Cytof antibody panel The top section of each panel shows the CyTOF measurements as ‘rain dots’. Each subpopulation is depicted as a pale green circle with an edge of a characteristic color scheme. Recent Mass Cytometry/CyTOF Citations: High-dimensional immune profiling of total and rotavirus VP6-specific intestinal and circulating B cells by mass cytometry. Search Our CyTOF-ready Antibodies . Target Clone Tag Note CD45 HI30 89Y MD-IPA CD196 /CCR6 G034E3 141Pr MD-IPA CD123 6H6 143Nd MD-IPA CD19 HIB19 144Nd MD-IPA CD4 RPA-T4 145Nd MD-IPA CD8a RPA-T8 146Nd MD-IPA CD11c Bu15 147Sm MD-IPA CD16 3G8 148Nd MD-IPA CD45RO UCHL1 149Sm MD-IPA Table 1 | CyTOF Antibody Panel and Validations Commercial validation In-house validation Identification Marker Metal conjugation CyTOF Flow cytometry Expression construct Etoposide-induced Sep 1, 2022 · In addition, the number of probes used in one reaction is significantly increased from 18 (for FACS) to 40–50 panels (for CyTOF) [6], and some studies have shown that a CyTOF panel can include as many as 100 channels [12]. and Maxpar antibody conjugation kits to conjugate your own antibodies. [14] Four to five polymers are bound to an antibody, resulting in about 100 isotope atoms per antibody. Imaging pricing from $1,000 USD per slide antibody conjugation methods facilitate the seamless incorporation of specific antibodies into the analysis panel, enabling the comprehensive interrogation of a wide range of cellular features [14,15]. Understanding the immune landscape in cancer patients undergoing these therapies is essential for optimizing treatment strategies and improving outcomes. All metal-conjugated antibodies were purchased from Fluidigm. Initially, 52 cellular targets, of interest for the field of cancer immunology, were selected for potential implementation in the panel. CyTOF® has several advantages that enable simplified high Optimize your panel using Maxpar Panel Designer, a free online tool for building panels with Standard BioTools metal-conjugated antibodies or custom conjugates. CyTOF Panel design . common lineage markers such as CD3, CD19, and CD11b) should be titrated individually and separately. May 17, 2019 · For CyTOF, purified unconjugated antibodies used were Biolegend MaxPar Ready antibodies or custom-made with no additional protein carrier from Biolegend or Thermo Fisher. CyTOF technology facilitates the use of more markers that can be detected simultaneously in a single cell and far less sample in a single panel. CyTOF is a technology that consists of cytometry by inductively coupled plasma time-of-flight mass spectrometry; it is a single-cell method that enables highly multiplexed and quantitative measurements of proteins and their modifications. edu R&D Systems antibodies lyophilized in a CyTOF-ready format. Maxpar OnDemand Antibodies are a collection of carefully selected clones and metal options that give you more choice in your panel design for optimal flexibility and performance. Here we present a reference panel for id … Easily build on the Maxpar Direct™ backbone panel with individual antibodies or Expansion Panels, designed to enable deeper profiling of specific immune cell subsets that are relevant in immune-mediated disease, such as infectious disease, cancer, autoimmunity or allergy. Custom conjugation service is available with a reduced price for antibody service projects. From the MaxPar Panel Desinger User Guide: Low-abundance antigens: Choose high sensitivity channels. Where feasible multiple resources for the same process are Apr 13, 2021 · The protein panel measured by CyTOF included 15 metabolic proteins, six of which are listed. 1 May 31, 2024 Optimize your panel using Maxpar Panel Designer, a free online tool for building panels with Standard BioTools metal-conjugated antibodies or custom conjugates. Antibody performance and reproducibility of our methodology was demonstrated by analysis of CyTOF data from technical and biological replicates (Experimental Procedures; Figure S2). Eight different panels were used to span the antibodies needed for the single CyTOF panel. The Human Cytokine Core CyTOF™ Panel, 5 Antibodies (201344) enables evaluation of 5 key cytokines produced and regulated by T cells in immune responses. bwh. These reagents were used to Feb 13, 2018 · Using a CyTOF antibody panel to interrogate features of HGSOC biology, combined with unsupervised computational analysis, we identified notewor … We have performed an in-depth single-cell phenotypic characterization of high-grade serous ovarian cancer (HGSOC) by multiparametric mass cytometry (CyTOF). 