pass. In the sensing zone each particle displaces its own volume of electrolyte. Volume displaced is measured as a voltage pulse; the height of each pulse being proportional to the volume of the particle. The quantity of suspension drawn through the aperture is precisely controlled to allow the system to count and size particles for an exact reproducible volume. Some disadvantages using beads for counts. Most importantly: 1.Beads tend to aggregate 2.bead concentration may vary (depends on bead aggregation, how well you vortex the beads, whether or not evaporation has occurred in the bead stock, pipetting issues)

34 Image based alternatives
Cell counting (2) Countess™ Automated Cell Counter (Invitrogen) Tali™ Image-Based Cytometer (Invitrogen) TC20 Automated Cell Counter (BioRad) etc... Image based alternatives @ UIC SOON @ UIC Image based alternative: -Countess (we have at UIC) -TC20 from bio-rad -Tali Are automated cell counters, provide the total cell count & assess cell viability via trypan blue exclusion In case of Tali & Muse (son deliver) more complex system Actually MINI cytometer, Evaluate GFP/RPF expression Run CELL CYCLE, Anexin and other apoptis assays, among other like the others, Easy to work with, Get comprehensive data with graphic reports right from your bench Coulter principle The Coulter method of sizing and counting particles is based on measurable changes in electrical impedance produced by nonconductive particles suspended in an electrolyte. A small opening (aperture) between electrodes is the sensing zone through which suspended particles pass. In the sensing zone each particle displaces its own volume of electrolyte. Volume displaced is measured as a voltage pulse; the height of each pulse being proportional to the volume of the particle. The quantity of suspension drawn through the aperture is precisely controlled to allow the system to count and size particles for an exact reproducible volume.

35 Today’s Future Applications Field of Flow cytometry always progressing
Not just in terms of reagents / fluochromes etc Also new technologys & equipments

36 Amnis Image Stream Merck Millipore
The ImageStream and FlowSight from Amnis (bought by Merck) are flow cytometers that combine flow cytometry with microscopy they take fluorescent pictures of every cell that runs through the instrument. resolution obviously not of confocal quality but can provide very useful information about Co-localization studies, cell cycle and so on... just a few examples: -regular plots -select pop. And then visualize it In addition to the cell cycle analysis based on DNA content, image analysis features can be used to distinguish mitotic events from G2 cells based on nuclear morphology.

37 Amnis Image Stream power of this technology has in the last years been confirmed by the increasing number of applications being published, including co-localization studies, morphology analysis, cell cycle and mitosis, DNA damage and repair, cell-cell interaction, etc... 

38 CyTOF – Mass Cytometer 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) The CyTOF instrument based on atomic mass spectrometry uniquely applied to single cell analysis. Cells are labeled with antibodies containing metal tags, introduced individually into the system, just like regular flow cytometer atomized & ionized with high temperatures plasma By time-of-flight is possible to separate  by mass and counted. It can measure 34 parameters simultaneously in a single cell presence of the tag element indicates that the antibody found and bound the target biomarker, and the intensity of the signal is directly proportional to the nr of Abs bound per cell. It’s stil a new technology a lot of optimizations are needed, still undergoing imense potential   specially for evaluation and modeling of molecular networks at single cell level It is important to note that cells without any tagging elements cannot be detected by mass cytometry: Only element tags can be registered with high sensitivity and specificity, and therefore there is no “auto-fluorescence”-like effects. Elemental tags are chosen from rare elements whose natural concentration in a biological sample is below the detection limit. Unstained cells are “transparent” to the mass cytometer. Omatsky et al. JAAS (2008), Bendall et al. (2011)

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 With the recent advances in flow cytometry we are heading further into the path of single cell analysis -many advances have been made in microfluidics and lab-on-a-chip systems; -there are now instruments (Amnis) that can image a cell at the point of laser interrogation; And there is now the -measure at least 34 parameters simultaneously with CyTOF. This is indeed area constantly evolving With still a lot exciting techniques & possibilities to be discovered Hope I was able to pass along that message and maybe interested you into looking forward to test some of these applications any help we can give is also an oportunity for us to gain more experience in Flow Cytometry So don’t be afraid to ask!

40 Available Laser Lines at IGC’s Flow Cytometry Lab
Summary Flow Lab UIC Available Laser Lines at IGC’s Flow Cytometry Lab Bench Top Analyzers Cell Sorters FACScan FACScalibur CyAn ADP LSR Fortessa MoFlo FACSAria 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 applications @ 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

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