Maeckerlab Weblog

Welcome to the NEW Maeckerlab Weblog!

I've redesigned this weblog a bit:  I've posted a new set of protocols, and simplified the categories.  See the list at left to quickly find posts of interest to you.

I've now joined Stanford as director of their Human Immune Monitoring Center, which is part of the Institute for Immunity, Transplantation, and Infection.

If you'd like to learn more about my flow cytometry consulting services, please visit my consulting page.

Please e-mail me at [email protected] with any questions or comments.  Thanks for visiting!

New Links of Interest

Many of you may already know about MyCyte, a "hub" for information on all things to do with flow cytometry.  Sponsored by the makers of FlowJo software, MyCyte has a news and discussion forum, job board, event calendar, reagent database, and more.  They even have a link with abstracts from my blog!

My research group has worked with CytoAnalytics, a consulting firm run by Janet Siebert that specializes in complex data analysis.  Using a variety of statistical and visualization tools, Janet allows investigation of trends in data that might otherwise be difficult to gleen.  For examples related to cytokine signature profiling, see our paper in Cytometry 73A: 289-298.

Forms for Flow Cytometry

Here are some Excel spreadsheets that may be useful to you in your daily lab work:

Form for designing multicolor Ab panels:  Download Ab panel config sheet

(delete rows for colors you don't use or that don't apply to your cytometer)

Form for hemacytometer cell counting & cell dilutions:  Download Cell Calculation Form

(enter information in the clear cells; yellow cells are auto-calculated for you)

MIFlowCyt Templates

MIFlowCyt (Minimum Information about a Flow Cytometry Experiment) is a new ISAC-approved standard for what information should be included when publishing flow cytometry data (see Cytometry A, 2008, 73(10):926-30 and online supporting material).

The Flow Core at Baylor Institute of Immunology Research has several multicolor instruments for which I've created MIFlowCyt templates:

Download MIFloCyt-Aria 

Download MIFloCyt-Aria II 

Download MIFloCyt-Canto-upstairs 

Download MIFloCyt-Canto-downstairs 

Download MIFloCyt-LSRII 

You can use these templates, which contain all default instrument information, to fill in the remaining information pertinent to your experiment.  Note that the templates contain information specific to the BIIR instruments; your cytometer may be different, but you can still use these as a starting point.

Literature Updates

I'm no longer posting literature updates to this blog.  Old updates (March 2006 - April 2008) can be found here.

Instead, I'm writing for Faculty of 1000 Medicine, and I would recommend using their service to find articles of interest and reviews.  In fact, you can visit this page to see the articles that I've recently evalutated for Faculty of 1000 Medicine.

Another useful updating service is provided for free by AMEDEO, which provides weekly updates of abstracts from the medical literature.  Your updates are customized by topic and journals of interest.  Check it out!

New Flow Cytometry Protocols

Here are some new protocols designed to cover basic areas of flow cytometry.  These are intended as general guides, not specific SOPs, which you can generate according to your own needs.

Cryopreservation of PBMC:  Download Protocol-Cryopreservation.pdf

Cell-surface staining for flow cytometry:  Download protocolsurface_staining.pdf

Activation for functional assays:  Download Protocol-Activation.pdf

Intracellular staining:  Download protocolic_staining.pdf

Experiment setup (for BD digital instruments):  Download protocolexp_setup.pdf

NEW Experiment setup (for BD analog instruments):  Download protocolexp_setupanalog.pdf

Technical Presentations

Here are some powerpoint presentations of potential training interest:

Basics of flow cytometry:  Download 1basics_of_flow.ppt

Multicolor flow cytometry:  Download 2008_07_maeckerdavis.ppt

Immune function assays:  Download immune_function_assays.ppt

Multicolor ICS Working Group

The Multicolor ICS Working Group is an informal association of key multicolor flow labs that I brought together in order to discuss and help standardize multicolor ICS assays.  Members of the group are listed here:  Download multicolor_ics_working_grp_members.doc

The group's accomplishments to date include:

• A compilation of instrument configurations (lasers and filter sets) in use by the various labs:  Download multicolor_ics_working_group_filter_summary_110905.xls
• A summary of a survey ranking markers as to their importance for use in multicolor ICS:  Download ics_marker_surveysummary.doc
• A compilation of validated panels in use by the various labs for multicolor ICS, with comments:  Download multicolor_ics_working_group_panel_summary_022306.xls 

