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flow cytometry

Transcript: By: Getsemani theoretically, any specimens from which a single cell suspension can be generated are suitable for flow cytometry analysis Blue Blind Working on flow cytometry flow cytometry Common specimens suitable for flow cytometry analysis include ---------------------------------------- what is flow cytometry Mark Zuckerberg By:Corey Red Blind *peripheral blood *bone marrow *body fluids *cerebrospinal fluids *urine *lymph node(cells or fresh tissue) *any fine-needle aspirates. prepared by: Mirko kamal Lina adnan Tawar tahseen Michel. "Color Blindness Simulator." Colblindor. N.p., n.d. Web. 28 Apr. 2014. <>. There are certain contacts and lenses with effects that help people affected with colorblindness. The certain lenses help people who suffer from color blindness see basic colors well. flow cells measurement "Color Blindness (simulations of Deficiencies)." Youtube. N.p., 7 June 2011. Web. 29 Apr. 2014. < Original what inside flow cytometer 1930's Moldavan in Science described the counting of blood cells filing through a capillary tube using a photoelectric sensor. 1940's US Army constructed a device that detected bacteria in a stream of air, using a Ford headlight as a light source and a photomultiplier tube as a detector. 60% 0.6 um 1950's Caspersson measured nucleic acid and protein metabol ism for normal and abnormal cell growth usinga Cadmium spark as a UV light source. Coulter constructed a cell counter based on the fact that the electrical conductivity of cells is lower than that of saline. Saline conducts, cells impede. 1960's Kamentsky at IBM developed the Rapid Cell Spectrophotometer. It used an arc lamp source and measured nucleic acid content and cell size. A computer (!!!!) measured and analyzed the data. IBM loaned Herzenberg a prototype and he developed the first real FACS. 1970's Industry takes over technical development: BD, Beckman, Ortho etc Specimens suitable for flow cytometry * flow cytometers have 3 key system Recent developments or research however, lack of distinct antigens or makers in the cells of interest or tissues limits the diagnostic value of flow cytometry flow cyto metry 1-fluidics 2-optics 3-electronics Mr. Zuckerberg was born May 14, 1984 He is one of the youngest billionaires. He created Facebook as blue because he is colorblind. He dropped out of college his sophomore year. He got married to Priscilla Chow on May 19, 2012 Has red/green colorblindness history of flow cytometry Flow cytometry * measurement of single cells,identification of sub-populations. * wide area of application for analysis and diagnosis. * maybe controlled remotely. benefits of flow cytomtry

Flow cytometry

Transcript: laser source convert eosinophil beam Reading II Past Perfect scattered light particles přeměnit principles of FC illuminate label osvítit turnaround time fluid světélkování lobularity fluid Flow Cytometry Definitions dispense antibody scatter Flow cytometry in practice direct 1 Have you already worked with the flow cytometer? When? What did you measure? sheath fluid sample physician zpracovat electronics fluorescence scatters Flow cytometry flow Reading III - gap-fill scattered light 3 Associated words 1 Week 6 Flow cytometry laser beam Reading process determined 2 What are the main uses of flow cytometry? Vocabulary revision size optical bench focusing event log optická lavice antigen nozzle emit converts 6 1 particles laser source When I arrived, they had run the flow. chemiluminescence 3 scatter rate use a new tense tekutina, kapalina illuminate nozzle 2 positive/negative sample structure process rozptýlit fluorescent electronics 5 5 these sheath fluid photometer Flow Cytometry - Vocabulary složení transports Vocabulary measures explain where it is used When I arrived, they were running the flow. 4 granularity flag Unit outline describe how fc works sample systems Flow cytometry is a technology that _____________ (1) and then ___________ (2) physical characteristics of single ___________ (3), usually cells, as they ___________ (4) in a ____________ (5) stream through a beam of light. The properties measured include a particles’ relative _____________ (6), relative granularity or _________________ (7) complexity, and relative fluorescence _____________ (8). These characteristics are ______________ (9) using an optical-to-electronic system. This system records how the cell or particle _____________ (10) laser light and emits fluorescence. past perfect tense 3 on granules optical absorbance photometer fluorescence When I arrived, they ran the flow. paprsek level detection intensity a plot lobes fluorescence vyzařovat Grammar Point complexity focusing GOALS 2 when practical uses analyses 4 by A flow cytometer is made up of three main ____________ (11): fluidics, optics and ______________ (12). The fluidics system ___________ (13) particles in a stream to the laser beam. The optics system _______________ (14) of lasers to ____________ (15) the particles in the sample stream and __________ (16) filters to ____________ (17) the resulting light signals to the detectors. The ___________ (18) system _____________ (19) the detected light signals into electronic signals. The computer can then ____________ (20) these signals. cell rinse units sample predilution complexity internal consists

