Below are some topics that should be considered when designing flow experiments.
Fluorescent dyes: Multi-parameter flow cytometry is made possible because there are dyes that are excited at different wavelengths or emit light at different wavelengths. This information about dyes is described by their excitation and emission spectra. Knowledge of these spectra is useful when designing staining panels and is useful for understanding the potential interactions between reagents.
There are several useful web resources that display spectra.
Becton Dickinson offers this spectral viewer: http://www.bdbiosciences.com/research/multicolor/spectrum_viewer/index.jsp
Invitrogen offers this spectral viewer: http://www.lifetechnologies.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html
Choice of reagents: Typically, monoclonal antibodies are used to identify cell markers and these must be either conjugated to a fluorochrome (often referred to as a dye) or to biotin, which is then detected by a fluorochrome-conjugated streptavidin. Often the same monoclonal antibody is conjugated to several different dyes and therefore there are multiple options to choose from when designing a staining panel. The choice of reagent to use in any particular panel can be determined by a combination of theoretical and empirical considerations. Based on our experience, we can provide guidance in choosing reagents. In addition, we can provide samples of reagents that we have in our large inventory.
Some antibody-dye conjugates are not available commercially. In particular, quantum dots are currently only available as streptavidin conjugates or as kits for in-laboratory conjugation. We have considerable experience with conjugations and can provide guidance. We can also provide small samples of these conjugates for testing.
Cell surface marker expression: In our laboratory, we focus on examination of T cell subsets, and multiple markers can be used to precisely define numerous cellular subpopulations. When determining which combination of markers to use in any individual staining panel, we start by choosing markers that identify the major lineage. In our panels these are CD3, CD4 and CD8. Then we add any other markers of interest.
Intracellular surface marker expression: Markers expressed inside the cell can be labeled by permeabilizing cells before staining. There are several different protocols for fixation and permeabilization. Intracellular staining is often used to identify cytokine-producing cells following a period of in vitro stimulation with various stimulants. This assay is often referred to as intracellular cytokine staining (ICS). We have had considerable experience using ICS to detect vaccine-induced responses. We recently validated an 8-color assay and this is described in the Journal of Immunological Methods paper referenced below.
Other functional FACS assays: FACS has the advantage of examining cells at the single cell level as opposed to in bulk. As indicated above, ICS is a method of determining cytokine production. There are several other functional FACS assays. CFSE staining examines proliferation. CD107 examines degranulation, which may be a correlate of cytotoxic potential. Other functional assays have been described in the literature. Many of the reagents used for these assays are sold by Molecular Probes/Invitrogen and the Molecular Probes Handbook is a useful resource for information about these assays and reagents. http://probes.invitrogen.com/handbook/
Sorting: The experiments referred to above analyze cells but do not sort cells. Some FACS instruments can also sort cells. The more advanced cell sorters, such as the BD Aria can sort based on multiple parameters (8 to 12). For specific technical information concerning sorting capabilities, contact one of the local flow cytometry shared facilities.
Fluorescence compensation: This is an advanced topic, but a minimal amount of knowledge is needed especially for experiments using 5 or more colors. There are several useful resources for learning more about compensation. Mario Roederer, a leading FACS expert, has written about this topic (see references below) and has useful information on a website. http://www.drmr.com/
Protocols: Note that these are protocols used by the HIV Vaccine Trials Network (HVTN) for the specific assays and procedures developed by the HVTN. The HVTN protocols would need to be modified for other applications.
The HVTN-general-ICS-SOP.pdf describes the ICS procedure, but does not include any of the details that are specific for the particular HVTN trial. The general ICS SOP is used in conjunction with a study specific procedure that includes specifics of the natibody reagents and stimulation reagents. The HVTN-ICS-study-specific.pdf is an example of what can be used to include the details of the particular HVTN trial and is used in conjunction with the general ICS.
The HVTN-LSRII-SOP.pdf is the procedure used for collection of the samples using the Becton Dickinson High Throughput Sampler (HTS) and the LSRII. This procedure makes use of a predefined LSRII collection template within the DiVa software. This template is not described and is only relevant for the plate layout used by the HVTN.
The HVTN-Thawing-PBMC-Samples-SOP.pdf is the procedure used for thawing cryopreserved peripheral blood mononuclear cells (PBMC).
The HVTN-Reagent-Titration-SOP.pdf is the procedure for titrating new lots of fluorochrome-conjugated antibody reagents and the cell viability marker for the use in the ICS assay.
The CFSE Proliferation Assay SOP.pdf describes the procedure for running the CFSE proliferation assay.
The following are links to local flow cytometry shared facilities:
The flow facility at the Fred Hutch http://sharedresources.fhcrc.org/core-facilities/flow-cytometry
University of Washington Department of Immunology Cell Analysis Facility http://depts.washington.edu/flowlab/
Other useful links:
Purdue University Cytometry Laboratories, host of the popular cytometry email list
The following two references are particularly useful as educational tools concerning advanced flow cytometry technology:
Multicolor flow cytometry reference including the description of the fluorescence-minus-one (FMO) control
Baumgarth, N. and Roederer, M. (2000) A practical approach to multicolor flow cytometry for immunophenotyping. J Immunol Methods 243, 77-97.
Practical and theoretical discussion of compensation
Roederer, M. (2001) Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry 45, 194-205.
Several chapters in the "Handbook of experimental immunology" provide general information on several topics related to multicolor flow. Those authored by members of the Herzenberg laboratory at Stanford University are available at this website: http://www.herzenberglab.com/Publications/publications.html
Relevant references from our group:
Multicolor flow described in "new technology" section with focus on naive/memory identification
De Rosa, S.C., Brenchley, J.M. and Roederer, M. (2003) Beyond six colors: a new era in flow cytometry. Nat Med 9, 112-7.
A discussion of usefulness of >6-color flow cytometry
Roederer, M., Brenchley, J.M., Betts, M.R. and De Rosa, S.C. (2004) Flow cytometric analysis of vaccine responses: how many colors are enough? Clin Immunol 110, 199-205.
A discussion of choice of reagents and planning multicolor experiments:
De Rosa, S.C. (2004) Multicolor Immunophenotyping: Human Mature Immune System. In: Z. Darzynkiewicz, M. Roederer and H.J. Tanke (Eds), Methods in Cell Biology, Vol. 75. Elsevier Academic Press, London, p. 577-594.
Available as a reprint from the core director
Quantum dots used in multicolor flow
Chattopadhyay, P.K., Price, D.A., Harper, T.F., Betts, M.R., Yu, J., Gostick, E., Perfetto, S.P., Goepfert, P., Koup, R.A., De Rosa, S.C., Bruchez, M.P. and Roederer, M. (2006) Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry. Nat Med 12, 972-7.
Validation of an ICS assay for vaccine evaluation
Horton, H., Thomas, E.P., Stucky, J.A., Frank, I., Moodie, Z., Huang, Y., Chiu, Y.L., McElrath, M.J. and De Rosa, S.C. (2007) Optimization and validation of an 8-color intracellular cytokine staining (ICS) assay to quantify antigen-specific T cells induced by vaccination. J Immunol Methods 323, 39-54.