Flow cytometry Mgr. Ondřej Vymazal Flow cytometry, Ondřej Vymazal, Department of Biology1 Introduction ̶ What is the Flow cytometer? ̶ For which application it can be used? ̶ How does it work? Flow cytometry, Ondřej Vymazal, Department of Biology2 Flow cytometry, Ondřej Vymazal, Department of Biology3 Analytical cytometry is a technique that allows us to examine cells in detail. Cells are the basic building blocks of all living things, including ourselves. Imagine you have a big box of LEGO bricks of different colors and shapes and you want to find out how many red bricks are in them. You could of course dump them out and count them one by one, but that would take a long time. Instead, you can use a special device that scans all the dice and tells you how many of them are red. This is similar to what analytical cytometry does, but instead of LEGO bricks, we're looking at cells. When we want to find out something specific about cells, for example how many of them have a certain molecule, we can mark them with a special dye that will only attach to cells with this molecule. Then we use a machine called a cytometer, which quickly looks at all the cells and tells us how many of them have this dye. This technique is very important in many fields of science and medicine. For example, it can help us find out what types of cells are present in the blood of a patient with a certain disease, or how the cells respond to a new drug. Question for chatGPT:explain what analytical cytometry is as if you were explaining it to teenagers in the third year of general grammar school. Try to use simple language and examples that a teenager could easily understand. Introduction ̶ What is the Flow cytometr? ̶ For which aplication it can be used? ̶ How does it work? Introduction Introduction ̶ What is the Flow cytometer? The machine that counts the particles/cells and measures their parameters at the single-cell level. ̶ For which application it can be used? ̶ How does it work? Flow cytometry, Ondřej Vymazal, Department of Biology6 Introduction Introduction ̶ What is the Flow cytometer? The machine that counts the particles/cells and measures their parameters at the single-cell level. ̶ For which application it can be used? Phenotyping, cell cycle analysis, cell viability, functional analysis. ̶ How does it work? Flow cytometry, Ondřej Vymazal, Department of Biology8 Flow cytometry, Ondřej Vymazal, Department of Biology9 Introduction ̶ What is the Flow cytometer? The machine that counts the particles/cells and measures their parameters at the single-cell level. ̶ For which application it can be used? Phenotyping, cell cycle analysis, cell viability, functional analysis. ̶ How does it work? Stream of single cells in suspension is flowing through the illuminated part, where parameters of cells are measured based on light scattering. Flow cytometry, Ondřej Vymazal, Department of Biology10 11 Flow cytometry, Ondřej Vymazal, Department of Biology Flow cytometry, Ondřej Vymazal, Department of Biology12 Brief history ̶ 1940-1950 Development of principles used in flow cytometry, fluorescent dyes, particles and spores in air counting. ̶ 1953 – Sheath flow principle. ̶ 1956 Wallace Coulter – Measuring the change in conductivity during the passage of cells in suspension through a small hole. ̶ 1960-1970 Flow cytometry development. ̶ Flow cytometry expansion and advancement. Flow cytometry, Ondřej Vymazal, Department of Biology13 Technical components Cells in suspension flow individually across illuminated part where they scatter light and emit fluorescence, which is detected, filtered and converted to digital values analyzed and stored on your computer. Fluidics Optics Electronics 14 Cells in suspension flow individually across illuminated