The increasing use of flow cytometry fcm for analyses of environmental samples has resulted in a large variety of staining protocols with varying results and limited comparability. One of the fundamentals of flow cytometry is the ability to measure the properties of. Online tutorials thermofisher was invitrogen tutorials on fluorescence, spectra, optical filters, flow, and data analysis online tutorial and quiz on compensation from dr. Fluorescence signal from pacific blue, alexa fluor 488, and alexa fluor 647 clickit edu flow cytometry assay kits. Attune nxt flow cytometer, invitrogen ebioscience flow cytometry antibodies, and invitrogen flow cytometry assays and reagents. Articulate the leader in rapid elearning and communications. Resuspend cells in an appropriate volume of flow cytometry staining buffer. The pdf is completely searchable and its printable too. The reagents and procedures highlighted here will give the investigators an indication of the health status and volume of mitochondria in primary cells and cell lines through the use of a number of cellpermeable dyes. Detection of factor viii fviii in cells by flow cytometry is controversial, and no monoclonal fluorescent antibody is commercially available. Apr 03, 2018 we have developed a simple, costeffective, and laborefficient twostep protocol for preparing adherent cells for highthroughput flow cytometry. The invitrogen attune nxt flow cytometer combines precision with performance in a true benchtop flow cytometer with up to 4 lasers and 16 parameters of detection. Multiparameter analysis of cytokine, transcription factor, and phosphorprotein expression by flow cytometry. A pipeline for developing and testing staining protocols for.
The software used to deconvolute the dna content frequency histograms, to estimate the proportions of cells in the respective phases of the cycle, is available. Invitrogen flow cytometry capabilities guide pages 1 50. We are focused on advancing meaningful discoveries and partnerships to make tools for cellular analysis widely accessible, affordable, and powerful for all life science scientists. This retains the target protein in the original cellular location.
Live and dead cells distinguished by flow cytometry. Especially leukemia diagnostics is done by flow cytometry surface. This increased level of profiling allows for a better understanding of the biology and greater discovery. Detection of mitochondrial mass, damage, and reactive oxygen. We typically use alexafluor 488 conjugated phalloidin invitrogen, as it is. The first is based on the simultaneous analysis of proliferationspecific marker ki67 and cellular dna content, which discriminate restingquiescent cell populations g0 cell and quantify cell cycle distribution g1, s, or g2m, respectively. Wash the cells by adding 1 ml or more if many samples of 1x pbs to the flow tube. Flow cytometry of bacterial membrane potential and. In this study, we optimized such an assay and successfully used it as a platform to study the functional properties of phosphoglycerate kinase pgkfviii lentiviral vectortransduced cells by directly visualizing fviii in cells after different gene. The attune nxt flow cytometer was developed with the goal of removing barriers associated with flow cytometry and enabling new. Adherent cells were grown on microplates, detached with 2. Wash the cells by adding 2 mltube of flow cytometry staining buffer. Flow cytometry is a powerful method, which is widely used for.
Cells were incubated at 37c for 15 min and then pipetted up and down to ensure single cells for flow cytometry analysis. Please read the following cell viability protocol in its entirety before beginning. Sep 12, 2017 beadassisted flow cytometry can be used as a highly sensitive semiquantitative method for ev analysis. Purdue cytometry core lab this site is an access point for many resources for flow and image. This modified protocol was then implemented with the automated flow cytometry device and used to monitor the cell cycle kinetics of a cho culture using 10. M inimize nonspecific interactions using super bright staining buffer introduction polymerbased dyes, such as invitrogen super bright fluorochromes and brilliant violet fluorochromes, have been reported to exhibit nonspecific interactions when using more than one polymer dyeconjugated antibody together in the same flow cytometry. The following methods have been optimized for monitoring cell proliferation in populations of human b and t lymphocytes. Viability staining protocol for flow cytometry invitrogen. In this chapter, we will address the methodology to assess the dynamics of mitochondrial redox status and vascular oxidative stress in response to. Brdu staining protocol for flow cytometry invitrogen ebioscience reagents staining cells with efluor proliferation dyes for flow cytometry invitrogen ebioscience reagents not for resale. Phosphospecific flow cytometry phosphoflow getting started with phosphoflow. Seroblock have been developed which, when added to a staining protocol, can ensure that only antigen. Cellbased flow cytometry assay to measure cytotoxic activity.
The dna of mammalian, yeast, plant or bacterial cells can be stained by a variety of dna binding dyes. For each sample, you will need between 105 and 106 cells. Super bright polymer dyes are sold under license from becton, dickinson and company. This approach has been complicated by a lack of specific markers for treg cells and lack of uniformity in quantification of treg cells. Inappropriately regulated apoptosis is implicated in disease states, such as alzheimers disease and cancer. Cell cycle analysis by quantitation of dna content was one of the earliest applications of flow cytometry. Molecular probes tutorial seriesintroduction to flow.