7 Protocol | 400272; CyTOF Software v6. It is also possible to conjugate an antibody that is already tagged with a fluorescent dye, to identify a region of interest by fluorescence microscopy The recent launch of the CyTOF platform means that there are some limitations in the choices of antibodies available. Most of the same techniques and reporter molecules can be used in CyTOF assays as in fluorescence flow cytometry. Leukocyte lineage markers were used to perform viSNE-automated analysis to study live CD45 + single cells in blood (online supplementary figure 1A). Mark M. Furthermore, differences in cell isolation and enrichment protocols, antibody reagent preparation, sample staining and Jan 26, 2021 · Mass cytometry (CyTOF) represents one of the most powerful tools in immune phenotyping, allowing high throughput quantification of over 40 parameters at single‐cell resolution. Davis, Shai S. The iterative approach builds upon a well-characterized backbone panel of antibodies and analysis using bioinformatic tools such as SPADE. 4 Analyze using post-analytical imaging and secondary analysis software tools. Shen-Orr, in Handbook of Systems Biology, 2013 Cytometry by time-of-flight (CyTOF) is a recently introduced technology that measures the abundance of metal isotope labels on antibodies and other tags (such as peptide-MHC tetramers for labeling specific T cells) on single cells using mass spectroscopy. The CyTOF technique helps to define the multifaceted contributions of immune defense within circulation and plaques (Table 4). Our results showed that increased frequencies of circulating GrzB+ CD8 T cells are associated with patient response to pembrolizumab. Feb 23, 2023 · For example, using the Standard BioTools™ Maxpar® Direct™ Immune Profiling Assay™—a validated, dry-format antibody panel for use on the CyTOF XT™ and Helios™ mass cytometry systems—researchers can profile 30 immune markers from just 300 µL of blood, without having to use additional sample for staining controls. 1 is an interactive web-based application to build CyTOF® flow cytometry and Imaging Mass Cytometry™ panels directly in your browser. The panel builder is designed to prevent selection of incompatible fluorochromes by blocking channels with excessive spectral overlap. Maximum sensitivity is centered slightly higher than the middle of the In this protocol, we use a CyTOF™ mass cytometry to collect single-cell data on a large number of cytokines/chemokines as well as cell-surface proteins that characterize T cells and other immune cells. This panel includes 7 pathologist-verified antibodies that enable phenotyping of lymphocytes and myeloid cells, and is designed to complement the Human Lymphoid IMC Panel, 6 Antibodies (201512) and Human Myeloid IMC Panel, 7 Antibodies (201513) for enhanced cell phenotyping capabilities. Background Antibody-based immunotherapies have transformed cancer treatment by using the body’s immune system to target and destroy tumor cells. [14] Tagged antibodies may be in solution, conjugated to beads, or surface immobilized. Unlabeled antibodies were conjugated in-house using the MaxPAR antibody labeling kit (Fluidigm) according to the manufacturer’s protocol. 0. Jul 19, 2024 · Instead, it can be applied to quantify the strength of information transfer between all possible pairs of proteins (e. B , Aortic leukocytes were stained with an anti–CD45-89Y antibody and spiked with anti-CD45. Panel design at your fingertips . For this, it is Mar 31, 2022 · Conjugated intracellular CyTOF antibodies (Supplementary Data 1) diluted in the permeabilization buffer from the FoxP3/Transcription Factor Staining Buffer Set were added into each tube and Oct 2, 2018 · Despite the liberal use of the term “proteomics” in conjunction with cytometry, CyTOF® and other antibody-based methods are not actually dealing with a proteome-wide profiling the cells, but instead a carefully selected panel of proteins (limited to proteins for which probes such as monoclonal antibodies exist) and physicochemical features After processing, samples were subjected to a CyTOF antibody panel that simultaneously measured the expression of 40 neutrophil surface markers . The Novus flow cytometry panel builder tool streamlines the panel design process by enabling researchers to quickly search for validated Bio-Techne antibodies that will work with a specific cytometer. Oct 28, 2019 · To develop the 40 marker imaging mass cytometry antibody panel, antibody performance was assessed by IHC. Prior to algorithm Jan 16, 2021 · The CeD panel was designed to yield maximal resolution of the cell subsets by consideration of metal utilization across the different cell types. Apr 17, 2017 · To identify myogenic progenitors in vivo within skeletal muscle, we capitalized on the CyTOF technology and assembled a panel of 23 isotope-conjugated antibodies. CyTOF Antibody Panel Guide 3. For example, in tumor CyTOF we used (metal channel 103) Rhodium as our Cell-ID™ to avoid overlap with the custom barcodes (metal channels 112 With over 450 unique conjugations, any user can create their own panel from our inventory. Additions or substitutions in this panel are more difficult, due to the requirement for surface marker antibodies to work well on fixed epitopes. , 2012). There are currently 44 metals available to conjugate to antibodies, so the maximum number of markers in a single panel is 44. 