Isolated mitochondria only: 15-mL polypropylene tubes 1.5- and 2-mL Eppendorf tubes Dounce homogenizer with Teflon pestle . In the presence of your therapeutic candidate, the exhausted T cells can be plugged into a functional assay of your choice; T cell cytotoxicity, T cell activation or mixed lymphocyte reactions Figure 1. ProMab Biotechnologies provides in vitro T cell trafficking assays to visualize organ-specific homing of T cells. The role of USP30, and by inference mitochondria, in CTL function was intriguing because there is an increased reliance on glycolysis in activated effector T cells ( 11 ). Further, the ability of ACC inhibition to block glycolysis was not impaired by ETO, as measured by the seahorse assay . As the frequency of T cells responding to tumor antigens within a healthy individual is very low, to model antigen-specific T cell activation, we utilize a panel of peptides derived from viruses or pathogens to which many people will have been vaccinated against or have been infected with. The translational machinery of human CD4(+) T cells is poised for activation and controls the switch from quiescence to metabolic remodeling. bioassay for T cell co-stimulating biologics----- 2-Cell Assay----- 1-Cell Assay----- 2-Cell + Assay. *(May require strong stimulation such as TCR and CD28 co-stimulation). assay, human Treg cells are purified from a leukapheresis sample. SEB interacts with T cell Receptors and class II MHC Molecules. Human primary T cells were isolated from PBMCs using negative selection, cultured in RPMI + 10% FCS and then activated using either anti-CD3/CD28 coated beads (Miltenyi Biotec) or anti-CD3/CD28 tetramer complexes (STEMCELL Technologies) following each manufacturer's protocol, or with PHA at a concentration of 10 µg/ml. Optimization of bead-to-cell ratio for an XF T cell Activation Assay titration of bead-to-T Cell ratio prior to, and 30 minutes post bead injection normalized to 100 % of pre-activated control. T cells pretreated with LY294002, Akti-1/2, or Rap were used in the Agilent Seahorse XF Real-Time T cell activation assay, which showed the immediate-early glycolytic switch in EM CD8+ T cells depends Isolated mitochondria only: 15-mL polypropylene tubes 1.5- and 2-mL Eppendorf tubes Dounce homogenizer with Teflon pestle . Example: CD3 CD28 Beads activate T cells. T Cell Migration Assays | ProMab. The responder cells from the same leukapheresis sample are labeled with proliferation tracking dye. Each Kit provides reagents for 2 full plate assays with XFe96/XF96 Analyzers. In addition to cell proliferation, a variety of signaling pathways are activated . This is accomplished via measuring proton efflux rates (PER) associated with glycolytic pathway-dependent energy production, and is built on the principle that T cell activation is . 5 •Incubate at 37°C. surrogate marker . This utility is now extended by the Agilent . The XF T Cell Activation Assay delivers a convenient, consistent solution to study T cell activation and associated metabolic reprogramming. Benchmarking of T cell activation assays • DC:CD4 re-stimulation assay with IFNγ& IL-5 FluoroSpot readout • Graph shows % of donors with a significant T cell response (IFNγand/or IL-5) The uniform 4.5 µm diameter, inert, superparamagnetic beads are similar in size to antigen-presenting cells, and are covalently coupled to anti-CD3 and anti-CD28 antibodies. We are among the few groups who can generate and demonstrate the use of dendritic cells and macrophages (including M1 and M2) from monocytic cell lines and T cells in activation studies. For Research Use Only. Seahorse XFe24 FluxPak, which includes sensor cartridges, cell culture microplates and Seahorse XF Calibrant (Agilent 102340-100). The simultaneous binding of SEB outside of the MHC on antigen presenting cells (APC) and to T cell receptors expressing certain Vβ results in superantigen like properties in elicting polyclonal T cell activation and cytokine responsesCD3 CD28 bead stimulation may provide a more . Both cell types are critical for the development of several diseases. Using the XF Real-Time T cell activation assay to probe upstream signaling