Measuring phosphorylation events by flow cytometry. This is a great starting phosphoflow experiment because it is simple stimulate a. Resuspend the cells in approximately 500 l of permwash buffer i prior to flow cytometric analysis. Each phospho specific antibody has been tested in three different flow cytometry buffer systems. Validation for phospho specific antibodies for flow cytometry invitrogen ebioscience phospho flow antibodies and related buffer systems are rigorously tested using the methods shown below to help give scientists confidence that they will perform as expected. Introduction flow cytometry is routinely used for the identification of cellular populations based on a surface phenotype and also used for cellular based assays such as cytotoxicity, viability, and apoptosis, among others. Principles and applications of flow cytometry table of.
Flow cytometry offers a quick and effective way to measure signaling at a specific time point in individual cells. Note that certain abs may benefit from using slight variations to this technique see results. Please see the productspecific flow protocol on the product webpage for appropriate fixation and permeabilization. Bd vacutainer cpt cell preparation tube with sodium heparin cpt tube. An optimized multiplex flow cytometry protocol for the analysis of intracellular signaling in peripheral blood mononuclear cells. Cv values for the multiplexed assays measured in complete process triplicates, grouped according to cell type analyzed upper panel. Many phosphorylation events can be analyzed simultaneously in each cell, along with cell surface markers, enabling complex biochemical signaling networks to be resolved in heterogeneous cell populations.
Titration is the process of identifying the correct concentration of antibody to use for a given assay. Bd phosflow protocol for human pbmcs protocol i detergent method. Nonspecific antibody binding is best eliminated by optimizing the amount. An optimized protocol to quantify signaling in human. Staining intracellular antigens for flow cytometry thermo fisher. Pbmcs and quantify signaling by measuring protein phosphorylation in cell subsets using flow cytometry. Add 2 ml of flow cytometry staining buffer to each tube. 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. Phospho flow cytometry is thus a powerful approach to characterize signaling aberrations, identify and validate biomarkers, and assess pharmacodynamics.
Multicolor flow cytometry bd phosflow protocol for human pbmcs procedure for manual setting of compensation for multicolor flow cytometric analysis immunohistochemistrytissue section staining uses of fc block western blotting faq. Flow cytometric analysis of hela cells, untreated blue or treated with htnf. Flow cytometry staining buffer and acquire samples on a flow cytometer. In this report, we devised a simple, sensitive, and rapid protocol to screen drug libraries for their effect on the ghdriven jakstat activation by flow cytometry. Phospho flow cytometry is a powerful technique for the detection of protein phosphorylation events that, like western blotting, relies on phospho epitopespecific antibodies. Cell preparation for flow cytometry protocols invitrogen ebioscience reagents red blood cell lysis protocols using. Finally, the approach can be a model for analysis of signaling in other. An air cooled argon laser operating at 20mw was used for excitation. Flow cytometry, methanol permeabilization protocol for directly conjugated antibodies a. Here, the optimized protocol for analysis of cll patient samples by phospho flow cytometry is provided figure 1a. Experiment outline see next page for reagents and manual for detailed protocols for each step 1. Phosphoflow course overview, example results, and detailed. Two basic protocols alcohol methoddetergent method 2. The recommended buffer systems will be noted on the technical data sheet for each specific antibody.
L of the same buffer prior to flow cytometric analysis. Techniques for phospho protein analysis becton dickinson. Phospho flow cytometry with fluorescent cell barcoding for. Please refer to the applications section on the front page of product datasheet or product webpage to determine if this product is validated and approved. The basic protocol involves fixation and permeablization of cells followed by staining with cellsurface or intracellular marker antibodies. Staining intracellular targets by flow cytometry can be difficult and may require specific protocols depending on your targets of interest. Flow cytometric detection of pakt and foxp3 in jurkat t. Phosphoher4y1162 antibody product information application ihcp, wb, if,e primary accession q15303 other accession q62956, q61527 reactivity human predicted mouse, rat host rabbit. Titration is the process of identifying the correct concentration of antibody to use for a. Apr 20, 2006 with phospho specific flow cytometry, or phospho flow, it is possible to measure the phosphorylation status of proteins critical to intracellular signaling cascades at the singlecell level 3,4,5. Phospho specific flow cytometric analyses correlated well with western blotting, providing cross platform validation of the technique.