Flow Cytometry

Transcript: FLOW CYTOMETRY There are five basic parts to flow cytometry: the fludics system, the lasers, the optics, the detectors, and the electronics and computer system. The fludic system processes the cells that pass through which are then scanned by the lasers which are also the light sources for scatter and fluoresensce. The optics collect and direct the light to the detectors which recieve the light and send the information to be analyzed by the electronic computer system for useful information. (Invitrogen). Analysis of cells: immunophenotyping, ploidy analysis, cell counting, and GFP expression analysis. (Invitrogen). Analysis is performed by passing thousands of cells through a laser. The data that is gathered is statistically analyzed for different cellular characteristics like size, complexity, phenotype, and many others. (Invitrogen). Hydodynamic focusing is when you inject the sample cells into a flowing stream of liquid or saline solution. The combined flow is then reduced to roughly the diameter of a cell to ensure each cell is scanned by the laser. (Invitrogen, Nolla & Valeros) it will refract and scatter light in a bunch of directions. Forward scatter is the amount of light that is scattered in front when the laser strikes the cell. The magnitude of the forward scatter can be used to determine the size of the cell because it is proportional. As the cell crosses the laser, light is scattered around the obsecuration bar and is collected by the detector. (Invitrogen). The light received is collected and analyzed. It scattered light is translated into a voltage. The smaller the cell, the smaller the amount of forward scatter. The amount of forward energy is translated into voltage. This histogram represents the size distribution among the cell sample. (Invitrogen). Like we've shown before, a laser beam scatters light at all angles. THe light that is scattered to the side is granularity and structural complexity inside the cell. This is known as side scatter and this light is collected through a different detector. (Invitrogen). Below is an example of a side scatter map and a forward scatter map. You can overlay them and create a two dimensional graph that explains much more about an individual cell and the entire cell population. From our knowledge about the current sizes of different cells in the blood stream, we can deduce what three cells are located in this sample blood stream based on the data. This sample includes lymphocytes, monocytes, and neutrophils. (Invitrogen). But what about cell function and structure? Fluorophore labeled antibodies is an example of flow cytometry at its finest. These labeled antibodies are added to the cell sample and the antibody binds to a specific molecule on the cell surface or inside the cell. When the laser signal touches the cell, a light of a certain color is detected. As the light travels along the path, a series of filters and mirrors allow the wavelength ranges to be detected by specfic detectors. (Invitrogen). Information is virtually collected the same way. The cells with more antibodies attached to them will emit more light. All the voltage pulses are recorded and graphed. (Invitrogen). Flow Cytometry has a threshold for data collection. If we wanted to analyze every cell that passed through, we would have way to many data points, so instead we target a range that we want to see. A certain forward scatter pulse must be scanned by the laser to collect data. The below diagram shows that in front of the threshold, the data for those cells are not being collected. Behind the line are the cells we care about. It is sort of like a baseline. (Invitrogen). Study Name: Flow cytometry analysis of glucocorticoid receptor expression and binding in steroid-sensitive and steroid-resistant patients with systemic lupus erythematosus Systemic lupus erythematosus (SLE) is an autoimmune disorder that can affect the skin, joints, kidneys, brain, and other organs. Treatment for SLE is Glucocorticoid (GC) Therapy. Glucocorticoids are steroid hormones. Some people are resistant to these steroids due to abnormalities in the Glucocorticoid receptor (GR). This study looked at GRs on T-cells from humans with SLE using flow cytometry. The probe that was used was the monoclonal antibody FITC-DEX probes. (Du, et. al, 2009). Bibliography: Du, et. al. (2009). Flow cytometry analysis of glucocorticoid receptor expression and binding in steroid-sensitive and steroid-resistant patients with systemic lupus erythematosus. Arthritis Research & Therapy. 11(4). Retrieved from: Invitrogen. Flow Cytometry. Retrieved from: Nolla, H., & Valeros, A. Flow Cytometry. Retrieved from: Additional Sources that may help: Flow Cytometry. Retrieved from:

Flow Cytometry

Transcript: Resuspend cells in 100 µl of primary antibody (prepared in incubation buffer at the recommended dilution). See individual antibody datasheet or product webpage for the appropriate dilutions. Incubate for 1 hr at room temperature. Wash by centrifugation in 2–3 ml incubation buffer. If using a fluorochrome-conjugated primary antibody, resuspend cells in 0.5 ml 1X PBS and analyze on flow cytometer; for unconjugated or biotinylated primary antibodies, proceed to immunostaining. Light refracted off of several mirrors lights are defected by detector system What is it? What is it used for? Optic System Results and Conclusion Amplification System Go and Save the Human Race!!!! Provides a single wavelength that passes through the cells light scatters in different directions: forward and side forward- measures cell volume/size side- measures inner complexity of cell, shape of nucleus, amount and type of granules, and membrane roughness Immunostaining cont'd NOTE: This step is critical for many CST antibodies. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol. Alternatively, remove fix prior to permeabilization by centrifugation and resuspend in 90% methanol as described above. Incubate 30 min on ice. Proceed with immunostaining or store cells at -20°C in 90% methanol. Collect cells by centrifugation and aspirate supernatant. Resuspend cells in 0.5–1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%. Fix for 10 min at 37°C. Chill tubes on ice for 1 min. For extracellular staining with antibodies that do not require permeabilization, proceed to immunostaining or store cells in PBS with 0.1% sodium azide at 4°C; for intracellular staining, proceed to permeabilization a measurable indicator of some biological state or condition measured and evaluated to examine normal biological processes, pathogenic processes or pharmacological responses to a therapeutic intervention Diagnosis of health conditions: i.e. Leukemia Basic research Clinical trials Immunology Pathology Detection of Activated Platelets in Whole Blood Using Activation-Dependent Monoclonal Antibodies and Flow Cytometry By Sanford J. Shattil, Michael Cunningham, and James A. Hoxie Flow Cytometry Cell Counting The goal of this experiment was to assess the degree of platelet activation and the efficacy of antiplatelet therapy in clinical disorders. activated platelets participate in thrombus formation. Solutions and Reagents Materials and Methods The process of developing valuable proteins Synthetic Biology NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemocytometer or alternative method. Aliquot 0.5–1 x 106 cells into each assay tube (by volume). Add 2–3 ml incubation buffer to each tube and wash by centrifugation. Repeat. Protocol The ability to separate cells according to their properties intracellular: DNA, RNA, protein molecule interaction extracellular: size, morphology, surface protein expression allows for isolation and organization of different cell types within organs and tissues Biomarker Detection Protein Engineering Permeabilization Cell Sorting Main Components of a Flow Cytometer NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water. 20X Phosphate Buffered Saline- To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. 16% Formaldehyde (methanol free). 100% methanol. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin in 100 ml 1X PBS. Store at 4°C. Secondary Antibodies: Anti-mouse, Anti-rabbit, Anti-rat Optional DNA Dye Resuspend cells in fluorochrome-conjugated secondary antibody or fluorochrome-conjugated avidin, diluted in incubation buffer at the recommended dilution. Incubate for 30 min at room temperature. Wash by centrifugation in 2–3 ml incubation buffer. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain Stream of liquid that contains line of cells Passes in single file through light beam for sensing Amplifies digital signal Transfers to computer for analysis Usage in Medicine Questions? Immunostaining cont'd Fixation Real Life Example The flow of cell measurements A form of biotechnology A laser-based, biophysical technology employed in cell counting, cell sorting, biomarker detection, and protein engineering With aid of the aforementioned antibodies, scientists had 99% positive correlation to activated platelets. Flow cytometry differentiated platelets from WBC's and RBC's as well as activated platelets from rested platelets Flow cytometry is an effective technique to detect activated platelets as it detected as little as 0.8% activated platelets in a heterogeneous mixture Lasers Video Animation It is a vital laboratory process transplantation oncology hematology genetics prenatal diagnosis quantification of cells in life