events required for activation. for antibody responses. Treatment of T cells with monoclonal anti-CD3 antibodies and anti-CD28 antibodies provide a co-stimulatory signal that engages the TCR which can be used for antigen-induced activation. The new Agilent Seahorse XF Hu T Cell Activation Assay Kit is an optimized turnkey solution that detects T cell activation in minutes, providing a kinetic readout of T cell activation dynamics in real-time. How this rapid, simple assay enables researchers to understand and direct immune responses. The T Cell Activation Bioassay (TCRαβ-KO) is a bioluminescent cell-based assay for measuring the potency of transgenic TCR constructs to activate T cells without the constraints of endogenous TCR expression. Chemotaxis/ Migration assays. Agilent Seahorse XF technology delivers real-time insights into cellular energy metabolism, and is relevant to the study of T cell activation and function. We offer activated human peripheral mononuclear cell (Human PBMC) and T cell activation assays to help you understand the interaction between your compound or biologic and the immune system, taking you to the clinic faster. For all extracellular flux assays, CD8+ T-cells were plated on cell-tak-coated Seahorse XF96 cell-culture microplates at a density of 2 × 10 5 cells per well. 4. It can take account of non-uniform seeding density across the well and plate. Flow cytometry is capable of measuring a number of different T cell properties, including relative proportions of T cell subsets, expression of activation . T cell priming (de novo activation of T cell responses) requires concurrent activation of two signals in T cells: recognition of peptides bound to MHC molecules through the TCR, and CD28 co-stimulation via CD80/CD86 on antigen presenting cells (APCs). Bio-Glo™ Luciferase Assay System (Cat.# G7940, G7941) is the required reagent for use with T Cell Activation Bioassay, Propagation Model. Easy to set up and convenient Seahorse technology have recently been proposed by Agilent Technologies and it could be used to monitor T cells metabolic potential. The MLR assay is an in vitro simulation of this phenomenon: T cells (responders) are mixed with antigen presenting cells (stimulators) from different (allogeneic) donors. According to recent research tumor induced T cell exhaustion plays a role in cancer . A protocol for real-time bioenergetic responses to T cell activation allows for the analysis of immediate metabolic changes after T cell receptor stimulation. Cell cultures were imaged at successive timepoints and . A protocol for real-time bioenergetic responses to T cell activation allows for the analysis of immediate metabolic changes after T cell receptor stimulation. Specific substrate utilization can be determined by the use of differential assay media, or the injection of drugs that specifically affect certain metabolic processes. Monocyte differentiation into dendritic cells and macrophages. CD28 Superagonistic Activation of T Cells Induces a Tumor Cell-Like Metabolic Program Thilipan Thaventhiran,1 Wai Wong,1 Ahmad F. Alghanem,1,* Naif Alhumeed,1 Mohammad A. Aljasir,1,{ Simeon Ramsey,2 Swaminathan Sethu,1,{Han Xian Aw Yeang,1,x Amy E. Chadwick,1 Michael Cross,1 Steven D. Webb,1,** Laiche Djouhri,3 Christina Ball,4 Richard Stebbings,4,{{and Jean G. Sathish1,{ With over 20,000 genes, 200,000 proteins and thousands of pathways, you can't measure everything in a cell at once, but you can measure the energy that drives them. Upon T cell activation, CD8+ T cells increase both OXPHOS and glycolysis (15). The T Cell Activation Bioassay is also available in CPM format, containing cryopreserved cells that can be thawed, propagated and banked for long-term use. Antigen recall assays assess the capacity of memory T cells to respond to their cognate antigen. The connection between energy metabolism and T-cell activation. Dendritic cells (DCs) are major antigen-presenting cells that can efficiently prime and cross-prime antigen-specific CD4+ and CD8+ T cells. Seahorse XFe24 FluxPak, which includes sensor cartridges, cell culture microplates and Seahorse XF Calibrant (Agilent 102340-100). • Robust