BD FACSVerse Flow Cytometer Filter Guide. Commercial flow cytometry systems were usually equipped with argon-ion lasers emitting at the blue-green 488 nm wavelength, allowing the efficient excitation of fluorescein and other traditional fluorochromes. Flow cytometry is a powerful, high-throughput technology that is used to characterize suspensions of single cells or particles. Fully utilize fluorescent proteins and reporter applications that require the violet, blue, or yellow laser, such as mCherry, GFP, and CFP. OPTICAL FILTERS The fluorescence emission of each fluorochrome is captured using optical filters, pieces of glass coated with specialized . It is a superior alternative to Pacific Blue, Alexa Fluor 405, or BD Horizon V450. We offer a wide variety of fluorochromes across multiple lasers line for flow cytometry. 10 000 particles per second) allowing quick and detailed analysis of cells. The best flow cytometry panels always include a viability dye. The laser consists of material, called the lasing medium, typically through which electrical energy is pumped. adequately excited by the 488 nm blue laser and can be used as an alternative for PE-Texas Red. Lasers with discrete wavelengths in the UV-visible spectrum are used to excite fluorophores. It emits at 455 nm and is best used for markers that are highly expressed due to its dim nature. In the image above, the pink box highlights a population that should not be biologically possible. . Table 1. Spontaneous and stimulated emissions. PE For example, while PE/Cyanine7 is primarily excited by the blue or yellow/green laser and APC/Cyanine7 is excited by the red laser, the emitted photons from each will end up at different detectors as a result of being separated into their respective dedicated paths. The current configuration allows for 3 colors off the blue laser, 5 colors off the yellow-green laser, 3 colors off the red and 6 off of the [] The CytoFLEX LX Flow Cytometer is an expansion of the CytoFLEX Platform. Expands the utility of violet laser flow cytometry eFluor ORGANIC DYES Direct conjugates for flow cytometry Robust performance for all work flow scenarios . Product Description MLL-FN/N-473 series laser with the characteristics of low noise and ultra compact, are ideal for use in DNA sequencing, flow cytometry, digital imaging, analytical chemistry, particle measurements, confocal microscopy, Raman Spectroscopy and many other applications. The basics of flow cytometry technology. Access is available 24 hrs a day and 7 days per week once investigators are registered in Stratocore and approved by the core director. In this chapter, we review the lasers available for flow cytometry, and provide guidance in choosing laser wavelengths and characteristics to best ma Overview of Lasers for Flow Cytometry The LSR Fortessa is is equipped with four laser lines (488nm, 405nm, 561nm, and 640nm) and is capable of detecting 18 parameters; 2 scatter and 16 fluorescent detectors. Nicole Brandon Senior Flow Cytometry Technologist. Flow cytometry made easy. Equipment Description: Beckman Coulter FC500 Flow Cytometer, product # 626554 Cytometry UPS, product # 178242 (220 V, 15 Amps, 3300 Watts) Computer works. . Introduction to Flow Cytometry Stephen De Rosa University of Washington, Department of Laboratory Medicine Fred Hutchinson Cancer Research Center HIV Vaccine Trials Network January 30, 2020. . Blue Laser Written by Tim Bushnell, PhD The most common laser type in a flow cytometer. Lasers are critical elements of all flow cytometers. = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1). Clinical Diagnostics; . They are large lasers and produced copious amounts of waste heat, requiring a complex cooling system. Multi-instrument alignment Harmonize data with collaborating labs to ensure reproducibility. The most advanced flow cytometry platform with a focus on speed from setup, to the acquisition, and analysis. The guava easyCyte flow cytometry systems are cost-effective, easy to use and deliver complete and comprehensive cell analysis- right on your benchtop. Frequently used fluorophores excited and detected by this laser are FITC, Alexa Fluor 488, PE, PerCP, and their tandems. Tens of thousands of cells can be interrogated by a flow cytometer in a single second. PE has an emission maxima of 575nm. Grey-filled histograms show microsphere peaks with the 100% population normalized to a fixed fluorescence intensity, with different voltage settings for each analysis (FACSVantage data only). StarBright Blue 700 PE-A750 PE-Cy7 s s Overview The blue laser is versatile due to its ability to excite both FITC (and similar fluorophores), PE, and PE tandems. 