Flow Cytometry

Transcript: The basic principle is the shining of a beam of light {containing a single wavelength} through a hydrodynamically- focused stream of liquid. They are several detectors that pick up both scattered and flourescent light. During WWII, it was used to help detect bacteria spores aerosols for defense against germ warfare. Instead of a liquid sheath fluid, this instrument used air to visualize particles What is it? By: Amanda Howell, Jennilyn Robles, and Greg Winkler a technique for counting and examining micrscopic particles such as cells and chromosomes. The original name of the flow cytometry technology was "pulse cytophotometry" & technique background How is it used allows for the simultaneous analysis of the physical and chemical characteristics of up to thousands of particles per second. History By analysing fluctuations in brightness at each detector it is then possible to determine various types of information about the physical and chemical structure of each individual particle. Flow Cytometry Flow cytometry has become a popular technique for phenotyping, cell cycle analysis, apoptosis assays, assessing gene expression, calcium flux measurements, and much more. Why is it important? Flow cytometry is routinely used in the diagnosis of health disorders, especially blood cancers, but has many other applications in both research and clinical practice Mark Fulwyer invented the first flow cytometer, which he called a "cell sorter". This was the basis of today's flow cytometers. The first was made in 1968.

Flow Cytometry

Transcript: References BD Biosciences (2000) states, "The design of the flow chamber causes the sample core to be focused in the center of the sheath fluid where the laser beam will then interact with the particles" (p. 9). What is the Optics System? Electronics Thank you! Lasers Lenses Optical mirrors Optical filters BD FACSJazz Cell Sorter Side-scattered light (SSC) Point where the laser light hits the particle/cell in the stream. Flow Cytometers Flow Cytometry Parts of the Fluidics System Flow Chamber BD Biosciences. (2000). SH800Hz Cell Sorter (Sony) BD Biosciences. (2000). Introduction to Flow Cytometry: A Learning Guide. Manual Part Number: 11-11032-01. Retrieved from: BD Biosciences. (2000). SY3200 Cell Sorter/Analyzer (Sony) Forward and Side- Scattered Light According to BD Biosciences (2000), "The purpose of the fluidics system is to transport particles in a fluid stream to the laser beam for interrogation" (p. 9). Optical Filters What is the Fluidics system? BD FACSCanto II Flow Cytometry System Interrogation Point Optics BD Biosciences. (2000). According to BD Biosciences (2000), "The optical system consists of excitation optics and collection optics" (p. 19). Excitation optics: Made up of lasers and lenses that help center the laser beam. Collection optics: Made up of lens to gather emitted light from the interrogation point and optical mirrors & filters to direct specific wavelengths to the correct detectors. The Three Systems of Flow Cytometry Hydrodynamic Focusing According to BD Biosciences (2000) hydrodynamic focusing is where, "The flow of the sheath fluid accelerates the particles and restricts them to the center of the sample core" (p. 9). According to BD Biosciences (2000), "Flow Cytometry is a technology that simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light" (p. 5). What is Flow Cytometry? Parts of the Optics System Fluidics Sample Sheath Fluid Flow Chamber Hydrodynamic focusing Laser beam Interrogation point Fluidics Optics Electronics According to BD Biosciences (2000) forward-scattered light (FSC), "is a measurement of mostly diffracted light and is detected just off the axis of the incident laser beam in the forward direction by a photodiode" (p. 13). BD Biosciences (2000) states that side-scattered light (SSC), "is a measurement of mostly refracted and reflected light that occurs at any interface within the cell where there is a change in refractive index" (p. 14). Forward-Scattered light: cell size. Side-Scattered light: cell granularity and internal structures. A part of the electronics system is, "Light signals are generated as particles pass through the laser beam in a fluid stream. These light signals are converted to electronic signals (voltages) by photodetectors and then assigned a channel number on a data plot" (BD Biosciences, 2000, p. 24). Flow? Flow Cytometry 101 Resources Forward-scattered light (FSC)