T cell activation and expansion without the use of magnetic beads, feeder cells or antigens. T cell development was normal in Usp30 −/− (KO) mice, but CTLs generated after activation of naïve CD8 + T cells showed reduced killing of target cells. Conclusions Based on these results we conclude that 1:10 diluted WB assays can be used as an alternative immunological assay. Optimization of bead-to-cell ratio for an XF T cell Activation Assay titration of bead-to-T Cell ratio prior to, and 30 minutes post bead injection normalized to 100 % of pre-activated control. The benefits of measuring T-cell activation using the XF T-cell activation assay. This delivers a unique, early window of T cell biology, enabling researchers to quantify activation kinetics and profile modulators of T . Product Description Unit Product Content Compatible Analyzer 103592-100 Seahorse XF Real-Time ATP Rate Assay Kit EA 6 pouches1 XFe96, XF96, XFe24 103325-100 Seahorse XF Cell Energy Phenotype Test Kit EA 12 pouches1 XFe96, XF96, XFe24, XF24 103015-100 Seahorse XF Cell Mito Stress Test Kit EA 6 pouches1 XFe96, XF96, XFe24, XF24 The assay plates were spin seeded for 5 min at 1000 rpm and incubated at 37 °C without CO 2 prior to performing the assay on the Seahorse Biosci-ence XFe96. For the unstimulated control wells, add 50 µl of sterile PBS. Cell counting is a non-destructive method to generate data for Seahorse™ normalization. T cells can be problematic to characterise because they have a wide variety of subtypes and because of the technical difficulties of studying the membrane-bound T cell receptor, but there are situations where you want to be able to do this such as analysing the degree to which immunological memory has been induced to measuring how well an individual mounts a response to a particular antigen. T cell activation via the αβ-T cell receptor (TCR complex) is required for in vitro expansion. This utility is now extended by the Agilent . XF Glycolysis Stress Test Assesses Glycolysis and Glycolytic Capacity in Cells . NETosis studies. This delivers a unique, early window of T cell biology, enabling researchers to quantify activation kinetics and profile modulators of T . The T Cell Activation with anti-CD3 Antibodies Protocol describes the process of activating human peripheral blood mononuclear (PBMC) cells or isolated T cells using anti-CD3 antibodies. Data were collected in Wave software and analyzed using GraphPad Prism software. A T cell activation assay from Agilent Technologies assesses kinetics in real time by monitoring the extracellular acidification rate, an indicator of glycolysis. Cells were plated on Cell-Tak (Fisher)-coated XF 96-well plates at 2 × 10 5 cells per well in . Figure 1. IFN-γ . ProMab has developed a systematic approach to T cell activation and proliferation assays for IO products discovery. Monitor the activation of the immune system. Cells were washed three times, plated at 0.7 or 1 × 10 6 cells (CD8 + and CD4 + T cells, respectively) per well in a 96-well Seahorse assay plate, precoated with Cell-Tack Cell and tissue . Request A Quote. RLU [AbX] AbX induced strong T cell activation signals in 2-cell . Thus, our T-cell-activation assay is not aCD3 HD specific, but rather a "catch-all" method for detecting all T-cell-activating impurities present in TDB drug substance or product. XF Assay Tutorial: A rapid, real-time assay to monitor T cell activation using the Agilent Seahorse XFp Analyzer. Figure 3. I have been having trouble using murine T cells to do Seahorse assay. Benchmarking of T cell activation assays • DC:CD4 re-stimulation assay with IFNγ& IL-5 FluoroSpot readout • Graph shows % of donors with a significant T cell response (IFNγand/or IL-5) Our platform can evaluate gene therapy-based IO drug candidates with possible properties that can affect the migration of immune cells, and in turn influence their anti-tumor ability. 5 PBMC test population. For other assay media options, refer to XF buffer factor protocol quick reference guide; Data analysis tool, the Seahorse XF Glycolytic Rate Assay Report Generator, is provided to simplify and transform . I have tried using (1mg/ml) poly-L-lysine to coat the plate to let my T cell adhere but they seemed to affect my anti-CD3 . 