Resources
- DNA Content for Cell Cycle Analysis of Fixed Cells With Propidium Iodide
- Apoptosis Assays (Sub Diploid, Annexin V, TUNNEL, Caspase Activity)
- Simultaneous analysis of DNA content and surface immunophenotype using gentle ethanol fixation techniques.
- CFSE Labeling
- Intracellular Cytokine Staining Protocol
- Measurement of GFP Expression and DNA Content in Permeabilized Cells
- 7-AAD Staining of Dead Cells
- Critical Aspects of Staining Cells for Flow Cytometry
- Preparation of 2% Formaldehyde Solution
- Fluorochrome Conjugation of Monoclonal Antibodies
- Staining Protocol for the Cell Permeant DNA Dye DRAQ5
- Reducing Cellular Autofluorescence
- Dead Cell Discrimination by EMA
- Protocol for LPS Activation of MAPK Signaling in Whole Blood Samples
- Protocol for Antibody Titration
- Mass Cytometry Methods and Protocols
- Immunophenotyping of Human Peripheral Blood Mononuclear Cells by Mass Cytometry (Heck S, Bishop CJ, Ellis RJ
Methods Mol Biol. 2019, 1979, 285-303)
- Preparing Viable Single Cells from Human Tissue and Tumors for Cytomic Analysis (Leelatian N, Doxie DB, Greenplate AR, Sinnaeve J, Ihrie RA, Irish JM. Curr Protoc Mol Biol. 2017)
- Phenotyping of Live Human PBMC using CyTOF Mass Cytometry (Leipold and Maecker 2015)
- Intracellular Cytokine Staining on PBMCs Using CyTOF Mass Cytometry (Lin, Gupta, and Maecker 2015)
- Multiparameter Phenotyping of Human PBMCs Using Mass Cytometry
- A Deep Profiler’s Guide to Cytometry
- Mass Cytometry Selected Publications
- DVS Sciences – Metal-Conjugated Antibodies (anti-Human)
- Panel Design Guidelines
- Experiment Planning Worksheet
- Cell Surface Staining Protocol
- Cytoplasmic Staining Protocol
- Intercalator Ir-125 Protocol
- Intercalator Rh500 uM (Live Dead) Protocol
- Phospho-Protein Staining Protocol
- Nolan Lab Signaling based Resource (Fluorescence & Mass)
- BD Buffer/Fixative Resource
NIH Recommendations for Rigorous Resources for Rigorous Research
ScientificScientific Premise in NIH Grant Applications
Enhancing Reproducibility through Rigor and Transparency
Clearinghouse for Training Modules to Enhance Data Reproducibility
Click here for training modules
Below are links to Optimized Multicolor Immunophenotyping Panels (OMIPs). These panels have been validated and published and are a useful resource for developing immunophenotyping panels for a variety of applications.
The following article contains background and explanation as the development of the OMIPs as a separate type of publication within the Cytometry Part A journal:
http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.20916/pdf
Below are links to the publications for established OMIPs thus far:
OMIP-001: Quality and Phenotype of Ag-Responsive Human T-Cells
OMIP-002: Phenotypic Analysis of Specific Human CD8+ T-Cells Using Peptide-MHC Class I Multimers for Any of Four Epitopes
OMIP-003: Phenotypic Analysis of Human Memory B Cells
OMIP-004: In-Depth Characterization of Human T Regulatory Cells
OMIP-005: Quality and Phenotype of Antigen-Responsive Rhesus Macaque T Cells
OMIP-006: Phenotypic Subset Analysis of Human T Regulatory Cells via Polychromatic Flow Cytometry
OMIP-007: Phenotypic Analysis of Human Natural Killer Cells
OMIP-008: Measurement of Th1 and Th2 Cytokine Polyfunctionality of Human T Cells
OMIP-009: Characterization of Antigen-Specific Human T-Cells
OMIP-010: A New 10-Color Monoclonal Antibody Panel for Polychromatic Immunophenotyping of Small Hematopoietic Cell Samples
OMIP-011: Characterization of Circulating Endothelial Cells (CECs) in Peripheral Blood
OMIP-012: Phenotypic and Numeric Determination of Human Leukocyte Reconstitution in Humanized Mice
