AllCells Flow Cytometry Webinar 09-26-2013
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The biotechnology and pharmaceutical industry is continuing to implement more flow cytometry-based assays in research and drug discovery due to the flexibility in assay development. The variety of assays which can be performed continues to expand, giving research laboratories options in data collection ranging from detection of cell signaling pathway activation to immunophenotyping. This webinar will provide an introduction to flow cytometry, a technique that uses fluorescently conjugated antibodies and dyes to identify characteristics in heterogeneous populations of cells. In addition we will present case studies on how flow cytometry can be used in drug discovery.
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AllCells Flow Cytometry Webinar 09-26-2013
- 1. Your Reliable Source of Human Primary Cells Flow Cytometry 101: How Flow Cytometry Can Help Advance Your Research
- 2. What is Flow Cytometry? Flow = Motion Cyto = Cell Metry = Measurement
- 3. Components of Flow Cytometry • Fluidics • Lasers • Optics • Data Analysis • Fluorescence- activated Cell Sorting
- 4. Fluidics System Transports Cells Laser Hydrodynamic Focusing Sheath Fluid Light Scatter Fluorescent Signal Sample
- 5. Sample Flow Rate Impacts Alignment High Sample Flow Rate =Small difference = Larger Core Low Sample Flow Rate =Large difference = Smaller Core
- 6. Lasers are ideal for flow cytometry http://notproperlydone.com Light Amplification by Stimulated Emission of Radiation 1. Stable 2. Focused 3. Single Wavelength
- 8. Optics Detect Emission of Fluorescence http://medicine.umich.edu •Dichroic Mirrors •Filters •Detectors
- 9. Multiparametric analysis is possible http://www.bdbiosciences.com
- 10. Sample Preparation •Incubate with Antibody •Wash to remove unbound Antibody •Analyze
- 11. Data presented in Histograms and Dotplots
- 13. How can I use Flow Cytometry? • Immunophenotyping • Intracellular proteins • Transcription Factors • Phosphorylation Status • Extracellular proteins • Receptor Density • Viability/Apoptosis • Caspase activation • Annexin-V presentation • TUNEL • Proliferation • CFSE • Cell Cycle Analysis • Isolation/Sorting
- 14. Case Study – Immunophenotyping Request: Characterize AML samples for expression of specific markers so customer could purchase samples of interest Approach: Multiparametric analysis of CD47, CD147, CD9 and proprietary protein Results: Identified specific patient samples that could be used in downstream analysis for customer
- 15. Case Study – Pathway Activation Request: Treat MM samples with proprietary compound and determine phosphorylation status of protein Approach: After treatment, fix and permeabilize cells and quantify phosphorylation of protein in cell subpopulations Results: Compound inhibited phosphorylation of proteins associated with proliferation/ apoptosis in CD38- and CD138-expressing cells 0 1000 2000 3000 4000 CD38 CD138 MeanFluorescent Intensity Untreated Treated
- 16. Case Study – Apoptosis Request: Treat CLL samples with proprietary compound and determine apoptotic affect Approach: After treatment, quantify apoptosis and necrosis using Annexin-V and 7-AAD Results: Compound induced apoptosis/necrosis in CLL samples
- 17. Case Study – Receptor Density Request: Determine receptor density of specific antigens in AML patients Approach: Measure Mean Fluorescent Intensity (MFI) of antibodies compared with beads bound with absolute levels of IgG Results: Quantified antigen binding capacity in multiple AML samples
- 18. Case Study – Multiparametric Sorting Request: Isolate CD14+CD16+ and CD14+CD16- subpopulations from patient with RA undergoing treatment with proprietary compound Approach: Samples sent from doctor at regular intervals were sorted based on CD14- and CD16- expression for RNA extraction and transcriptional analysis. Results: CD14+CD16+ and CD14+CD16- fractions were isolated from fresh PB MNC.
- 19. Case Study –Sorting based on MFI Request: Sort CD56-bright and CD56-dim populations for downstream analysis Approach: Isolate CD56+ cells using immunomagnetic isolation and sort CD56 subpopulations based on MFI Results: Isolated CD56-expressing cells based on relative expression level for RNA analysis
- 20. Flow Cytometry is a powerful research tool Cell Isolation Proliferation Apoptosis (Annexin V/PI) Pathway Activation
- 21. Basha Stankovich, Ph.D. Bioservices Manager bstankovich@allcells.com www.allcells.com/technical-support/ www.allcells.com/support/webinars/ Contact Information
- 22. Please use the prompt on the right of your screen to send a question to the organizer Questions will be read and answered Any offline questions may be sent to sales@allcells.com Thank you! Q&A
Notes de l'éditeur
- Thank you for joining us today. Here at AllCells, we use flow cytometry on a daily basis to aid in research at a variety of levels for groups in academic institutions and biotech and pharmaceutical companies. Today, I would like to share with you some of the basic principles of flow cytometry and how this powerful yet flexible platform can be used to help advance your research projects.