Growth dynamics of mammalian cells monitored with automated. Free exosomes alone are too small to be detected by most flow cytometry instruments. Suspensions of live untreated and dead alcoholtreated staphylococcus aureus panels a and c and escherichia coli panels b and d were stained and analyzed by flow cytometry according to the kit protocol. Flow cytometry is a powerful method, which is widely used for highthroughput quantitative and qualitative analysis of cells. Bacterial detection and livedead discrimination by flow. The application of flow cytometry or fluorescenceactivate cell sorting facs has enabled a quantitative approach to assess mitochondrial redox status in terms of m and mto 2. Spin down cell suspension at rpm for 5 minutes and decant supernatant.
Immunofluorescent staining of live cells for nuclear visualization 1. Add an appropriate volume of staining buffer generally 50 ul per 1 x 10e6 cells. The majority of flow cytometers are now digital systems. Apr 20, 2018 this video is an explanation of flow cytometry, it contains a full explanation about flow cytometer and data obtaned from it. In contrast, flow cytometry quantitates the total fluorescence intensity of. Description flow cytometry provides a rapid and reliable method to quantify viable cells in eukaryotic and prokaryotic cell suspensions. This is a flow cytometrybased protocol to measure glucose uptake of mouse embryonic fibroblasts mefs and. Pdf identification of ros using oxidized dcfda and flow. Bacterial detection and livedead discrimination by flow cytometry introduction accurate determination of live, dead, and total bacteria is important in many microbiology applications. Designing a panel for flow cytometry is a highly complex process requiring a lot of time and effort. Intracellular staining flow cytometry tested during development recommended assay procedure. Experimental protocols labeling cells for analysis in flow cytometry follow the guidelines below for labeling cells with celltrace violet for analysis using flow cytometry. The annexin v apoptosis assay university of kansas. Clickit edu protocol for flow cytometry thermo fisher.
Calcium flux is a rapid and sensitive measure of cell activation whose utility could be enhanced with better techniques for data extraction. Flow cytometry intracellular staining protocol fix cells before intracellular staining to fixing and permeabilization ensure stability of soluble antigens or antigens with a short halflife see the special recommendations below for exceptions. Cells are usually stained in polystyrene round bottom 12 x 75 mm 2 falcon tubes. Simplified protocol for flow cytometry analysis of fluorescently. Beckman coulter have placed it on their new flow cytometry website. Glucose is a ubiquitous energy source in most organisms and plays a pivotal role in cellular metabolisms and homeostasis.
Ratiometric analysis of fura red by flow cytometry. Instead, we present a guide that will be invaluable to beginners in flow cytometry and act as a factpacked synopsis for those of you interested in teaching. Propidium iodide is a suspected carcinogen and should be handled with care. Life technologies attune with plate handler protocol for. Practical flow cytometry, fourth edition protocol online. Flowcytometry platform for intracellular detection of. In this unit, two protocols are described for analyzing cell cycle status using flow cytometry. On paper, it should work better than brdu of course. This flow cytometry guide aims to give you a basic overview of all the important facets of flow cytometry without delving too deeply into the complex mathematics and physics behind it all. Current protocols in cytometry online access to through lane library. This webinar covers the basic components of a flow cytometer, how to interpret a dye excitationemission spectrum, how data is displayed, basic gating demonstration, and.
Fitc annexin vdead cell apoptosis kit with fitc annexin v. Viability assessment with fcm is in this context of particular interest because incorrect staining could severely affect the outcomeinterpretation of the results. Invitrogen dapi 4,6diamidino2phenylindole, dihydrochloride. A schematic of staining protocol and data acquisition by flow cytometry. How to use the invitrogen flow cytometry panel builder. Exosomehuman cd9 flow detection reagent from cell culture allows easy visualization of exosomes by flow cytometry using this magnetic separation technology. Protein and cell analysis brochure fisher scientific. Simplified protocol for flow cytometry analysis of. We describe a technique to monitor calcium flux by flow cytometry, measuring fura red calcium dye by ratiometric analysis. Jurkat human tcell leukemia cells were treated with 10. This method was derived from protocols of flow cytometry based cytotoxic assays in vivo in mice 4,5 and in vitro in humans 6. Analyze samples by flow cytometry, or if staining for intracellular targets, proceed with best protocols. Attune nxt flow cytometer thermo fisher scientific.
This tutorial on flow cytometry looks at the systems that make up the flow cytometer and how those systems work together to collect information on cells. During that time, the culture was subjected to recovery from nutrient deprivation and a nutrient upshift. Edu is an alternative to brdu from life technologies. This chapter describes reliable flow cytometric methods for assessment of two important physiologic characteristics of bacteria, membrane potential and membrane permeability, which can provide indications of the effects of antimicrobial agents on microorganisms. Livedead sperm viability kit flow cytometry protocol. Traditionally, viability in bacteria is synonymous with the ability to form colonies on solid growth medium and to proliferate in liquid nutrient broths.