OMIP-013: Differentiation of Human T-Cells
OMIP-014: Validated Multifunctional Characterization of Antigen-Specific Human T Cells by Intracellular Cytokine Staining
OMIP-015: Human Regulatory and Activated T-Cells Without Intracellular Staining
OMIP-016: Characterization of Antigen-Responsive Macaque and Human T-cells
OMIP-017: Human CD41 Helper T-cell Subsets Including Follicular Helper Cells
OMIP-018: Chemokine Receptor Expression on Human T Helper Cells
OMIP-019: Quantification of Human cdT-Cells, iNKT-Cells, and Hematopoietic Precursors
OMIP-020: Phenotypic Characterization of Human cd T-cells by Multicolor Flow Cytometry
OMIP-021: Simultaneous Quantification of Human Conventional and Innate-Like T-Cell Subsets
OMIP-022: Comprehensive Assessment of Antigen-Specific Human T-Cell Functionality and Memory
OMIP-023: 10-Color, 13 Antibody Panel for In-depth Phenotyping of Human Peripheral Blood Leukocytes
OMIP-024: Pan-Leukocyte Immunophenotypic Characterization of PBMC Subsets in Human Samples
OMIP-025: Evaluation of Human T- and NK-Cell Responses Including Memory and Follicular Helper Phenotype by Intracellular Cytokine Staining
OMIP-026: Phenotypic Analysis of B and Plasma Cells in Rhesus Macaques
OMIP-027: Functional analysis of human natural killer cells
OMIP-028: Activation Panel for Rhesus Macaque NK Cell Subsets
OMIP-029: Human NK-Cell Phenotypization
OMIP-030: Characterization of Human T Cell Subsets via Surface Markers
OMIP-031: Immunologic Checkpoint Expression on Murine Effector and Memory T-Cell Subsets
OMIP-032: Two Multi-Color Immunophenotyping Panels for Assessing the Innate and Adaptive Immune Cells in the Mouse Mammary Gland
OMIP-033: A Comprehensive Single Step Staining Protocol for Human T- and B-Cell Subsets
OMIP-034: Comprehensive Immune Phenotyping of Human Peripheral Leukocytes by Mass Cytometry for Monitoring Immunomodulatory Therapies
OMIP-035: Functional Analysis of Natural Killer Cell Subsets in Macaques
OMIP-036: Co-inhibitory Receptor (Immune Checkpoint) Expression Analysis in Human T Cell Subsets
OMIP-037: 16-Color Panel to Measure Inhibitory Receptor Signatures from Multiple Human Immune Cell Subsets
OMIP-038: Innate Immune Assessment with a 14 Color Flow Cytometry Panel
OMIP-039: Detection and Analysis of Human Adaptive NKG2C+ Natural Killer Cells
OMIP-040: Optimized Gating of Human Prostate Cellular Subpopulations
OMIP-041: Optimized Multicolor Immunofluorescence Panel Rat Microglial Staining Protocol
OMIP-042: 21-Color Flow Cytometry to Comprehensively Immunophenotype Major Lymphocyte and Myeloid Subsets in Human Peripheral Blood
OMIP-043: Identification of Human Antibody Secreting Cell Subsets
OMIP-044: 28-Color Immunophenotyping of the Human Dendritic Cell Compartment
OMIP-045: Characterizing Human Head and Neck Tumors and Cancer Cell Lines with Mass Cytometry
OMIP-046: Characterization of Invariant T Cell Subset Activation in Humans
OMIP-047: High-Dimensional Phenotypic Characterization of B Cells
OMIP-048 MC: Quantification of Calcium Sensors and Channels Expression in Lymphocyte Subsets by Mass Cytometry
OMIP-049: Analysis of Human Myelopoiesis and Myeloid Neoplasms
OMIP-050: A 28-color/30-parameter Fluorescence Flow Cytometry Panel to Enumerate and Characterize Cells Expressing a Wide Array of Immune Checkpoint Molecules
OMIP-051 – 28-Color Flow Cytometry Panel to Characterize B Cells and Myeloid Cells
OMIP-052: An 18-Color Panel for Measuring Th1, Th2, Th17, and Tfh Responses in Rhesus Macaques
OMIP-053: Identification, Classification, and Isolation of Major FoxP3 Expressing Human CD4+ Treg Subsets