2. upregulation of activation markers on the cell surface 3. differentiation into effector cells 4. induction of cytotoxicity or cytokine secretion 5. induction of apoptosis One of the most common ways to assess T cell activation is to measure T cell proliferation upon in vitro stimulation of T cells via antigen or agonistic antibodies to TCR. The nuclear factor of activated T cells (NFAT), IL-2 or other kinds of factors will be detected by bioluminescent methods or ELISA kits. As such, mitochondria play a crucial role in virtually all aspects of tumor progression. The Agilent Seahorse XF T-cell activation assay offers you improved insight into early T-cell activation dynamics with the ability to . 895-906.e5. In vitro tools: DC-T cell assays: procedure. Direct cell counting can significantly reduce the amount of time required to perform normalization. Cytokine assays. The XF T Cell Activation Assay delivers a convenient, consistent solution to study T cell activation and associated metabolic reprogramming. Seahorse XF Hu T Cell Activation Assay Kit. We present methods for using inhibitors to alter cellular metabolism, measurement of intracellular ATP in NK cells, assessment of real-time glycolysis and oxidative phosphorylation by an extracellular flux assay from Seahorse Biosciences, and some basic protocols for stimulation of NK cells via cytokines and receptors. . Illustration of iQue ® Human T Cell Killing Kit assay principles.. CD8+ T cell status can be profiled for the expression of activation marker CD25 and exhaustion marker PD-1. This product is designed for cell therapy research applications, but may be qualified for use as an ancillary material (AM) following . CD8+ T cell activation can be measured by cytokine production. 4 •Add target & APCs. Briefly, stimulated T cells were plated (10 5 viable cells/well) in Cell-Tak (Corning) coated Seahorse Analyzer XFe96 culture plates in Seahorse assay media supplemented with glucose (10 mM), Gln (2 mM), and pyruvate (1 mM). The primary consideration when gathering materials to run this assay is that the antigen-specific T cell line you use must be specific to your antigen of choice. Analysis of T cell responses is a frequently used tool to monitor the activation of the immune system.The Epibase® in vitro T cell assays take into account multiple components of the vaccine, including the protein component and also other factors that may contribute to immunogenicity, such as adjuvants. 103575-100) or Seahorse XF RPMI medium, pH 7.4 (Cat No. This might play an essential role in optimal T-cell activation and secretion of IFN-γ against the antigens and mitogen in 1:10 diluted WB assays. 2. Agilent Seahorse XF technology delivers real-time insights into cellular energy metabolism, and is relevant to the study of T cell activation and function. T-cell and B-Cell Activation T-cell and B-Cell Activation - Components of the adaptive immune system with the main function to secrete antibodies upon activation. T cell activation and differentiation. As a result, T cells are activated and thus they can . Agilent Seahorse XF Hu T Cell Activation Assay Kit measures human (Hu) T cell activation response within several minutes of stimulation using Seahorse XF Analyzers. 4. 0:32. T-Cell Activation Assay Kit. SEB is also a known as superantigen, as it can trigger polyclonal T cell activation and massive cytokine release resulting in toxic shock syndrome. Dispense 50µl of the antibody solution to each microwell of the 96-well assay plate. T cell activation/proliferation assays using human PBMC can be used as a . 