part where they scatter light and emit fluorescence, which is detected, filtered and converted to digital values analyzed and stored on your computer. Fluidics Optics Electronics Flow cytometry, Ondřej Vymazal, Department of Biology15 ̶ Laminar flow. Fluidics Flow cytometry, Ondřej Vymazal, Department of Biology16 ̶ Hydrodynamically focused cells in fluid system are analyzed using light/laser rays. ̶ Laminar flow. Fluidics Flow cytometry, Ondřej Vymazal, Department of Biology17 Technical components Cells in suspension flow individually across illuminated part where they scatter light and emit fluorescence, which is detected, filtered and converted to digital values analyzed and stored on your computer. Fluidics Optics Electronics Flow cytometry, Ondřej Vymazal, Department of Biology18 Optics ̶ UV/Arc lamps ̶ Lasers Light/laser sources Flow cytometry, Ondřej Vymazal, Deparmetment of Biology19 Optics Optical channels ̶ Way of the light from the point of cell illumination to the detector. ̶ Optical parts separate certain wavelengths. 20 Flow cytometry, Ondřej Vymazal, Department of Biology 21 Optics Filters ̶ Used for wavelength separation. ̶ Placed in 45° angle as a dichroic mirror. 22 Flow cytometry, Ondřej Vymazal, Department of Biology 23 Optics Channels ̶ Information about surface properties and cell size. ̶ Can distinguish live and dead cells. ̶ Information about inclusions in cells and cell density. ̶ Can distinguish granular cells. Flow cytometry, Ondřej Vymazal, Department of Biology 24 Optics ̶ Fluorescence emitted from each fluorochrome is detected by specific fluorescence channel. ̶ The specificity of the detection is provided by selectivity of the filters and mirrors. Channels Flow cytometry, Ondřej Vymazal, Department of Biology 25 Optics Channels Flow cytometry, Ondřej Vymazal, Department of Biology 26 Detectors Optics ̶ Diodes (can by used in forward scatter). ̶ Only for high signal, better linearity. ̶ Photomultipliers (PMT detectors for fluorescence). ̶ Sensitive, but can be harmed by overexposure by high laser power. Flow cytometry, Ondřej Vymazal, Department of Biology Flow cytometry, Ondřej Vymazal, Department of Biology27 BD Facs Canto II 28 Použití textu v prezentaci ̶ Máme zde několik úrovní odrážek, konkrétně tři. U všech textů by se mělo používat zarovnání odstavce vlevo. ̶ Položka odrážkového seznamu druhé úrovně. ̶ Druhá položka odrážkového seznamu druhé úrovně. Položka odrážkového seznamu třetí úrovně. Má velikost fontu 16 a doporučujeme nepoužívat menší velikost fontu z důvodu zajištění čitelnosti. ̶ Samozřejmě lze použít i běžný text bez odrážek. Flow cytometry, Ondřej Vymazal, Department of Biology 29 FACSCanto Laser Detector Fluorochromes 405 450/50 Pacific Blue, eFluor 450, V450, BV 421, Cascade Yellow, (DAPI) 510/50 AmCyan, BV 510, Krome Orange, V500, Cacsade Blue 488 530/30 FITC, Alexa Fluor 488, BB 515, CFSE, Fluo-3, GFP 585/42 PE, PI 670LP PerCP, PerCP-Cy 5.5, PC5, 7-AAD 780/60 PC7 633 660/20 APC, Alexa Fluor 647, eFluor 660 780/60 APC-Cy7, APC-H7, APC - Alexa Fluor 750, APC-eFluor 780 Flow cytometry, Ondřej Vymazal, Department of Biology 30 Sources of fluorescence Optics ̶ Fluorophores conjugated antibodies. ̶ Autofluorescence. ̶ Fluorescent dyes. ̶ Other chemicals with fluorescence properties (drugs,…). Flow cytometry, Ondřej Vymazal, Department of Biology Flow cytometry, Ondřej Vymazal, Deparmetment of Biology31 Použití textu v prezentaci ̶ Máme zde několik úrovní odrážek, konkrétně tři. U všech textů by se mělo používat zarovnání odstavce vlevo. ̶ Položka odrážkového seznamu druhé úrovně. ̶ Druhá položka odrážkového seznamu druhé úrovně. Položka odrážkového seznamu třetí úrovně. Má velikost fontu 16 a doporučujeme nepoužívat menší velikost fontu z důvodu zajištění čitelnosti. ̶ Samozřejmě lze použít i běžný text bez odrážek. Flow cytometry, Ondřej Vymazal, Deparmetment of Biology32 Multicolor panel building ̶ Pair bright fluorophores (e.g. PE) with low expressing markers. ̶ Dimmer fluorophores (e.g. Pacific Blue) with highly expressed markers. 