- When you think of flow cytometry, you might think of large, complicated instruments with multiple lasers; You might think of the histograms and dotplots that are the output of flow cytometry assays; or you might think of fluorescent dyes and conjugated antibodies that are used in the set-up of the assays.Let’s start with the meaning of the term “flow cytometry”Flow describes something in motionCyto = Greek root referring to a living organism or CellMetry = metric or a measurement Put it together = measurement of cells in motion
- There are 5 main components to flow cytometry that we will discuss todayThe Fluidic system which uses liquids to transport and align cells for interrogationLasers which activate fluorescent signals for detection of specific proteins on or in the cellOptics for detection of these fluorescent signalsData analysis which can be performed on the instrument or using additional software capable of depicting information in histograms or dotplotsAnd finally, specialized flow cytometers are capable of sorting specific populations based on their fluorescent signature.
- Our first component, Fluidics is used to move the cells and also to line them up through a process called hydrodynamic focusing. This is a result of a differential velocity between the sheath fluid and the sample fluid.The movement of the sheath fluid through the flow cell forces the cells into a single file, allowing us to interrogate single cells individually in a large population. The results are thousands to hundreds of thousands events that may be recorded with each event being a separate and unique cell.
- The alignment of these cells into a single file is dependant on the difference between the flow rates of the sheath fluid (in light blue) and the sample fluid (in dark blue). Since the Sheath Flow rate is fixed at a high velocity, the only control we have in setting up the assays are of the sample flow rate. A high sample flow rate can increase the speed at which we analyze our samples. However, this results in a smaller difference in velocity between the sheath and sample fluids. There our “core” becomes larger. The core being the diameter of the sample stream. A larger core allows more cells through simultaneous and decreases our confidence in only collecting information from single cells.On the other hand, a low sample flow rate means a larger difference between the sheath and sample fluid velocities and a smaller core. This means that, although it is taking us more time to collect the information, we can feel confident that our samples are aligned in single file. This is especially important if our goal is to sort cells with high purity.
- The second component important in flow cytometers is lasers.Laser is an acronym forLight Amplification by Stimulated Emission of RadiationWhile I won’t be getting into any of the physics of lasers, they are important in flow cytometry for a couple of reasonsthey are relatively stable and consistent in their output and can last many years depending on usageBecause of the alignment of the light waves, they can be focused into very small point which allow us to interrogate our cells depicted here in this drawing.They can be limited to emitting only a single wavelength of light (as opposed to xenon or mercury lamps you might be familiar with in fluorescent microscopy)
- The specific wavelength of the laser light is used to excite the fluorophore (for example FITC is excited using 488nm light) into a higher energy level. As the fluorophore spontaneously releases the energy to return to its ground state, fluorescent light is emitted.If we look at the excitation/emission spectrum for FITC (a commonly used fluorophore in flow cytometry), we can see that this fluorophore is robustly excited using a 488 nm laser and has maximum emission at approximately 525nm. We can take advantage of the energy released by this fluorophore by capturing the signal using our optic system
- The 3rd component of flow cytometers is the optical bench.The optics of a flow cytometer are probably the most complicated part of the instrument. Optics consist of filters, dichroic mirrors and detectors.-There are 2 basic types of detector, size and fluorescence. They are all based on light emission.-FSC and SSC detectors measure the light blocked or refractory to the cell. Both of these detectors measure 488 nm light. This is not associated with a fluorescent signal. Rather it is used to detect the size of the cell. Unfortunately, we won’t have time to discuss this aspect in more detail today. We can only say that FSC is a measurement of overall relative size of a cell (large or small) while SSC measures the complexity of a cell. This correlates with the presence of structures such as nuclei or other organelles in a cell.-All signal that is a result of fluorescent emission passes through or is refracted from dichroic mirrors (DM) at specific wavelengths. For example here we have a 560 nm SP DM. This means, that all light with a wavelength less than 560 nm is allowed to pass through the mirror. While all light with a wavelength of higher than 560nm is refracted. This allows us to collect only specific wavelengths of light while collecting additional information from the remaining light. This is what allows us to see more than one color at the same time on each cell. -The light that is passed through is then filtered through a 530/30 BW filter. The filter will allow through all light in a 30 nm range with 530 nm as the peak. In other words, it allows through all light between 515-545nm. Finally, the detector behind it collects the signal and it is converted into a digital signal and sent to the computer. We have a similar pattern of mirrors, filters and detectors for other wavelengths allowing us to collect multiple fluorophores simultaneously.