1. Lasers - the most commonly used wavelengths of lasers used in flow cytometers are 488nm (blue), 633-637nm (red), 405-407nm (violet), and 514nm (green). This metric is termed a sensitivity index or staining index (SI) and accounts for error and peak spreading in the background fluorescence distribution. No longer. Flow: To issue or move in a stream. In fact, the term "Blue" laser is often interchanged with "488" laser. Parallel vs co-linear laser arrangements Knowledge of excitation light sources is crucial for making decisions concerning the fluorophores you may use in your experiment. The CytoFLEX Flow Cytometer, the first introduction to the CytoFLEX Platform, provides the performance you need in an easy to use system allowing you to focus on the science, not the instrumentation. Argon- and krypton-ion lasers make excellent laser sources for flow cytometry. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. Pacific Blue: 488 Laser A: 685: 710/50: . Six spatially separated lasers allows panels to be spread across the spectrum reducing cross talk and spectral overlap. Instrument capabilities are dictated by the wavelengths and characteristics of its laser sources. A number of lasers are commonly used and are named after the emission wavelength or colour: 488nm (Blue argon laser . The WOLF utilizes typical Forward- (FSC) and Back- (BSC) scatter detectorsas well as 3 fluorescent channels excited by the blue (488 nm) laser. Sample flow rate: positive displacement. In the most basic sense, this is an accurate description. flow cytometer lasers iFluor dyes Answer Flow cytometers are commonly equipped with one or more of the following laser lines: Ultraviolet (355 nm) Violet (405-407 nm) Blue (488 nm) Green (532 nm) Yellow (561-568 nm) Red (633-647 nm) Refer to Table 1 below for common fluorophores used with each laser line and their respective applications. Until recently, developing fluorescence-based flow cytometry assays with 40 colors has been merely aspirational, with many turning to competing technologies for high-parameter applications. At this writing, no laser source with a wavelength of less than 320 nm has been reportedly used for traditional flow cytometry. They produce high-quality Gaussian TEM 00 beams with low noise and good power stability, particularly in the higher-power water-cooled units. The core is equipped with 1 upgraded FACScan flow cytometer that is equipped with a low-power (15mW) argon laser that emits blue light at 488 nanometers as well as a low-power (30mW) helium-neon diode laser that emits red light at 633 nanometers. The UV-visible spectrum. Blue Laser (488nm) FITC (GFP . The WOLF uses laser-based flow cytometry technology for detecting and sorting a variety of cell types, including mammalian, plant, fungal, and bacterial cells. Previously available VLDs had a maximum available power of less than 25 mW; this was sufficient for excitation of densely labeled cell surface antigens using fluorochromes such as Cascade Blue or Pacific . with minimal excitation by the 488-nm Blue laser. Flow Cytometry, then, can be defined as the characterization and measurement of cells and cellular constituents as they travel in a stream. In order to take measurements, a laser beam is focused on the stream as cells travel single-file through the stream. Several attempts have therefore been made to minimize the counting effort by combining the analysis with a flow cytometry (FCM) technique for high-throughput analysis. For use in flow cytometry, PE and APC are extracted from red algae and purified. CytoSense is scanning the scatter and fluorescence properties of EVERY particle passing the laser beam (even with the high particle loading, i.e. This metric is displayed on the histograms, and is designated SI-1. . With 64 fluorescence detectors and only 5 lasers, the Cytek Aurora now has the capability to resolve up to 40 colors in combination. MACS Flow Cytometry - Antibodies 1.1.1 Violet laser (405 nm) VioBlue Dye (Em max 452 nm) The VioBlue Dye is a coumarin-based fluorochrome with excitation and emission wavelengths of 400 nm and 455 nm, respectively. UV Laser - 355nm 60 mW PMT Bandpass Long Pass Fluorochrome A 740/35 690LP BUV737 B . FQY = fluorescence quantum yield in aqueous buffer (pH 7.2). NovoCyte is a high-performance benchtop flow cytometer designed for all levels of users and all types of laboratories. Laser: blue 488 nm (50mW) Flow cell dimension: standard square capillary with ID of 100 m. PerCP-Cy5.5 is not subject to photobeaching like PerCP and