6% Aug 18, 2020 · We thus asked whether we could detect an expansion of eNePs and subset N1 in cancer patients. Conjugating antibodies to metals allow users to customize and/or expand CyTOF panels and assures that the whole project can be stained with the same lot of validated antibody conjugated metals to reduce batch effects. Standard projects utilize more than 50 pre-optimized panel kits and over 900 individual catalog antibodies. Don’t miss the unexpected. May 26, 2020 · CyTOF utilizes next-generation, Samples were processed and antibody staining was performed using a panel of metal-conjugated antibodies specific for lineage markers and other markers of To design an immunometabolic CyTOF panel, Hartmann et al. Highlights Comprehensive—Profile 37 immune cell populations from PBMC or whole blood with an optimized 30-marker panel. CyTOF® Software v9. 1-145Nd–stained splenocytes (1×10 6 ) from age-matched CD45. Apr 14, 2022 · panel design, antibody conjugations, sample staining, sample acquisition, and data pre- processing and analysis. anti-FITC) that can be used with existing fluorescent reagents. 3 to 4 weeks from receipt of antibody: 5 to 6 weeks from receipt of antibody: Flow Cytometry Antibodies: Scale: standard Antibody input: 200 µg Buffer: purified, protein-free buffer: PN 3999999-2 Minimum yield: 40 µg: PN 3999999-1 Yield: 100 tests (1–3 x 10 6 cells) Scale: bulk Antibody input: variable mass Buffer: purified, protein-free Oct 28, 2016 · Panel Design and Consultation Harvard Medical Area CyTOF Antibody Resource and Core o Large collection of Human and Mouse antibodies labeled with rare-earth metal isotopes – o Strategically labeled to reduce and prevent spillover between strong and weak signals o CyTOF staining panels Prepare antibodies (antibody plate) Mark out 8 wells of a 96-well plate; Place 122. CyTOF antibodies were labeled with metal-tag at the SickKids-UHN Flow and Mass Cytometry Facility using the MaxPar Antibody Labeling kit from Fluidigm (catalog #201300). 5 uL FACS). Supplementary Table 5. CyTOF antibody conjugation is notably more streamlined, permitting the attachment of a broad spectrum of metal isotopes via a consistent Jan 11, 2024 · CyTOF antibodies in our panel (Supplementary Table 4) were validated using methods previously described, including the use of human lymphoid aggregate cultures generated from tonsils 2,3,4,5,18,19. CyTOF Assays. 0 Release Notes | FLDM-00223 3 to 4 weeks from receipt of antibody: 5 to 6 weeks from receipt of antibody: Flow Cytometry Antibodies: Scale: standard Antibody input: 200 µg Buffer: purified, protein-free buffer: PN 3999999-2 Minimum yield: 40 µg: PN 3999999-1 Yield: 100 tests (1–3 x 10 6 cells) Scale: bulk Antibody input: variable mass Buffer: purified, protein-free Optimize your panel using Maxpar Panel Designer, a free online tool for building panels with Standard BioTools metal-conjugated antibodies or custom conjugates. Jan 7, 2025 · These features allow for the measurement of more than 50 parameters simultaneously without the need to compensate between different metal-coupled antibodies. B) Two-dimensional visualization of major leukocyte populations in human bone marrow (random sampling a total 50,000 cells in which 10,000 cells Jun 26, 2020 · deployment of CyTOF-based immune phenotyping studies are limited by complex experimental workflows and the need for specialized CyTOF equipment and technical expertise. Dec 5, 2023 · The mass cytometry workflow is shown in Figure 1. The CyTOF is most sensitive to metal tags in the 153-176 Da range; Choose mass tags in channels which receive little or no crosstalk from other metals in the panel; Consider amplifying low abundance signal: Nov 1, 2024 · However, the sensitivity of mass spectrometry results in the measurement of metal impurities and oxide formations, which need to be carefully considered in antibody panel design (e. Nov 25, 2023 · Using our CyTOF workflow as previously described 11 with some modifications (see Materials and Methods for more detail), whole blood samples were characterized using an antibody panel of 37 Oct 26, 2020 · Our validated antibody panel contained 30 immune markers (Supplementary Table 1) 17. 9 for S100), for the CyTOF IMC platform. 1 offers instrument control and data acquisition functionality for both imaging and flow applications with the Hyperion XTi™ Imaging System and CyTOF XT™. Designed as a simple workflow, the solution brings together CyTOF® technology, a 33-antibody reagent kit and Maxpar Pathsetter templates, enabling identification of 38 mouse immune cell populations. Oct 22, 2021 · A routine CyTOF antibody panel targeting the T lymphocytes subsets was established that allowed us to profile the extracellular expression of T cell subsets among AAD patients (N=45) and age-sex . Guidelines for Panel Design. Flow pricing from $300 USD per sample. Jun 5, 2015 · The eight phospho-specific antibodies were chosen to represent major signaling nodes responsive to cytokine, TLR, and antigen receptor signaling. Table S3. , through antibody concentrations and coupling of antibodies to neighboring metals). Jan 12, 2023 · While some antibodies demonstrated high background and limited positive signal by CyTOF, one antibody in particular provided an excellent signal-to-noise ratio and was therefore selected for the panel. We use a CyTOF™ (Cytometry by Time-Of-Flight) mass cytometer to acquire the ICP-MS data. Notably, this antibody has been used in other senescence studies for flow or mass cytometry 30,31. Antibody Panel for CyTOF. 