- Before we move on to the Data Analysis section, I would like to spend a couple of minutes discussing sample preparation and choosing the best fluorophores.If you are interested in multiparametric analysis or cell sorting, it is important to choose antibodies that have different emission spectra. This allows you to analyze multiple parameters simultaneously. For example, if you have an antibody specific to CD34 that is directly conjugated with FITC and you also would like to analyze the population of cells expressing CD38. You would choose a color such as PE-Cy7. This is an example of 4 colors that are possible to use simultaneously with a 488 nm laser. The inclusion of additional lasers, such as the red laser (633nm) or the violet laser (405 nm) further increases the selection of fluorophores.
- Once you have identified the fluorophores you would like to use, the actual sample prep is relatively simple. You will incubate your cell sample with the antibody or antibodies of interest. These antibodies can be directly conjugated, or you can come in with a secondary antibody if there is not a fluorescently conjugated antibody commercially available.After incubation, you will wash away any unbound antibody and you are ready to run the samples on the flow cytometer.
- Your data output will fall into 2 basic types; histograms or dotplotsHistograms present the data as a single variable. The output is a curve with the relative fluorescent intensity on the x-axis and the number of cells with that specific intensity represented by the height of the curve. -You will notice that the data is represented here relative to an isotype control. This is a sample of cells that have been incubated with a control which contains the fluorescent dye bound to a protein that does not react with the antigen we are analyzing. A positive signal is determined as being more fluorescent than the negative/isotype controls. -A second type of output used in flow cytometric analysis is the dotplot. This is a bivariate plot depicting the fluorescence on both axes. For example, we show here the CD71 (FITC) fluorescence on the x-axis and CD36 (PE) fluorescence on the y-axis. This allows us to analyze the expression of multiple antigens on the surface of the cells simultaneously. For example, all events in the upper right hand corner are expressing both CD71 and CD36 on the surface of the cells. However, events in the lower left corner of the dotplot express neither CD71 or CD36. Similarly, events in the lower right or upper left quadrants express only one or the other of these antigens, but not both. This is useful because many proteins are expressed on more than one cell type. So, if you can identify multiple proteins in you cell of interest, you are more likely to be identifying a pure/homogeneous population.-Drawing gates on either of these types of plots allows us to quantify the number of cells expressing the antigen of interest relative to the rest of the population.
- The final component in flow cytometry that I would like to discuss today, before we move into some practical applications for research projects, is sorting of the cells based on fluorescence. The principal is the same as we have previously discussed. The only difference is that in sorters, there is an additional component which applies an electrical charge to the cells you specify and deflects these cells into one of multiple positions. For example, you will see in this diagram that all cells which are green are deflected to the right hand tube. Similarly, purple cells are deflected to the left. Cells which are not of interest continue to waste. Based on the charge we give the cells, we can collect up to 4 separate cell populations simultaneously into individual tubes. Or we can sort specific cell types into individual wells of a 96 well plate for additional studies.
- Many of you may be asking how this is relevant to your project?There are many types of assays that have been adapted to flow cytometry. The strength of flow cytometry is in the numbers or quantitation. While you can use an ELISA to determine whether a protein is expressed, you can use flow cytometry to determine how many cells express that protein. Or whether a specific subset of cells express that protein. You can use flow cytometry for assays ranging from characterization of protein expression to activation of signaling pathways, such as caspase or phosphorylation status. You can use it to determine viability or apoptosis or cell proliferation. These are just a few of the many possible assays that are commonly used on the flow cytometer.Here at AllCells, we run these assays regularly for our customers. For the next few minutes, I’d like to share with you a couple of examples of how these assays can be used in research projects similar to yours.
- Here is an example of a recent experiment we performed for a customer using multiple antigens to immunophenotype a set of samples.
- In conclusion, I would like to reiterate to you the ability of flow cytometry to give you the data you are looking for in your research needs. Here at AllCells, we use flow cytometry to determine the effect that compounds may have on normal and diseased cells from patients. We analyze proliferative and apoptotic affects as well as cell signaling. Additionally, we isolate specific cell types using FACS based sorting. These cells may then be used in cell culture or in downstream assays such as transcriptional profiling. If you would like to discuss your specific project in further detail, I would be happy to discuss how flow cytometry can be incorporated into your studies.