can be . Intracellular flow cytometry Spectral properties for PE and APC. Multiple commercial computer programs in addition to the instrument provided software are available for analysis of flow cytometry data. Wild type GFP could not be excited on these systems. Cytek's Aurora Spectral Cytometer has a rapidly growing presence among flow cytometry research instruments. Introduction: In previous studies we and others have demonstrated the usefulness of violet laser diodes (VLDs) as replacement laser sources for krypton-ion lasers on stream-in-air cytometers. Acoustic Focusing Cytometers In these cytometers, ultrasonic waves are used to focus the cells for analysis. Pacific Blue dye Invitrogen Molecular Probes Pacific Blue dye is a bright blue-fluorescent dye optimally excited by the 405 nm line of the violet laser and with excitation/emission at 410/455 nm. Left column, InSpeck Blue microsphere mixtures (Molecular Probes Invitrogen) analyzed on the indicated instrument with the indicated laser. KIRAVIA is a coined term meaning "sparkling" in Japanese. This model expands research possibilities with up to six lasers and 21 color parameters. Flow cytometry combines the flexibility and sensitivity of fluorescence technology with high speed and data integration capabilities. All instruments in the platform utilize CytExpert for CytoFLEX . Table 1. Laser lines commonly used in flow cytometry are: 355 nm, 375 nm, 405 nm, 488 nm, 530 nm, 561 nm, and 640 nm. In this unit, we describe two protocols for analyzing cell cycle status using flow cytometry. For more information about fluorophores and immunophenotyping, refer to our flow cytometry resources below. How the flow cytometer is set up determines how the fluorophore is detected. Coeff. (BD Biosciences, San Jose, CA) equipped with a 20-mW solid-state blue laser (488 nm) and a 17-mW helium-neon (He-Ne) red laser (633 nm). FC500 MPL Flow Cytometer in excellent condition. Flow cytometry is a technique used to measure antigen expression on populations of cells. For instruments equipped with blue and violet lasers and configured for . Alexa Fluor 633 conjugates can be used in multi-color flow cytometry with instruments equipped with a second red laser or red diode. The mission of the Flow Cytometry Core at Penn State College of Medicine is to facilitate cutting-edge research by providing state-of-the-art fluorescence-activated cell sorting and analytical services at reasonable hourly rates. For example, in the characterization of subpopulations of stem cells, millions of cells are analyzed to achieve a statistically significant sampling of the subset of interest. UC Flow Cytometry Core. In the flow cytometry, fluorochrome associated with a cell is excited by a laser, and the fluorescence emitted by the fluorochrome is collected by the detector assigned to that fluorochrome 1.In the multicolour flow cytometry system, each detector accepts light in a particular range of . BST E1514 412.624.4434 nas260@pitt.edu. 50 mW at 488 nm (blue) 50 mW at 561 nm (yellow-green) 80 mW at 640 nm (red) 64 Fluorescence Emission Detection Channels (providing measurement of the full visible spectrum for each fluorochrome) Ultra-violet detector module: 16 channels; Violet detector module: 16 channels; Blue detector module: 14 channels; Yellow-Green detector module: 10 . Flow Cytometry Analysis; BD LSRII TAC5-UV; BD LSRII Green; BD LSRII at Amistad; BD LSRII TAC6-GS; FACSCalibur; BD LSR Fortessa . The budget-friendly instrument can detect up to 17 parameters with enhanced sensitivity and resolution. The octagon- and trigon-shaped optical pathways of collection optics maximize signal detection and increase sensitivity and resolution allowing you to identify dim and rare cell populations Can be configured with up to 5 lasersblue, red, violet, UV and yellow-green. Typically, this laser has a wavelength of 488nm in flow cytometers. . It has become the gold standard in cell analysis and now is being used as an analytic tool across numerous sectors of life sciences. PE is also excitable by the 561 nm laser and so PE based fluorophores appear on both lists. PE PE-eFluor 610, PE/Dazzle 594, PE-TxRed, PE-CF594, Live/Dead Red PE-Alexa Fluor 610 Zombie Red . Blue Laser Unique Signatures. Multiwavelength, white light, and wavelength tunable lasers are poised to enhance our analytical capabilities even further. Introduction. A step-by-step protocol for cell-surface and intracellular antibody staining from researchers, for researchers by enQuire Bio. . Single blue laser (488 nm) or dual blue (488 nm) and red (640 nm) excitation lasers