Pbmcs step 3 might help improve the signal for some phospho markers. The following cisplatinstaining protocol has minimal impact on the phosphorylation of multiple targets when performed before pma and ionomycin cell activation. Phosphospecific flow cytometric analyses correlated well with western blotting, providing cross platform validation of the technique. Phospho specific flow cytometry phosphoflow getting started with phosphoflow. Current protocols in cytometry wiley online library. This information can be used to individually sort or separate subpopulations of cells. Analysis of pbmcs shows excellent precision of normalized mfi values of phospho proteins. For the success of such a protocol, two critical parameters must be met. Automation of a phosphostat5 staining procedure for flow. For detection of some phosphorylated signaling molecules such as mapk and stat proteins, it may be preferential to use protocol c. Signaling and phosphoflow in addition to the expression of surface molecules, there is a lot of interest in the activation state and measurement of signaling cascades. Measuring phosphorylation events by flow cytometry provides a rapid and efficient way to measure kinase cascades in individual cells. Titration and antibodies the basics flow cytometry staining protocol background.
Protocol for phosphoflow cytometry preparation uab. Centrifuge samples at 300400 x g at room temperature for 5 minutes, then discard the supernatant. Pe antistat3 phospho tyr705 antibody tyrosine phosphorylation of stat3 at tyr705 occurs in response to lif, il6, leptin, osm, egf, pdgf, and hgf. This manual is ori ginally from the 2008 embo advanced flow cytometry. Flow cytometry requires only a small sample size and is ideal for performing rapid, quantitative, multiparameter analyses of single cells and distinct cell subpopulations. Chander raman phospho flow methanol perm cell stimulation do your regular cell stimulation procedure fix. For protocol questions or pdf reprint requests, email dr. An optimized multiplex flow cytometry protocol for the. However, the impact of cisplatinstaining should be evaluated individually for all phospho proteins and with different stimulation conditions.
Intracellular staining of phospho epitopes by flow perez and nolan. Pdf phospho flow cytometry methods for the analysis of kinase signaling in cell lines and primary human blood samples. Pbmc phospho flow protocol materials primary cell sample pbmcs stimuli. Flow cytometry is the technical process that allows for the individual measurements of cell fluorescence and light scattering. Bd phosflow protocol for human pbmcs protocol i detergent method reagents required. Strict adherence to time and temperature recommendations for fixation, permeabilization, and staining is necessary for optimal resolution of. Perform heat mediated antigen retrieval with citrate buffer ph 6 before commencing with ihc staining protocol. With phosphospecific flow cytometry, or phospho flow, it is possible to measure the phosphorylation status of proteins critical to intracellular signaling cascades at the singlecell level 3,4,5. Phosphoprotein detection by highthroughput flow cytometry. Biobits phospho staining and intracellular flow cytometry.
Gary nolan and colleagues pioneered a method using flow cytometry to look at cell signaling in single cells using antibodies targeting phosphorylated protein sites, termed phosphoflow cytometry or phosflow. Flow cytometry was done using an epic elite cell sorter beckman. Introduction flow cytometry is routinely used for the identification of cellular populations based on a surface phenotype. Training novices in generation and analysis of high. The sample was incubated with the primary antibody 35 ugmillion cells in 2.
Also used to ensure the antibody performs within acceptable parameters. Nov 15, 2010 phospho specific flow cytometry, or phospho flow, measures the phosphorylation state of intracellular proteins at the single cell level. Pbmcs can be prepared and then stored for up to 7 days for use at a later time see step 7. Flow cytometry flow cytometry is the technical process that allows for the individual measurements of cell fluorescence and light scattering. Resuspend stained cells in an appropriate volume of flow cytometry staining buffer and acquire samples on a flow cytometer. Recently, phosphospecific flow cytometry has emerged as a powerful tool to analyze. Our optimized phospho flow cytometry protocol can serve as a basis for quantifying b cell subset signaling in health and disease. Intracellular phosphoprotein staining techniques for flow cytometry.
Bd phosflow protocol for human pbmcs bd biosciencesus. All reagents required for this protocol may be efficiently purchased together in our. Phosphoflow course overview, example results, and detailed manual. Phospho her4y1162 antibody product information application ihcp, wb, if,e primary accession q15303 other accession q62956, q61527 reactivity human predicted mouse, rat host rabbit clonality polyclonal isotype rabbit ig phospho her4y1162 antibody additional information gene id 2066 other names receptor tyrosineprotein kinase erbb4. Please see the phospho flow cytometry antibody clone buffer selection guide or the datasheet for the individual antibody for more information. Here we have optimized a protocol to quantify phospho signaling in human trbs by flow cytometry. After testing various fixing and permeabilization conditions, the following protocol was adopted for its speed, reproducibility, and optimal staining for all tested abs. Pdf analysis of signaling events by dynamic phosphoflow. Intracellular staining of phosphoepitopes by flow perez and nolan. Pe antistat3 phospho tyr705 antibody antistat3 phospho. Phosphobar2s261 antibody protein information name adrb2 dot blot analysis of. Antibody protocols flow cytometry, immunohistochemistry. Cell preparation for flow cytometry protocols invitrogen ebioscience reagents red blood cell lysis protocols using ebioscience lysis buffers invitrogen ebioscience reagents staining cell surface targets for flow cytometry invitrogen ebioscience reagents. Phosphospecific antibodies for flow cytometry thermo.