Detecting CD107 and IFNg

Many labs are measuring degranulation of T cells by cell-surface expression of CD107 (see Betts et al., J Immunol Methods 281: 65 (2003), Link to PubMed abstract), and it's often desirable to simultaneously detect intracellular cytokines, such as IFNg.  Unfortunately, optimization of both of these readouts in the same stimulation is not trivial.  I suggest the following tips:

1.  Stimulate cells in the presence of 5 ug/mL EACH of brefeldin A and monensin.  Monensin helps maximize the CD107 readout, whereas brefeldin A helps maximize the IFNg readout.  To acheive these final concentrations without addition of an excessive amount of solvent, dissolve monensin (Sigma #5273) at 5 mg/mL in methanol, then dilute 1:10 in PBS on the day of use, followed by 1:100 dilution into the culture medium.  Ditto with brefeldin A, using DMSO as the initial solvent (or use the FastImmune brefeldin A from BD [Link to product description], which comes as a 5 mg/mL stock in DMSO).

2.  Use antibody to CD107a (use of CD107b antibody adds only slightly to the signal) in PE [Link to product description] or PE-Cy5 [Link to product description], as these give the brightest signals.

3.  Add the antibody, at 10 uL per 200 uL of cell stimulation culture, at the beginning of the activation period, and activate for 5-6 hours.  Continue with processing for surface and intracellular staining as usual.

Recent Maecker Publications

Here are some recent publications with which I've been involved.  Open Access articles are linked to the full text article, others are linked to their PubMed abstract.  Technical papers on flow cytometry are bolded.

Maecker, H. T. 2009. Multiparameter flow cytometry monitoring of T cell responses. In Methods Mol Biol. 375-391.  Link to PubMed asbstract

Butterfield, L. H., M. L. Disis, B. A. Fox, P. P. Lee, S. N. Khleif, M. Thurin, G. Trinchieri, E. Wang, J. Wigginton, D. Chaussabel, G. Coukos, M. Dhodapkar, L. Hakansson, S. Janetzki, T. O. Kleen, J. Kirkwood, C. Maccalli, H. Maecker, M. Maio, A. Malyguine, G. Masucci, A. K. Palucka, D. M. Potter, A. Ribas, L. Rivoltini, D. Schendel, B. Seliger, S. Selvan, C. L. Slingluff, Jr., D. F. Stroncek, H. Streicher, X. Wu, B. Zeskind, Y. Zhao, M. B. Zocca, H. Zwierzina, and F. M. Marincola. 2008. A systematic approach to biomarker discovery; Preamble to "the iSBTc-FDA taskforce on Immunotherapy Biomarkers". Journal of translational medicine 6:81.  Link to full text

Nomura, L., V. C. Maino, and H. T. Maecker. 2008. Standardization and optimization of multiparameter intracellular cytokine staining. Cytometry A.  Link to PubMed abstract

Chen, C. I., H. T. Maecker, and P. P. Lee. 2008. Development and dynamics of robust T-cell responses to CML under imatinib treatment. Blood 111:5342-5349.  Link to PubMed abstract

Jacobson, M. A., Q. X. Tan, V. Girling, C. Poon, M. Van Natta, D. A. Jabs, M. Inokuma, H. T. Maecker, B. Bredt, and E. Sinclair. 2008. Poor predictive value of cytomegalovirus (CMV)-specific T cell assays for the development of CMV retinitis in patients with AIDS. Clin Infect Dis 46:458-466.  Link to PubMed abstract

Janetzki, S., K. S. Panageas, L. Ben-Porat, J. Boyer, C. M. Britten, T. M. Clay, M. Kalos, H. T. Maecker, P. Romero, J. Yuan, W. Martin Kast, and A. Hoos. 2008. Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI). Cancer Immunol Immunother 57:303-315.  Link to PubMed abstract

Maecker, H. T. 2008. Measuring human cytokines. In Handbook of Human Immunology. A. Donnenberg, and M. O'Gorman, eds. CRC Press, Boca Raton, FL. 517-540.  Link to CRC press description