provide up to eight simultaneous detection parameters, including six fluorescent colors plus forward and side scatter Like other Alexa Fluor dyes, Alexa Fluor 633 exhibits uncommon photostability, making it an ideal choice for fluorescent microscopy. This module was small, air-cooled, single mode and could be operated in CW mode in the 10-20 mW range. Generally, the blue laser is also used to measure light scatter. . The 488-nm laser . Introducing the KIRAVIA Dyes, a brand new fluorescent chemistry to advance applications in multicolor flow cytometry and beyond. The iQue 3 combines a patented sampling method which allows for the fastest sample acquisition in the industry. . Therefore, the blue 488 nm laser, which is close to FITC's absorbance peak of 490 nm, is commonly used to excite this fluorophore. The way that lasers work is interesting. Filter Guide. For conventional instruments equipped with a Yellow-Green laser (561-nm), RY586 can be used as an alternative to PE and we recommend using an optical filter centered near 586-nm (eg, a 586/15-nm bandpass filter). Each . The cells flow single file past focused laser beams and light scatter or fluorescence data is collected. Quantification of Cytokines Using BD Cytometric Bead Array on the BD FACSVerse System and Analysis in FCAP Array Software. Result: classic flow cytometry data for small cells <5m and added morphological properties for particles >5m. It is excited efficiently by the 488nm blue laser, but is even brighter when excited by 561nm yellow lasers on some cytometers. A flow cytometer is an instrument that can count the number of various cell types in a blood sample. The robust SD is estimated as the (84 percentile of the background median minus the background median, divided by 0.995. Dead cells can bind antibodies nonspecifically, causing false positives and affecting cell frequencies. The Aurora is available with 3 or 5 laser configurations (355 nm, 405 nm, 488 nm, 561 nm and 633 nm), with the latter offering up to 64 different fluorescent parameters. Waste and cleaning vials . To accomplish this, the blood sample is first concentrated and then treated with a mixture of fluorochromesfluorescent chemicals that each preferentially bond to a specific protein target on the cell surface. To help you choose the right fluorochromes for your experiments, we have developed a fluorochrome atlas that allows you to view and compare dyes with attributes such as excitation/emission spectra, filter recommendations and relative resolution information. Flow Cytometry Reagents. Double-staining FISH . Pacific Blue (ex 410/em 455) Pacific Blue is a blue, fluorescent, coumarin based dye, excitable by the 405 nm laser. Example configurations for LSR instruments (L, M, Y) Laser Laser Power Channel Instrument Bandwidth Long Pass Old Detector Name . Flow cytometry allows researchers to ask complex questions, such as how the activity of 2 key signaling pathways in 3 immune cell types changes in response to a treatment, and answer the question within a few hours by labeling and running a single sample on a flow cytometer. To get you from samples to actionable results in record time. Housed in ultra compact. Ext. Red Laser Blue Laser. FITC emits fluorescence from 475 to 650 nm, peaking at 525 nm, which falls in the green spectrum. The Assembly, G532, 5051 Centre Avenue 412.624.4434 nrb18@pitt.edu. For example, if a blue laser is used to illuminate the molecule . Conjugates of this dye are strongly fluorescent even at neutral pH. Most commonly utilized in the Life Sciences, this application is now also used in microbiology, food quality control, plant and animal cytology, immunology, and other areas. . The sample is focused to ideally flow one cell at a time through a laser beam, where the light scattered is characteristic to . Cascade Blue, Pacific Blue, Sky Blue, Alexa Fluor 405, BFP, CFP, Hoechst Blue Laser - 488nm 50 mW PMT: Bandpass: Long Pass: . In this study, we have evaluated a newly developed solid-state 280 nm laser as a cytometry excitation source. Panels that lack a viability dye. It has the ability to handle 96, 384 well plates and . Flow cytometry measures phenotypic, biochemical and molecular characteristics of individual cells or particles suspended in a fluid stream. Fluorochromes have specific spectra of wavelengths with which they are maximally excited, so the more lasers an instrument has the more fluorescent colors one can measure. 2. Bio-Rad has dyes excitable by the common lasers used in flow cytometry, details of which can be found on our Fluorophores for Flow Cytometry page.