Phosphoepitope staining for flow cytometry optimized protocol. Phospho bar2s261 antibody is for research use only and not for use in diagnostic or therapeutic procedures. Measurement of map kinase activation by flow cytometry. Pbmcs can be prepared and then stored for up to 7 days for. Phospho bar2s261 antibody protein information name adrb2 dot blot analysis of anti phospho bar2ps261 antibody cat. Multicolor flow cytometry stem cell research tcell immunology. Intracellular phosphoprotein staining techniques for flow. This protocol combines fixation and permeabilization into a single step. Phosphospecific flow cytometry, or phospho flow, measures the phosphorylation state of intracellular proteins at the single cell level. Please refer to the applications section on the front page of product datasheet or product webpage to determine if this product is validated and approved for use in flow cytometry f. 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 phospho proteins and weve done it over and over, so we know what to expect. Request pdf singlecell phosphoprotein analysis by flow cytometry this protocol describes methods for monitoring intracellular phosphorylationdependent signaling events on a singlecell basis. Resuspend the cells in approximately 500 l of permwash buffer i prior to flow cytometric. Fluorescent cell barcoding in flow cytometry allows high.
Here we demonstrate application of tsa to detection of weakly expressed proteins with flow cytometry figure 1. Using phosphoflow cytometry to study signaling in human. Precision of flow cytometry based phospho protein measurements of pbmcs from 3 different donors. Fluorescence signals were collected using bandpass filters centered at 525 and 575nm, with fluorescence compensation set to suppress fitc spillover into the pe channel. Novel flow cytometric techniques for analysis of protein. Phosphospecific flow cytometry phosphoflow getting started with phosphoflow.
Flow cytometry protocol flow cell signaling technology. Abstract this article presents a single experiment designed to introduce a trainee to multiple advanced bench and analysis techniques, including high. Measurement of map kinase activation by flow cytometry using. Flow cytometry, kinase activation, phosphoproteins, proteomics, singlecell. Phosphoproteins by flow cytometry surface markers and. Protocol for phosphoflow cytometry preparation provided by donald j mcguire and dr. As a proof of concept, we optimized the pstat5 staining one of the most challenging phospho epitopes in flow cytometry in a cell line im9 stimulated with human gh hgh. This process is performed at rates of thousands of cells per. After testing various fixing and permeabilization conditions, the following protocol was adopted for its speed. Feb 28, 2012 moreover, to accurately measure signaling responses in tregs, the cells would need to be purified rapidly away from other cells following tcr stimulation. Flow cytometry requires only a small sample size and is ideal for performing rapid, quantitative, multiparameter analyses of single cells and distinct.
This process is performed at rates of thousands of cells per second. Phospho eif2s1 s51 antibody e90 recombinant ab32157. Weve created a number of resources to better help you choose the right intracellular flow staining protocol to remove the guesswork and help you generate clear and consistent results. Phosphobar2s261 antibody is for research use only and not for use in diagnostic or therapeutic procedures. Singlecell phosphoprotein analysis by flow cytometry. Gary nolan and colleagues pioneered a method using flow cytometry to look at cell signaling in single cells using antibodies targeting phosphorylated protein sites, termed phosphoflow cytometry or. Phospho flow cytometry is a powerful technique for the detection of protein phosphorylation events that, like western blotting, relies on phosphoepitopespecific antibodies. Outline see next page for reagents and manual for detailed protocols for each step. Examples of basal signaling characterization, antiigmb cell receptor. Finally, the approach can be a model for analysis of signaling in other rare immune cells.
Flow cytometry protocols thermo fisher scientific us. Flow cytometry, kinase activation, phospho proteins, proteomics, singlecell. Ic fixation and permeabilization, foxp3transcription factor buffer, and ic fixation buffermethanol. Please see the productspecific flow protocol on the product webpage for appropriate fixation and permeabilization conditions, and recommended. Protocol for phospho flow cytometry preparation provided by donald j mcguire and dr. Each phosphospecific antibody has been tested in three different flow cytometry buffer systems.
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