Maecker, H. T., J. Hassler, J. K. Payne, A. Summers, K. Comatas, M. Ghanayem, M. A. Morse, T. M. Clay, H. K. Lyerly, S. Bhatia, S. A. Ghanekar, V. C. Maino, C. Delarosa, and M. L. Disis. 2008. Precision and linearity targets for validation of an IFNgamma ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides. BMC Immunol 9:9.  Link to full text

Siebert, J. C., M. Inokuma, D. M. Waid, N. D. Pennock, G. M. Vaitaitis, M. L. Disis, J. F. Dunne, D. H. Wagner, Jr., and H. T. Maecker. 2008. An analytical workflow for investigating cytokine profiles. Cytometry A 73:289-298.  Link to PubMed abstract

Soares, A. P., T. J. Scriba, S. Joseph, R. Harbacheuski, R. A. Murray, S. J. Gelderbloem, A. Hawkridge, G. D. Hussey, H. Maecker, G. Kaplan, and W. A. Hanekom. 2008. Bacillus calmette-guerin vaccination of human newborns induces T cells with complex cytokine and phenotypic profiles. J Immunol 180:3569-3577.  Link to PubMed abstract

Ghanekar, S. A., S. Bhatia, J. J. Ruitenberg, C. D. Rosa, M. L. Disis, V. C. Maino, H. T. Maecker, and C. A. Waters. 2007. Phenotype and in vitro function of mature MDDC generated from cryopreserved PBMC of cancer patients are equivalent to those from healthy donors. Journal of immune based therapies and vaccines 5:7. Link to full-text

Inokuma, M., C. dela Rosa, C. Schmitt, P. Haaland, J. Siebert, D. Petry, M. Tang, M. A. Suni, S. A. Ghanekar, D. Gladding, J. F. Dunne, V. C. Maino, M. L. Disis, and H. T. Maecker. 2007. Functional T cell responses to tumor antigens in breast cancer patients have a distinct phenotype and cytokine signature. J Immunol 179:2627-2633. Link to PubMed abstract

Streitz, M., L. Tesfa, V. Yildirim, A. Yahyazadeh, T. Ulrichs, R. Lenkei, A. Quassem, G. Liebetrau, L. Nomura, H. Maecker, H. D. Volk, and F. Kern. 2007. Loss of receptor on tuberculin-reactive T-cells marks active pulmonary tuberculosis. PLoS ONE 2:e735. Link to full-text

Sinclair, E., Q. X. Tan, M. Sharp, V. Girling, C. Poon, M. V. Natta, D. A. Jabs, M. Inokuma, H. T. Maecker, B. Bredt, and M. A. Jacobson. 2006. Protective immunity to cytomegalovirus (CMV) retinitis in AIDS is associated with CMV-specific T cells that express interferon- gamma and interleukin-2 and have a CD8+ cell early maturational phenotype. J Infect Dis 194:1537-1546. Link to PubMed abstract

Murray, R. A., N. Mansoor, R. Harbacheuski, J. Soler, V. Davids, A. Soares, A. Hawkridge, G. D. Hussey, H. Maecker, G. Kaplan, and W. A. Hanekom. 2006. Bacillus Calmette Guerin vaccination of human newborns induces a specific, functional CD8+ T cell response. J Immunol 177:5647-5651.  Link to PubMed abstract

Nomura, L. E., B. Emu, R. Hoh, P. Haaland, S. G. Deeks, J. N. Martin, J. M. McCune, D. F. Nixon, and H. T. Maecker. 2006. IL-2 production correlates with effector cell differentiation in HIV-specific CD8+ T cells. AIDS Res Ther 3:18.  Link to pdf

Maecker, H. T., and J. Trotter. 2006. Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69:1037.  Link to PubMed abstract

Shu, S., A. J. Cochran, R. R. Huang, D. L. Morton, and H. T. Maecker. 2006. Immune responses in the draining lymph nodes against cancer: implications for immunotherapy. Cancer Metastasis Rev 25:233.  Link to PubMed abstract

Disis, M. L., C. dela Rosa, V. Goodell, L. Y. Kuan, J. C. Chang, K. Kuus-Reichel, T. M. Clay, H. Kim Lyerly, S. Bhatia, S. A. Ghanekar, V. C. Maino, and H. T. Maecker. 2006. Maximizing the retention of antigen specific lymphocyte function after cryopreservation. J Immunol Methods 308:13.  Link to PubMed abstract