1 •Prepare assay buffer. As well as tailored assay readouts, we also offer standard analysis options, such as cytokine levels by ELISA (e.g. The simultaneous binding of SEB outside of the MHC on antigen presenting cells (APC) and to T cell receptors expressing certain Vβ results in superantigen like properties in elicting polyclonal T cell activation and cytokine responsesCD3 CD28 bead stimulation may provide a more . This unit contains several protocols to determine the energy utilization of T cells in real-time using a Seahorse Extracellular Flux Analyzer (www.seahorsebio.com).The advantages to using this machine over traditional metabolic assays include the simultaneous measurement of glycolysis and mitochondrial respiration, in real-time, on relatively small numbers of cells, without any radioactivity. Report Generators - Single and Multi-file (if available) Seahorse XF Cell Energy Phenotype Test Report Generators Using their T cell receptors (TCRs), T cells scan the surface of the other cells, recognizing MHC-peptide complexes on allogeneic cells as foreign. Cell Metab, 28 (2018), pp. ImmunoCult™ Human CD3/CD28 T Cell Activator can be used on the Seahorse XF Analyzer to measure T cell activation response and is also available as part of the Agilent Seahorse XF Hu T Cell Activation Assay Kit. If cytokine production is used as a read out, the antigen-presenting cells do not need to be inactivated. The T Cell Activation Bioassay reflects the mechanism of action (MOA) of biologics designed to engage the TCR and induce TCR-mediated T cell activation. We use myeloid and lymphocytic cell lines for various immune response assays such as dendritic cell maturation, T cell activation, phagocytosis, etc. Agilent Seahorse XF Hu T Cell Activation Assays measuring the proton efflux rate (PER) upon the injection of a soluble CD3/CD28 activator in the standard assay design (A) and the modulation assay design (B). XF Glycolysis Stress Test Assesses Glycolysis and Glycolytic Capacity in Cells . 6 •Add Steady-Glo and read plates. Pre-activation represents the ECAR measured 1 hour after conventional activation of T cells, and is normalized to 100 %. The new Agilent Seahorse XF Hu T Cell Activation Assay Kit is an optimized turnkey solution that detects T cell activation in minutes, providing a kinetic readout of T cell activation dynamics in real-time. Dynabeads® Human T-Activator CD3/CD28 offer a simple method for activation and expansion of T cells that does not require feeder cells (antigen-presenting cells) or antigen. Data analysis strategy of XF Hu T Cell Activation Assay Figure 2. Thus, we 14, analyzed the metabolic consequence of C1qbp ablation in naïve or in vitro-activated CD8 + T cells by using Seahorse Extracellular Flux Analyzer. Assay of Antigen Specific T Cell Activation. Different T cell phenotypes are profiled for the expression of 3 early/late activation markers: CD69 (early), CD25 (late), and HLA-DR (even later). Cytokine release assays/ Luminex Multiplexing assays. Figure 4. T cell assay readouts described in more detail below, including ELISA to measure cytokine levels in the culture supernatants and ELISpot to quantify the frequency of cytokine-producing cells. Proliferation and cytotoxicity. All Epibase® in vitro assays can be . This assay is best performed with Seahorse XF DMEM medium, pH 7.4 (Cat No. Pre-activation represents the PER measured 1 hour after conventional activation of T cells, and is normalized to 100 %. 2 •Serially dilute AbX. Figure 3. Antigen recall assays assess the capacity of memory T cells to respond to their cognate antigen. CAR-modified T cells can recognize tumor cells via binding of the CAR to its tumor-associated antigen (TAA) independent of TCR-MHC/peptide interactions, and the engineered signaling endodomains can help T cells be activated and persistent. Interferon gamma and TNF alpha are usually present after antigen stimulation. XF Assay Tutorial: A rapid, real-time assay to monitor T cell activation using the Agilent Seahorse XFp Analyzer. Specifically, anti-CD3 antibody-mediated luminescence is detected using either the TCR/CD3 Effector Cells (NFAT) or the TCR/CD3 Effector Cells (IL-2), and the signal is enhanced following the T-cell activation. Downstream assays are still possible after normalization. . Report Generators - Single and Multi-file (if available) Seahorse XF Cell Energy Phenotype Test Report Generators Specific substrate utilization can be determined by the use of differential assay media, or the injection of drugs that specifically affect certain metabolic processes. 