Flow Cytometry

Transcript: Flow Cytometry What is it? - Analyzes single cell/particle in 2 directions (McKinnon, 2018) - Uses 3 methods: GFP, DNA, CD3 FTIC - Solid tissues can be analyzed as single cells - Powerful data analysis - 2 parameter histogram or other algorithims such as PCA, SPADE, tSNE used What it is Notable History 1974: 1 laser 2 light detector system Notable History Now: 3 laser sorter with multiple detectors (Herzenberg, Parks, Sahaf, et. al, 2002) Pros Cons Pros/Cons - Subpopulation analysis - Highlights non-uniformities - Takes off debris or dead cells when providing final data - Expensive to the counterparts of radioimmunoassay and enzyme-linked immunosorbent assay - Could be a slow process (Robison, 2019) Immunophenotyping Immunophenotyping - CD numbers - ex) CD3 - CD markers - T cell markers: CD3 CD4 and CD8 Isolating Dedritic Cells in Spleenocytes 05 04 - Spleenocyte extraction - CD4 for T-cells - CD11c for DC's - Using Cytek Arora : Intensity vs. Channels - Steps: - Set up plate - Run Flow - Data Analysis 03 Isolating Dendretic Cells in Spleeonocytes 02 01 Results - ~70% purity - Ways to Improve - Optimize media/conditions - 2 kits: first time negative, second time postive selection Results/ Other Things I Learned Other Things I Learned! Other Things I Learned - Skin Transplant on Mice - Using Basic Science Lab equipment - Basic Immunology Conclusion & Discussion Conclusion & Discussion Conclusion Conclusion Discussion discussion References References Herzenberg, L. A., Parks, D., Sahaf, B., Perez, O., Roederer, M., Lamp; Herzenberg, L. A. (2002, October 1). History and future of the Fluorescence ACTIVATED Cell sorter and Flow Cytometry: A view from Stanford. OUP Academic. McKinnon, K. M. (2018, February 21). Flow cytometry: An overview. Current protocols in immunology. Robison, C. (2019, March 2). The disadvantages of gel electrophoresis. Sciencing.