5 (anti-CD45 PcP-Cy5. For example, in tumor CyTOF we used (metal channel 103) Rhodium as our Cell-ID™ to avoid overlap with the custom barcodes (metal channels 112 Complete Panel (Phosflow) Complete Panel (Phosflow) Marker Clone Antibody Vendor Catalog # CD45 CD45 (PAN) 30-F11 Fluidigm 3089005B 115Ln CD11b CD11b CD11b CD11b M1/70 Biolegend 101249 IL6 IL-6 MP5-20F3 UBC Antibody Lab AB0000920 141Pr MHCII MHCII MHCII MHC-II M5/144. Barcoding live Jurkat cells using Pd-tagged CD45 antibodies. Table 2 lists the 34 antibodies that were finally chosen for inclusion into the IMC antibody panel. Each panel indicates the antibodies and their fluorescent conjugation . Nair, N. Optimize your panel using Maxpar Panel Designer, a free online tool for building panels with Standard BioTools metal-conjugated antibodies or custom conjugates. Dec 22, 2016 · A repository of well-characterized metal conjugated antibodies; Metal labeling reagents for conjugation of antibodies to these metals: 145Nd, 148Nd, 158Gd, 160 Gd, 164Dy, 165Ho, 171Yb, 173Yb; Metal conjugated anti-fluorochrome antibodies (e. 5) addition on the detection of BV711 Jan 26, 2021 · This whole blood sample processing workflow leverages the commercially available Fluidigm MaxPar Direct Immune Profiling Assay (MDIPA), which incorporates a dry tube containing a 30-marker broad immunophenotyping panel. Right: level of clustering analysis that antibodies were used in. Mass cytometry is advantageous because of its ability to precisely and accurately distinguish between reporter molecules. , edges in a signaling network topology) available in a CyTOF antibody panel. see Notes 15 and 16 for extensive description of the procedures needed for panel Design, antibody conjugation, antibody titration, and required controls. While antibody conjugates are the most common reagents, other reporters such as live/dead or cell cycle reagents are also possible to use. Furthermore, differences in cell isolation and enrichment protocols, antibody reagent preparation, sample staining and General Guidelines for Metal Selection for Antibody Labeling - Panel Development The CyTOF is currently tuned for a mass window approximately AW 103-203. Notably, this is designed to improve immunophenotyping standardization by allowing either PBMC or whole CyTOF XT Hyperion XTi Imaging System Biomark X9 System for High-Throughput Genomics Kits, Reagents, & Accessories Cytometry Tissue Imaging Genomics Antibody and Panel Kit Selector Imaging Mass Cytometry Jun 3, 2019 · a–d, Single-cell suspensions from lung cancer patients were stained with a 28-color mAb panel. “And, with the potential for over 100 distinguishable tags, CyTOF is seeing utility for applications spanning basic research through to clinical research and monitoring of disease treatment. Apr 1, 2024 · To ensure that a relevant comparison of the technologies could be made, we developed the identical panel for full spectral flow cytometry and mass cytometry encompassing the exact same antibodies. Apr 13, 2022 · Antibodies to primary antigens (e. Antibodies to primary antigens (e. By using atomic mass measurements, mass cytometry avoids autofluorescence signals from cells and tissues, enables fast panel design since all antibodies are used at once and facilitates barcoding to multiplex samples in large-scale acquisitions. Where feasible multiple resources for the same process are compared, allowing researchers experienced in flow cytometry but with minimal mass cytometry expertise to develop a data-driven and CyTOF XT Hyperion XTi Imaging System Biomark X9 System for High-Throughput Genomics Kits, Reagents, & Accessories Cytometry Tissue Imaging Genomics Antibody and Panel Kit Selector Imaging Mass Cytometry Sep 11, 2022 · When selecting purified antibodies for CyTOF, it is important that antibodies are carrier-free (bovine serum albumin (BSA)- and preferably azide-free). Mar 17, 2023 · To avoid significant spillover from critical markers in the pre-processing and de-barcoding stage, we recommend choosing the appropriate Cell-ID™ based on the surface/intracellular antibody panel. Jul 15, 2020 · Based on this we selected antibodies that displayed a clear signal-to-background ratio for potential inclusion in the final IMC antibody panel. Mass cytometry (CyTOF, Cytometry by time of fight) expands upon the principles of