103576-100). The allogeneic MLR assay is also a co-culture assay and permits measurement of DC-mediated T cell activation. SEB interacts with T cell Receptors and class II MHC Molecules. Immunogenicity assessment/ Cytokine storm: Multiplexing assays against multiple donors. Not for use in diagnostic procedures. The antigen-specific T cell assay measures T cell activation in vitro using cultured T cell lines, which ensures a quick response to the antigen stimulus and not any other antigens. For info: 800-227-9770. The assay consists of a genetically engineered Jurkat T cell line with endogenous TCR α and β chains knocked out using CRISPR/Cas9. Functional assays for T cell co-stimulatory molecules: Principles. The activation of naive T cells by antigen is one of the central events in the adaptive immune responses. Example: CD3 CD28 Beads activate T cells. Illustration of Human T Cell Activation Cell and Cytokine Profiling Kit assay principles. Introducing the Incucyte ® Immune Cell Clustering & Proliferation Assays. ECAR and OCR were measured using a Seahorse XFe96 Extracellular Flux Analyzer (Agilent Technologies). Figure 1. The glycolytic rate can be analyzed by the absolute PER (pmol/min) or by the Metabolic cell features are able to give reliable information on cell functional state. Mitochondrial function and glycolysis are key in a variety of vital cellular processes, including cellular activation, proliferation, differentiation, cell death, and disease progression. Assay Procedures. Given that naïve CD8+ T cells did not express C1qbp Its potency at low concentrations is due to simultaneously binding of SEB to the class II major histocompatibility complex (MHC) antigens expressed on professional antigen presenting cells (APCs . The responder cells are incubated with . As the frequency of T cells responding to tumor antigens within a healthy individual is very low, to model antigen-specific T cell activation, we utilize a panel of peptides derived from viruses or pathogens to which many people will have been vaccinated against or have been infected with. Meanwhile, the T cell activation and proliferation process can also be evaluated via cell . Product Ordering Information: Cat No. Both cytokine and proliferation can be measured from the same assay, if desired. Immune . Seahorse Cell Metabolism Technology. A Real-Time Cytotoxicity Assay as an Alternative to the Standard Chromium-51 Release Assay for Measurement of Human NK and T Cell Cytotoxic Activity Julien Fassy , Kyriaki Tsalkitzi , Maria Goncalves-Maia , Véronique M. Braud , Investigate the mechanisms of immune cell activation (proliferation), regulation and differentiation (clustering) inside your incubator and without the need to lift or label your cells. We recommend using a 2-mL homogenizer for small tissue samples (e.g., spinal cord or microdissected brain regions), a 7-mL homogenizer for . Visualize and quantify immune cell interactions and proliferation in real time. iQue® Human T Cell Activation Kit. The 2 effector cytokines (IFNγ and Granzyme B) are also quantified using 2-plex Qbeads in a sandwich immunoassay format in the same well. 3 •Add AbX dilutions. The responder (e.g., CD4+ T cells) and stimulator cells (e.g., monocyte-derived DCs or peripheral blood mononuclear cells [PBMCs]) must come from different donors since T cell activation is based on an allogeneic immune reaction. These cells are not anergic but are comparatively less proliferative, cytotoxic and able to produce IFNγ and IL-2 than resting T cells. Thus, metabolic potential assessment of T cells in malignancy setting represents a promising area, especially in adoptive cell therapy procedures. Binding of an antigen to the T cell receptor (TCR)/CD3 complex along with costimulatory signals initiates clonal expansion of both CD4 + helper and CD8 + cytotoxic T cells. Agilent. As the . DC-T cell assays are used to assess the potential immunogenicity of different product formulations that directly modulate T cell activation. Figure 1. We recommend using a 2-mL homogenizer for small tissue samples (e.g., spinal cord or microdissected brain regions), a 7-mL homogenizer for . 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