Flow Cytometry

Transcript: GOALS & AIMS: To engineer the most effective tumor targeting molecule for each clinical biomarker. Proteins are engineered to deliver diagnostic and therapeutic payloads to precise locations in the body such as metastatic tumors. Flow cytometry and directed evolution are used to identify the most effective proteins based on their ability to selectively target disease biomarkers. Acquisition Analysis Consultation Instrument Troubleshooting Repair Overview of Flow Cytometry Services Masonic Cancer Research Building (MCRB) 425 E River Pkwy, East Bank Campus Benjamin Hackel, Ph.D. Data Visualization need info from Ben INSERT VIDEO INTERVIEW OF INVESTIGATOR DISCUSSION RESEARCH AND INTERACTION WITH RESOURCE Paul Champoux... [insert catchy value-added phrase] Abstracts Subsidized rates on all services offered by Flow Cytometry Research Focus: Cancer & Cardiovascular Research Building (CCRB) 2231 6th St. SE, East Bank Campus Engineering Molecules for Tumor Targeting Grants --- Credits --- Music: "Raro Bueno" by Chuzausen ( Images: Various images taken from currently available UMN content Kruziki, M.A., Bhatnagar, S., Woldring, D.R., Duong, V.T., and Hackel, B.J., "A 45-amino acid scaffold mined from the Protein Data Bank for high affinity ligand engineering" Chem. Biol. in press. Woldring, D.R., Holec, P.V., Zhou, H., and Hackel, B.J., “High-throughput evolution reveals an optimal gradient of diversity in sitewise combinatorial library design of a hydrophilic fibronectin domain” submitted. Case, B.A., and Hackel, B.J. "Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor" submitted. Benjamin Hackel, Ph.D. Benjamin Hackel, Ph.D. NIH R21 Molecular Imaging of MET with Small Protein Ligands (PI: Hackel) MN Futures Award (PIs: Hackel, Panyam, Sachdev) DoD Grant: Prostate Cancer imaging (PI: Hackel) Additional knowledge and expertise provided by Flow Cytometry staff 2nd Location Flow Cytometry Services Publications *services used on this project Posters Research Recognition & Outcomes Benjamin Hackel, Ph.D. Flow Cytometry Resource Location Masonic Cancer Center Researcher Benefits 26. Kruziki, M.A., Holec, P.V., Woldring, D.R., Zhou, H., and Hackel, B.J.* “Rational identification of scaffolds for combinatorial discovery of ligands”, American Chemical Society, Denver, CO, March 2015. 24. Kruziki, M.A., Bhatnagar, S., Zhou, H., Easton, A., and *Hackel, B.J., “Evolving a 45-Amino Acid Ligand Scaffold for Enhanced Stability and Tumor Targeting”, American Institute of Chemical Engineers Annual Meeting, Atlanta, GA November 2014. 23. Woldring, D.R., Holec, P.V., Zhou, H., and *Hackel, B.J., “Optimizing Combinatorial Diversity with High Throughput Selections and Computation”, American Institute of Chemical Engineers Annual Meeting, Atlanta, GA November 2014. 22. Holec, P.V. and *Hackel, B.J., “ProtID: Systematic Identification of Protein Scaffolds for Molecular Recognition”, American Institute of Chemical Engineers Annual Meeting, Atlanta, GA November 2014. 20. *Woldring, D.R., and Hackel, B.J., “Gradient Diversity Enriches Combinatorial Protein Library Design”, Annual Symposium of the Protein Society, San Diego, CA, July 2014. 19. Hackel, B.J., “Engineering protein ligands for molecular imaging”, Gordon Research Conference: Peptides, Chemistry and Biology, Ventura, CA February 2014. 17. Woldring, D.R. and *Hackel, B.J., “Gradient diversity enriches combinatorial library design”, American Institute of Chemical Engineers Annual Meeting, San Francisco, CA November 2013. 16. Kruziki, M.A., Bhatnagar, S., and *Hackel, B.J., “Engineering picomolar affinity into a 5 kDa scaffold for tumor targeting”, American Institute of Chemical Engineers Annual Meeting, San Francisco, CA November 2013. 15. Case, B. and Hackel, B.J.*, “Engineering ligand biophysics to enhance tumor targeting”, American Institute of Chemical Engineers Annual Meeting, San Francisco, CA November 2013. INSERT VIDEO INTERVIEW OF INVESTIGATOR DISCUSSING THE VALUE OF THE CORE TO THIS PROJECT Insert video of shared resource leader/coordinator discussing involvement in project, interesting takeaways, etc. Flow Cytometry Flow Cytometry Leadership Insert visual(s) illustrating research results utilizing core support/resources/expertise Resource located in 1-209 CCRB space near research member laboratories 16. Protein Engineering General Summit, Boston, MA, May 4, 2015. 15. Department of Pharmacology, University of Minnesota, May 1, 2015. 14. Giuseppe Garibaldi Memorial Hematology Oncology Research Conference, Minneapolis, MN, March 6, 2015. 13. R&D Systems, Minneapolis, MN, March 2, 2015. 12. Chemical Biology Seminar, University of Minnesota, February 9, 2015. 11. International Conference on Biomolecular Engineering, Austin, TX, January 18, 2015. 10. Department of Physics and Astronomy, University of Minnesota, October

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