Protocols

References, protocols, and related reviews listed below.

Additional details of validation procedures, laboratory protocols, cell tracking and viability reagents, barcoding, bead normalization, and other details can be made available upon request for CIC users, collaborators, and sponsors.

Mass cytometry reviews and perspectives:

  • Mass cytometry:
    • EW Newell, Y Cheng.  Mass cytometry: blessed with the curse of dimensionality.  Nature Immunology 2016. DOI.
    • MH Spitzer, GP Nolan.  Mass cytometry: Single cells, many features. Cell 2016. DOI.
  • Comparison of single cell technologies, including mass cytometry:
    • Chattopadhyay PK, Gierahn TM, Roederer M, Love JC. Single-cell technologies for monitoring immune systems. Nature Immunology 2016. DOI.
  • Revealing ‘cyto incognito’, hidden and overlooked cells:
    • Irish JM. Beyond the Age of Cellular Discovery. Nature Immunology 2014 Nov 14. Pubmed. DOI.
  • Clinical applications of Systems Immune Monitoring (SIM) with mass cytometry:
    • Greenplate AR, Johnson DB, Ferrell PB, Irish JM. Systems Immune Monitoring in Cancer Therapy.  European Journal of Cancer NIHMS776476.  Pubmed.  DOI.
  • Design of human myeloid mass cytometry panels for MDSC, M1/M2, and TAMs:
    • Roussel M, Greenplate AG, Irish JM.  Dissecting complex cellular systems with mass cytometry. Human Innate Immunity. Experimental Approaches for the Investigation of Innate Immunity 2016. DOI.

Tissue preparation and viable cell cryopreservation methods:

  • Viable cells from human blood, PBMC Ficoll and cryopreservation protocol:
    • Leelatian N, Diggins KE, Irish JM. Characterizing phenotypes and signaling networks of single human cells by mass cytometry. Methods in Molecular Biology 2015, 1346:99-113. PMC4656023. Pubmed. DOI.
  • Viable cells from human solid tumors and tissues:
    • Leelatian N*, Doxie DB*, Greenplate AR, Lehman JM, Sinnaeve J, Mobley BC, Kauffmann RM, Werkhaven JA, Mistry AM, Weaver KD, Thompson RC, Massion PP, Hooks MA, Kelley MC, Chambless LB, Ihrie RA, Irish JM. Single Cell Analysis of Human Tissues and Solid Tumors with Mass Cytometry. Cytometry B. PMCID in progress. Pubmed. DOI.

Staining methods:

  • Mass cytometry surface staining & mass cytometry phospho-flow protocols:
    • Leelatian N, Diggins KE, Irish JM. Characterizing phenotypes and signaling networks of single human cells by mass cytometry. Methods in Molecular Biology 2015, 1346:99-113. PMC4656023. Pubmed. DOI.
  • Solid tumor mass cytometry & extensive tissue disaggregation testing for mass cytometry:
    • Leelatian N*, Doxie DB*, Greenplate AR, Lehman JM, Sinnaeve J, Mobley BC, Kauffmann RM, Werkhaven JA, Mistry AM, Weaver KD, Thompson RC, Massion PP, Hooks MA, Kelley MC, Chambless LB, Ihrie RA, Irish JM. Single Cell Analysis of Human Tissues and Solid Tumors with Mass Cytometry. Cytometry B. PMCID in progress. Pubmed. DOI.

Data analysis methods:

  • Data annotation, sharing, and analysis:
    • Kotecha N, Krutzik PO, Irish JM. Web-based Analysis and Publication of Flow Cytometry Experiments.  Current Protocols in Cytometry 2010 Jul, Chapter 10, Unit10.17.  PMCID: PMC4208272. Pubmed. DOI.
  • viSNE:
    • Amir el-AD1, Davis KL, Tadmor MD, Simonds EF, Levine JH, Bendall SC, Shenfeld DK, Krishnaswamy S, Nolan GP, Pe’er D. viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nature Biotechnology 2013. DOI.
  • Modular bioinformatics workflow:
    • Diggins KE, Ferrell PB, Irish JM. Methods for discovery and characterization of cell subsets in high dimensional mass cytometry data. Methods PMCID: PMC4468028. Pubmed. DOI.
  • MEM:
    • Diggins KE, Greenplate AR, Leelatian N, Wogsland CE, Irish JM. Characterizing cell subsets using marker enrichment modeling. Nature Methods PMCID: in progress. Pubmed. DOI.

Panel development references (CIC Panels):

  • CIC-001 (Human B cell emphasis) & CIC-002 (Human T cell emphasis):
    • Validation work with these panels in graft vs. host disease (GVHD): Polikowsky HG, Byrne MT, Maier CM, Greenplate AR, Jagasia M, Irish JM. Systems Immune Monitoring with Mass Cytometry Characterizes Altered Peripheral Immune Cell Environments in Patients with Chronic Graft Versus Host Disease. Blood ASH Abstracts 2016 DOI.
  • CIC-002 (Human T cell emphasis) and CIC-003 (Human T cell emphasis):
    • Mass vs. fluorescence comparison for many clones: Nicholas KJ, Greenplate AR, Flaherty DK, Matlock BK, San Juan J, Smith RM, Irish JM, Kalams SA. Multiparameter analysis of stimulated human peripheral blood mononuclear cells: a comparison of mass and fluorescence cytometry. Cytometry 2015 PMCID: PMC4808335.  Pubmed. DOI.
    • Initial systems immune monitoring panel with a T cell emphasis: Greenplate AR, Johnson DB, Roussel M, Savona MR, Sosman JA, Puzanov I, Ferrell PB*, Irish JM*.  Myelodysplastic Syndrome Revealed by Systems Immunology in a Melanoma Patient Undergoing Anti-PD-1 Therapy, Cancer Immunology Research 2016. NIHMS774078. PMCID in progress. Pubmed. DOI.
  • CIC-004 (Human myeloid cell emphasis):
    • Review of key myeloid cell features: Roussel M, Greenplate AG, Irish JM.  Dissecting complex cellular systems with mass cytometry. Human Innate Immunity. Experimental Approaches for the Investigation of Innate Immunity 2016. DOI.
    • Myeloid panel for acute myeloid leukemia (AML): Ferrell PB, Diggins KE, Polikowsky HG, Mohan SR, Seegmiller AC, Irish JM.  High-dimensional analysis of stem and progenitor cell phenotypes during acute myeloid leukemia therapy response. PLOS One 2016 Apr 13;11(4). PMCID: PMC4830605. Pubmed. DOI.