OMIP-054: Broad Immune Phenotyping of Innate and Adaptive Leukocytes in the Brain, Spleen, and Bone Marrow of an Orthotopic Murine Glioblastoma Model by Mass Cytometry
OMIP-055: Characterization of Human Innate Lymphoid Cells from Neonatal and Peripheral Blood
OMIP-056: Evaluation of Human Conventional T Cells, Donor-Unrestricted T Cells, and NK Cells Including Memory Phenotype by Intracellular Cytokine Staining
OMIP-057: Mouse γδ T-Cell Development Characterized by a 14 Color Flow Cytometry Panel
OMIP-058: 30-Parameter Flow Cytometry Panel to Characterize iNKT, NK, Unconventional and Conventional T Cells
OMIP-059: Identification of Mouse Hematopoietic Stem and Progenitor Cells with Simultaneous Detection of CD45.1/2 and Controllable Green Fluorescent Protein Expression by a Single Staining Panel
OMIP-060: 30-Parameter Flow Cytometry Panel to Assess T Cell Effector Functions and Regulatory T Cells
OMIP-061: 20-Color Flow Cytometry Panel for High-Dimensional Characterization of Murine Antigen-Presenting Cells
OMIP-062: A 14-Color, 16-Antibody Panel for Immunophenotyping Human Innate Lymphoid, Myeloid and T Cells in Small Volumes of Whole Blood and Pediatric Airway Samples
OMIP-063: 28-Color Flow Cytometry Panel for Broad Human Immunophenotyping
OMIP-064: A 27-Color Flow Cytometry Panel to Detect and Characterize Human NK Cells and Other Innate Lymphoid Cell Subsets, MAIT Cells, and γδ T Cells
OMIP-065: Dog Immunophenotyping and T-Cell Activity Evaluation with a 14-Color Panel
OMIP-066: Identification of Novel Subpopulations of Human Group 2 Innate Lymphoid Cells in Peripheral Blood
OMIP-067: 28-Color Flow Cytometry Panel to Evaluate Human T-Cell Phenotype and Function
OMIP-068: High-Dimensional Characterization of Global and Antigen-Specific B Cells in Chronic Infection
OMIP-069: Forty-Color Full Spectrum Flow Cytometry Panel for Deep Immunophenotyping of Major Cell Subsets in Human Peripheral Blood
OMIP-070: NKp46-Based 27-Color Phenotyping to Define Natural Killer Cells Isolated From Human Tumor Tissues
OMIP-083: A 21-marker 18-color flow cytometry panel for in-depth phenotyping of human peripheral monocytes
Useful Links
FlowRepository.org is a Web-based flow cytometry data repository provided by the International Society for Advancement of Cytometry (ISAC). It supports storage, annotation, analysis, and sharing of flow cytometry datasets. A fundamental tenet of scientific research is that published results should be open to independent validation and refutation. With FlowRepository, researchers can annotate their datasets in compliance with the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard, thus greatly facilitating third-party interpretation of their data.
Tools and Tutorials
- BenchSci
- Current Protocols in Cytometry
- FCS Express Tutorial Videos
- Fluorofinder 2.0 Flow Cytometry Panel Builder
- Purdue University Cytometry Laboratories
- Antibody Resource Page
- Intracellular Cytokine Staining Protocol
- Counting Statistics – How Many Cells Do I Collect?
- Practical Flow Cytometry (4th edition-Free Online Viewing)
- Beginner Flow Cytometry Book (Flow Cytometry A basic introduction)
Flow Cytometry Vendor Links
- BD Biosciences (reagents) Susan Gigler (susan.gigler@bd.com)
- Beckman Coulter (reagents)
- BioLegend (reagents) Hank Beggs (hbeggs@biolegend.com)
- Bio-Techne Ty Vaivoda (bulk@novusbio.com)
- Cytek Biosciences (reagents)
- Luminex (Amnis)
- Miltenyi Biotec (reagents) Christina Stillwell (christinas@miltenyi.com)
- Phoenix Flow (Accutase/reagents/software)
- Spherotech (Beads)
- Thermo Fisher Scientific (Caltag Reagents, Molecular Probes Reagents, Quantum Dot Reagents, Dynal Beads) Alycia Curlee (alycia.curlee@thermofisher.com)