flow cytometry by employing antibodies that are labeled with metal isotopes instead of fluorochromes. Apr 14, 2022 · Included in this review are experiment and panel design, antibody conjugations, sample staining, sample acquisition, and data pre-processing and analysis. Table S2. Live Jurkat cells were barcoded using three strategies for combining CD45 antibodies tagged with six palladium isotopes. This panel is optimized for use in FFPE human tissue Sep 26, 2018 · To validate the measurement accuracy of bismuth-tagged antibody in high-dimensional CyTOF assays, the same antibodies were conjugated with both bismuth and lanthanide mass tags: (i) human anti-CD3 May 22, 2023 · CyTOF antibody panel. “Because CyTOF detects mass, it is highly precise,” reports Ellis. Maxpar panel kits Human Immune Jan 1, 2024 · Our CyTOF antibody panel simultaneously measured surface and intracellular protein targets enabling a systems-level view of how immune cell phenotypes respond to therapeutic pressure. Don’t see what you need in our catalog? Build a custom project. , SP142 for PD-L1 and 4C4. CyTOF is, however, complementary to RNA-seq in providing proteomic information Panel design for cytometry experiments is the art of pairing probes (generally antibodies) with detected-molecules (fluorophores for flow or conjugated lanthanides and cadmiums for mass cytometry) so that expression differences are maximised and emphasised. Metal conjugated anti-fluorochrome antibodies (e. This panel is optimized for use in FFPE human tissue sections CyTOF Software v6. Allograft outcomes of DSAPOS recipients, stratified by development of ABMR together CyTOF® technology, a dry 30-marker antibody panel and automated analysis with Maxpar Pathsetter™ software so you can easily quantify 37 immune cell populations with performance you can trust. It aims to find the concentration of antibody that best resolves a positive signal from the background and ensures saturation of all binding sites with minimal antibody excess. Flexible: Integrate barcodes with CyTOF panels without compromising panel size. broad immunophenotyping panel. This antibody panel is used with 8 standard stimulation conditions (unstimulated, IFNa, IL-6, IL-7, IL-10, IL-21, LPS, PMA+ ionomycin), although other stimuli can be added. We were unable to confidently identify eNePs in our Protocols for antibody conjugation, cell surface, viability, cytoplasmic targets, nuclear targets, signaling proteins and phosphoproteins can be found on Fluidigm's website by scrolling down to the Protocol section, under Resources. Purified unlabeled antibodies were purchased from the vendors as shown in table S1. CyTOF immune phenotyping. This panel consists of antibodies that identify Th1/Th2/Th17 cytokines, which have important implications in immunopathology, including autoimmune diseases and cancer. BSA competes with the primary antibody to attach to the label of interest, which greatly reduces the conjugation efficiency. first screened over 100 commercial antibodies against a broad range of metabolic factors and came to a selection of 41 metabolic antibodies that passed all biological controls and yielded robust reproducible results in a proof-of-concept study of human white blood cells (Hartmann et al Aug 27, 2024 · Here, we used a CyTOF panel of 15 Vβ antibodies, 4 Vα antibodies and 23 lineage/phenotyping antibodies to follow TCR usage and T cell response simultaneously. Metal cross-talk & antibody sensitivity knowledge is used to carefully design each panel. The assay enables comprehensive identification and characterization of 37 immune cell populations. The concentration will be 1/50 vol/vol. Jan 1, 2020 · Fluidigm commercializes antibodies conjugated with yttrium, bismuth and heavy metals of the lanthanide family (Table 1) and MaxPar® X8 antibody-labelling kit for custom antibody conjugation with lanthanide isotopes. Streamline your multicolor flow cytometry workflow by optimizing your antibody choices with our flow cytometry panel builder Save time with optimized antibody recommendations, tailored to your experimental setup A) CyTOF antibody panel of human immune cell lineage markers and proteins associated with neutrophil function and maturation. The CyTOF workflow. Jun 8, 2018 · A, Markers of the 35-marker pan-leukocyte CyTOF antibody panel (full panel shown in Online Table VI). Improve Molecular Profiling : Simultaneous detection and resolution of more targets than is feasible with conventional flow cytometry, making TOFMS ideal for testing rare or precious samples. Staining Procedure Systems Immunology. CyTOF was performed on fresh human bone marrow from healthy donors (n=5). A defining characteristic of senescent cells is expression of cell cycle inhibitors, in particular p16 or p21 29, so we carefully We constructed and validated a comprehensive CyTOF antibody panel to include markers for both cell identity and senescent phenotype (Table 1). 