33 FACSCanto Laser Detector Fluorochromes 405 450/50 Pacific Blue, eFluor 450, V450, BV 421, Cascade Yellow, (DAPI) 510/50 AmCyan, BV 510, Krome Orange, V500, Cacsade Blue 488 530/30 FITC, Alexa Fluor 488, BB 515, CFSE, Fluo-3, GFP 585/42 PE, PI 670LP PerCP, PerCP-Cy 5.5, PC5, 7-AAD 780/60 PC7 633 660/20 APC, Alexa Fluor 647, eFluor 660 780/60 APC-Cy7, APC-H7, APC - Alexa Fluor 750, APC-eFluor 780 Flow cytometry, Ondřej Vymazal, Department of Biology 34 Technical components Cells in suspension flow individually across illuminated part where they scatter light and emit fluorescence, which is detected, filtered and converted to digital values analyzed and stored on your computer. Fluidics Optics Electronics Flow cytometry, Ondřej Vymazal, Department of Biology Flow cytometry, Ondřej Vymazal, Deparmetment of Biology35 ̶ When cells go through laser scattered light makes pulse at the sensor. Electronics Signal processing 36 Signal processingElectronics Lin or log signal amplification Analog/digital conversion Flow cytometry, Ondřej Vymazal, Department of Biology 37 Data analysisElectronics Data visualization ̶ histogram ̶ dot plot ̶ isometric display ̶ contour plot ̶ chromatic (color) plots ̶ 3D projection Flow cytometry, Ondřej Vymazal, Department of Biology 38 Data analysisElectronics Gating Back gating Flow cytometry, Ondřej Vymazal, Department of Biology Ukázková prezentace LF MU v jednotném vizuálním stylu39 Compensation Ukázková prezentace LF MU v jednotném vizuálním stylu40 Compensation ̶ Single stained control for each fluorophore in antibody cocktail. ̶ Cells or compensation beads. ̶ New compensation after any changes in cocktail, same compensation for comparable experiments. 41 Compensation ̶ Video with compensation in BD software ̶ Compensation of a 7 color panel on the BD LSR II: https://youtu.be/5UHw4DIArx4 Flow cytometry, Ondřej Vymazal, Department of Biology 42 Phenotyping Applications Baumer, Y., Gutierrez-Huerta, C.A., Saxena, A. et al. Immune cell phenotyping in low blood volumes for assessment of cardiovascular disease risk, development, and progression: a pilot study. J Transl Med 18, 29 (2020). https://doi.org/10.1186/s12967-020-02207-0 Flow cytometry, Ondřej Vymazal, Department of Biology 43 Cell cycle analysis Applications ̶ Fluorescent dyes binding DNA. ̶ Cells have to be fixed and permeabilized. ̶ Quaternary benzo[c]phenanthridine alkaloids (QBAs) ̶ I. Slaninova, J. Slanina and E. Taborska, "Quaternary benzo[c]phenanthridine alkaloids--novel cell permeant and red fluorescing DNA probes," Cytometry A, vol. 71, no. 9, pp. 700-708, 2007. ̶ Can be used to label viable cells. Flow cytometry, Ondřej Vymazal, Department of Biology 44 Cell cycle analysis Applications ̶ Software for analysis of distribution of cells in individual phases. Flow cytometry, Ondřej Vymazal, Department of Biology 45 Viability Applications ̶ Based on detection of membrane integrity. ̶ Some fluorescence dyes (PI,EBr) don´t leak into live cells. Flow cytometry, Ondřej Vymazal, Department of Biology 46 Doxorubicin accumulation Applications ̶ Chemotherapeutic drug with fluorescent properties. ̶ Cells get rid of it from intracelular space using ABC-transporters in cytoplazmic membranes. Cytoplasm Outside ABC-transporter Doxorubicin Flow cytometry, Ondřej Vymazal, Department of Biology 47 -DOXO +DOXO Verapamil Lignans Flow cytometry, Ondřej Vymazal, Department of Biology 48 Thanks for attention! Flow cytometry, Ondřej Vymazal, Department of Biology