Explore the optics, software, fluidics and key design features of the instruments. Assessing mitochondrial redox status by flow cytometric. You must be connecting from a stanford ip address or using a vpn connection to stanford. Critical aspects of staining for flow cytometry from givan, a. Propidium iodide cell viability flow cytometry protocol. Modifications to the basic flow cytometry cell surface staining protocols enables simultaneous detection of surface molecules, intracellular antigens, cell signaling phosphorylation events, and rna transcripts at the singlecell level. Invitrogen life technologies, carlsbad, ca, usa that has been widely validated in the literature on.
This is the job of the signal processing electronics. When staining proteins inside the cell, it is important to consider their location as this may dictate the protocol and buffer system that will perform optimally. Flow cytometric analysis of bd pharmingen calcein am fluorescence in jurkat. Flow cytometry stepbystep protocol prepare your cell suspensions for flow cytometry although most flow cytometry experiments involve labeling populations of cells that are relatively abundant, the number of cells required will vary depending upon the rarity of your cells. M edu for 2 hours and detected according to the recommended staining protocol. Multiparameter flow cytometry analysis of t regulatory treg cells is a widely used approach in basic and translational research studies. However, its straightforward applicability for extracellular vesicles evs and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles exosomes. Molecular probes flow cytometry thermo fisher scientific. M inimize nonspecific interactions using super bright.
Twostep protocol for preparing adherent cells for high. Tips and tricks decreasing the serum content of the culture medium to invitrogen ebioscience reagents cell viability protocols for flow cytometry. Fitc annexin vdead cell apoptosis kit with fitc annexin v and pi, for flow cytometry catalog no. Analysis of cell cycle by flow cytometry 303 cytometers are becton dickinson immunocytometry systems, beckmancoulter inc. Annexin v is commercially available, conjugated to most common fluorochromes. Flow cytometry protocols thermo fisher scientific us. Staining intracellular antigens for flow cytometry thermo fisher.
We routinely use this protocol to measure relative polymerized actin content. Practical flow cytometry, fourth edition free online version from invitrogen, free download from beckman coulte jan272009 pages. Let the invitrogen flow cytometry panel builder guide you in designing your next. Mar 31, 2015 the current best and most widely used protocol for beadsfree flow cytometry of exosomes uses ultracentrifugation uc coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye pkh67 and antibodies, and an optimized version of commercial highend cytometer for analysis. Intracellular flow cytometry thermo fisher scientific. General procedure for flow cytometry using a conjugated primary antibody. Analysis of bacterial cultures using the livedead baclight bacterial viability and counting kit. Harvest, wash the cells and adjust cell suspension to a concentration of 15 x 10 6 cellsml in ice cold pbs, 10% fcs, 1% sodium azide. Invitrogen flow cytometry reagents with ebioscience antibodies over 10,000 conjugated antibodies specifically developed, validated, and manufactured for flow cytometry. Muellerhinton broth gibco invitrogen corporation, carlsbad, ca with. An optimized multiparameter flow cytometry protocol for. If you will be using a different system, consult the appropriate protocol in our flow cytometry protocols for multiplex mirna assays page, or contact our technical support. Livedead baclight bacterial viability and counting kit.
Tips and tricks decreasing the serum content of the culture medium to flow cytometry cell cycle analysis using propidium iodide dna staining. Can anyone share a edu staining protocol for flow cytometry. V242 introduction apoptosis is a carefully regulated process of cell death that occurs as a normal part of development. Lipofectamine 3000 transfection reagent hepg2 liver. Stepbystep detailed flow cytometry protocol enquire bio. Life technologies attune with plate handler protocol for multiplex mirna assays this protocol contains instruction for setup a life technologies attune with plate handler. The following protocol has been optimized for cell concentrations up to 106 cellsml. Each of the livedead fixable dead cell stain kits was used to differentially stain a mixture of live left peak and heattreated jurkat cells right peak according to the protocol provided in this document panel a, livedead fixable blue stain kit with uv excitation. This is a great starting phosphoflow experiment because it is simple stimulate a suspension cell line with a few different cytokines, measure a few abundant phosphoproteins and weve done it over and over, so we know what to expect. Flow cytometer with 405 nm laser and 450 nm emission filter. Relative quantitation of polymerized actin in suspension cells by. Flow cytometry for intracellular staining conjugated. Invitrogen dynabeads exosomehuman cd9 flow detection reagent. Evs were isolated from culture supernatants of human primary tlymphoblasts or skmel103.