7: Reprocess IMD Files without Headers Protocol | 400261; CyTOF Software: Data Retention and Computer Management Recommendations | FLDM-400273; Maxpar Panel Designer v2. Healthy peripheral blood and bone marrow cells are stained with a pre-optimized "backbone" antibody panel in addition to the progressively diluted (titrated) antibodies. Mucosal Immunol. Profiling intracellular cytokines and immune cell subsets in whole blood provides valuable The launch of new Maxpar® OnDemand Antibodies adds flexibility to the Fluidigm Imaging Mass Cytometry™ (IMC™) antibody portfolio. Create an account for free and start exploring and designing new panels. Conjugation of antibodies and metal isotopes is an easily performed step that results in increased options for panel design, and has been previously described May 25, 2021 · The Maxpar® Direct™ Immune Profiling Assay™ is a dry 30-marker antibody panel designed and optimized for deep immune profiling of human peripheral whole blood and PBMC. Anti-mouse Ly6G IA8 141Pr Anti-mouse CD11c N418 142Nd Anti-mouse Ly6C H1. IMC technology uses cytometry by time-of-flight (on which CyTOF™ systems are based) to overcome the multiplexing limitations of traditional immunohistochemistry (IHC) and immunofluorescence. cells were frozen at −80 °C Nov 29, 2023 · These features allow for the measurement of more than 50 parameters simultaneously without the need to compensate between different metal-coupled antibodies. g. In a CyTOF mass cytometer, the signal intensity is lower for channels in the high end and low end of the mass window of the detector, while the maximum sensitivity is in the upper-middle range. List of antibodies used in the Cytof panel. Imaging Mass Cytometry technology generates high-dimensional spatial data at subcellular resolution. 2 Biolegend 107637 142Nd B220 B220 CD279 CD279 29F. panel. 4 162Dy Anti-mouse CD45 30-F11 147Sm Anti-mouse CD11b M1/70 148Nd Anti-mouse B220 RA3-6B2 176Yb Anti-mouse CD25 3C7 151Eu Anti-mouse CD3 145-2C11 165Ho Antibody titration is the first step in any panel optimization and needs to be conducted before using the reagents in a multicolor experiment. When designing a panel, one must consider several things, including antigen abundance, sensitivity of the system to certain probes, sources of background signal, and measurement of probe signal in undesired channels (crosstalk). The Human Cell Functional State IMC™ Panel, 5 Antibodies assesses the functional state of cells. Based on the expression pattern of this panel, CD8 + T cells are stratified into subpopulations with distinct metabolic profiles. Thirty-seven antibody targets (Supplementary Table 1) directed against cell surface and intracellular proteins were initially designed using the web-based panel designer Dec 6, 2023 · The drawback is that this technique utilizes a pre-defined antibody panel and hence requires a priori knowledge. The limit remains the number of available pure metal isotopes [ 7 ]. 1A12 UBC Antibody Lab To extend the antibody panel, metals and conjugation kits are commercially available for in-house conjugation of antibodies. CellCarta has knowledge and expertise in custom antibody conjugation and can build a CyTOF panel starting with purified, unconjugated antibodies, or custom reagents. Recently, Winkels's group demonstrated the phenotypic diversity of leukocytes from aortas of healthy and atherosclerotic mice via single‐cell RNA sequencing (scSeq) and CyTOF with a panel of 35 markers. In this webinar, we describe our development and optimization of a 37-marker lyophilized antibody panel to profile whole blood samples in a multi-site pediatric allergy immune monitoring study. Aug 12, 2024 · CyTOF Panel (9) PN 201341B/C. However, optimizing a panel that both explores the desired markers and accounts for isotope spillover and varying degrees of antibody signal intensity often requires a customized panel. 5 uL of FACS into well #1,and 75uL into wells #2-#8; Antibody volume to add: add 2. Maxpar® Panel Designer v2. Design panels using IHC-validated antibodies conjugated to metal tags. The internal reference cells were easily resolvable from the experimental sample based on Ta181 signal, and viSNE analysis of the lyophilized PBMC sample allowed clear identification of major immune subsets that can be used to evaluate the consistency of marker staining across samples Jan 25, 2018 · Mass cytometry, or CyTOF (cytometry by time-of-flight), uses metal-conjugated antibodies to enable a high-dimensional description of tens of markers at the single-cell level, thereby providing a Part 2. A similar process was followed for antibodies CyTOF Panel Optimization. 4. No intracellular antibodies are used. Easily characterize breadth and depth of immune cell subpopulations to gain deeper insights into complex biology. a, Impact of anti-CD45 PerCP-Cy5. The most widely available CyTOF, the Helios, is capable of detecting ions atomic masses from 75 to 209 Da . The current selected mass window in AW 103–203 Dec 19, 2019 · Mixed samples were equally divided and stained with two panels (Panel A and B, Supplementary Tables 2 and 3) of metal-conjugated antibodies and acquired on the CyTOF instrument. anti-FITC) that can be used with existing fluorescent reagents; CyTOF Antibody Panel Guide Feb 17, 2022 · The samples were collected on two different CyTOF instruments using three antibody panels with slight variations in naming conventions, the number of proteins measured, and protein-metal labels Sample multiplexing with Maxpar® cell barcoding reagents for CyTOF® Sample-specific cell barcoding for simplified workflows and improved staining consistency . Pre-conjugated antibodies are available from Fluidigm and from the BWH Antibody Core. 1 Apoe −/− mice. conjugated antibodies. et al. Mass cytometry, or CyTOF, is a useful technology for high-parameter single-cell phenotyping, especially from suspension cells such as blood or PBMC. 1. 5 uL of fluorophore conjugated antibody to well #1 (containing 122. Following design & conjugation, all antibodies are validated on the CyTOF with a standard staining procedure. It should also be noted that CyTOF does not have the ability to discriminate cell size (FSC and SSC in flow cytometry), so all cells must have antibodies to define them. In total, 80 antibodies were tested, 43 of which performed well on frozen sections. By relying on time-of-flight mass spectrometry, this technique circumvents the limitations that spectral overlap places on conventional flow cytometry. 7 Release Notes | 400314; Custom Channel Filtering with CyTOF Software v6. The polymer terminates with a thiol or a maleimide that links it to reduced disulfides in the Fc region of the antibody. harvard. Baseline characteristics of DSAPOS recipients and their donors, stratified by development of ABMR. Panel design: 40+ channels available in CyTOF, increasing flexibility and ease in panel design. Scalable: Combine 35-plus samples at once to run larger experiments. 3 Image biomarkers using precise laser-directed protein capture and detection with CyTOF technology. DREMI-derived features are in principle easier to interpret in a biological context since they consider how information is transmitted between cells Apr 28, 2020 · By virtue of CyTOF, we differentiated CD45 + PBMCs into 31 clusters using metal-labeled antibodies (Supplementary Table 2) against 18 immune cell surface markers and observed obvious differences Apr 1, 2022 · To develop an antibody panel that could extensively characterize the melanoma microenvironment, we selected antibodies commonly used for immunohistochemistry and immunofluorescence applications, including clinically used clones (e. It is particularly appealing to monitor the systemic immune changes that could accompany cancer immunotherapy. However, wide deployment of CyTOF-based immune phenotyping studies are limited by complex experimental workflows and … Jan 26, 2023 · Advantage: large panels, distinct signals with low crossover, single tube multi-sample multi-plex, simple panel reconfigurations; Assay Support: broad array of commercially available antibodies as well as proprietary antibodies, amenable to formalin-fixed, paraffin-embedded or frozen tissue sections; Content. (2016) 9: 68. Jul 31, 2023 · We constructed and validated a comprehensive CyTOF antibody panel to include markers for both cell identity and senescent phenotype (Table 1). Start Designing Panels Phospho-CyTOF: Our 30-antibody phospho-CyTOF panel can be used with the indicated stimuli to read out a broad array of signaling pathways in many different cell subsets. Human TBMNK+G CyTOF Panel, 9 Antibodies: 201338B/C † Human Broad Immune Profiling CyTOF Panel, 20 Antibodies: 201339B/C: Maxpar® Cell Surface Staining Kit: 201601: Maxpar Cytoplasmic/Secreted Antigen Staining Kit: 201602: Maxpar Nuclear Antigen Staining Kit: 201603: Maxpar Phosphoprotein Staining Kit: 201604 Antibody conjugation for CyTOF is mostly standardized and follows an optimized protocol through Fluidigm. Cell identify markers Antibodies Clone Metal lab . Jul 31, 2023 · We next applied our CyTOF antibody panel to freshly isolated bone and marrow mesenchymal cells from INK-ATTAC mice: a transgenic model with an inducible caspase 8 cassette driven by the p16 Ink4a Sep 10, 2019 · Tantalum-labeled lyophilized reference PBMC were spiked into an experimental sample prior to staining with a CyTOF antibody panel. ” a repository of well-characterized metal conjugated antibodies; metal labeling reagents for conjugation of antibodies to these metals: 145Nd, 148Nd, 158Gd, 160 Gd, 164Dy, 165Ho, 171Yb, 173Yb. A defining characteristic of senescent cells is expression of cell cycle inhibitors, in particular p16 or p21 29, so we carefully Left: list of antibody panel used for CyTOF with conjugated unique metal isotopes. Pre-mixed dry antibody panels offer significant advantages over a conventional liquid antibody for flow cytometry workflows by significantly reducing the processing time, technical variation and potential errors associated with pipetting multiple individual antibodies, and these advantages are Jun 26, 2020 · deployment of CyTOF-based immune phenotyping studies are limited by complex experimental workflows and the need for specialized CyTOF equipment and technical expertise. Subsequently, antibodies directed against secondary and tertiary antigens can be titrated within the combined antibody panel, in the presence of the antibodies already optimally titrated against primary antigens. Due to the limited availability of antibodies, identical clones could only be allocated to the same antibody for part of the panel. They can also be accessed when logged into the panel design website. Figure 1. ” Figure 1. Catalog #s: MAB197, MAB170 Resources Panel Design Panel Design. The high end and the low end of the mass window have lower signal intensities compared to the middle of the mass range. We analyzed blood from healthy donors and melanoma patients with our CyTOF-antibody panel and identified subset N1 by gating on Lin − CD66b + CD16 − CD10 − CD71 + cells (Figures 7A and 7B). Many of these markers were taken from a standard fluorescence phenotyping panel (Maecker et al. Because CyTOF signals do not rely on fluorescence and thus are extremely discrete, the result is negligible signal overlap with no need for additional color validations and controls. The Maxpar® Direct™ Immune Profiling Assay™ is a dry-format, ready-to-go, validated 30-marker antibody panel for whole blood As for any type of fluorescent antibody panel, design of a high-dimensional panel requires the ability to distinguish all levels of Ag expression, including low expression. As various immune cell types are identified and distinguished based on their cell surface markers, CyTOF permits the Table S1 CyTOF antibody Panel . Nov 1, 2022 · The main workflow of IMC is composed of four steps: antibody panel design, staining, imaging and analysis. This panel includes 5 pathologist-verified antibodies that enable assessment of cell proliferation, cytotoxic T cell activation, T cell exhaustion, and identification of regulatory T cells . and Max-Par antibody conjugation kits to conjugate your own antibodies. Pre-mixed dry antibody panels offer significant advantages over a conventional liquid antibody for flow cytometry workflows by To avoid significant spillover from critical markers in the pre-processing and de-barcoding stage, we recommend choosing the appropriate Cell-ID™ based on the surface/intracellular antibody panel. Panel Designer calculates signal overlap and utilizes a specialized algorithm to adjust metal-tagged targets to maximize performance. The bottom sections show the biaxial contour plots of the indicated isotopes. The mass cytometry panel required five custom antibody conjugations: CD8a, α4 integrin, αE integrin, β7 integrin, and CD1c. See full list on ledererlab. v9. (CyTOF) using 34 antibody targets to characterize the integrated landscape of single-cell cell surface and intracellular signaling intracellular antibody panel, fixed with a fresh 1. 1 2 Nominal (in silico) Time required with a CyTOF antibody panel. This kit is provided with an optimized protocol (See ref 10 of Table 1 and Note 4) based on a procedure enabling to first chelate The final choice of antibodies relied upon the availability of high-quality antibody clones. Dec 31, 2019 · After staining with these tagged antibodies, single-cell suspensions are introduced into the CyTOF analyser where they are first nebulized into droplets and subsequently introduced into an ICP Oct 1, 2022 · In general, antibody panel design in mass cytometry, although more flexible, follows similar rules to flow cytometry. The internal reference cells were easily resolvable from the experimental sample based on Ta181 signal, and viSNE analysis of the lyophilized PBMC sample allowed clear identification of major immune subsets that can be used to evaluate the consistency of marker Successful large-scale CyTOF immune monitoring studies require several important considerations, ranging from panel design to data analysis. Supplementary Table 3 details the antibodies used for mfIHC. 15. Aug 23, 2022 · For CyTOF-based immunophenotyping studies, Selvanantham suggests starting with the Standard BioTools Maxpar® Direct™ Immune Profiling Assay™, a dry-format, single-tube assay measuring 37 cell populations from a 30-marker antibody panel. Cell staining with these CyTOF antibodies show excellent correlations in cross validation studies performed with fluorescently labeled antibodies analyzed by flow cytometry (Figure 2). Jun 26, 2020 · Mass cytometry (CyTOF) represents one of the most powerful tools in immune phenotyping, allowing high throughput quantification of over 40 single parameters at single-cell resolution. Fluidigm also provides pre-designed panel kits, sample prep reagents, barcoding kits. mxfjesb yvyyyw plnzb agdby aubs hiypti egz fwvwsyr ykiapgfn mtykvx