Maecker, H. T., J. Moon, S. Bhatia, S. A. Ghanekar, V. C. Maino, J. K. Payne, K. Kuus-Reichel, J. C. Chang, A. Summers, T. M. Clay, M. A. Morse, H. K. Lyerly, C. Delarosa, D. P. Ankerst, and M. L. Disis. 2005. Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT. BMC Immunol 6:17.  Link to pdf

Maecker, H. T., A. Rinfret, P. D'Souza, J. Darden, E. Roig, C. Landry, P. Hayes, J. Birungi, O. Anzala, M. Garcia, A. Harari, I. Frank, R. Baydo, M. Baker, J. Holbrook, J. Ottinger, L. Lamoreaux, C. L. Epling, E. Sinclair, M. A. Suni, K. Punt, S. Calarota, S. El-Bahi, G. Alter, H. Maila, E. Kuta, J. Cox, C. Gray, M. Altfeld, N. Nougarede, J. Boyer, L. Tussey, T. Tobery, B. Bredt, M. Roederer, R. Koup, V. C. Maino, K. Weinhold, G. Pantaleo, J. Gilmour, H. Horton, and R. P. Sekaly. 2005. Standardization of cytokine flow cytometry assays. BMC Immunol 6:13.  Link to pdf

Suni, M. A., V. C. Maino, and H. T. Maecker. 2005. Ex vivo analysis of T-cell function. Curr Opin Immunol 17:434.  Link to PubMed abstract

Campbell, M. J., J. Scott, H. T. Maecker, J. W. Park, and L. J. Esserman. 2005. Immune dysfunction and micrometastases in women with breast cancer. Breast Cancer Res Treat 91:163.  Link to PubMed abstract

Sun, Y., J. E. Schmitz, P. M. Acierno, S. Santra, R. A. Subbramanian, D. H. Barouch, D. A. Gorgone, M. A. Lifton, K. R. Beaudry, K. Manson, V. Philippon, L. Xu, H. T. Maecker, J. R. Mascola, D. Panicali, G. J. Nabel, and N. L. Letvin. 2005. Dysfunction of simian immunodeficiency virus/simian human immunodeficiency virus-induced IL-2 expression by central memory CD4+ T lymphocytes. J Immunol 174:4753.  Link to PubMed abstract

Ghanekar, S. A., H. T. Maecker, and V. C. Maino. 2005. Immune monitoring using cytokine flow cytometry. In Manual of Clinical Laboratory Immunology. N. R. Rose, ed. ASM Press, Washington, DC.  Link to ASM Press description

Maecker, H. T. 2005. The role of immune monitoring in evaluating cancer immunotherapy. In Immunotherapy of Cancer. M. L. Disis, ed. Humana Press, Totowa, NJ.  Link to Humana Press description

Walker, J. M., H. T. Maecker, V. C. Maino, and L. J. Picker. 2004. Multicolor flow cytometric analysis in SIV-infected rhesus macaque. Methods Cell Biol 75:535.  Link to PubMed abstract

Drohan, L., J. J. Harding, B. Holm, E. Cordoba-Tongson, C. L. Dekker, T. Holmes, H. Maecker, and E. D. Mellins. 2004. Selective developmental defects of cord blood antigen-presenting cell subsets. Hum Immunol 65:1356.  Link to PubMed abstract

Maecker, H. T., T. Frey, L. E. Nomura, and J. Trotter. 2004. Selecting fluorochrome conjugates for maximum sensitivity. Cytometry A 62:169.  Link to PubMed abstract

Maecker, H. T., and V. C. Maino. 2004. Analyzing T-cell responses to cytomegalovirus by cytokine flow cytometry. Hum Immunol 65:493.  Link to PubMed abstract

Dunne, J. F., and H. T. Maecker. 2004. Automation of cytokine flow cytometry assays. J Assoc Lab Automation 9:5.

Maino, V. C., and H. T. Maecker. 2004. Cytokine flow cytometry: a multiparametric approach for assessing cellular immune responses to viral antigens. Clin Immunol 110:222.  Link to PubMed abstract

Jacobson, M. A., H. T. Maecker, P. L. Orr, R. D'Amico, M. Van Natta, X. D. Li, R. B. Pollard, and B. M. Bredt. 2004. Results of a Cytomegalovirus (CMV)-Specific CD8+/Interferon- gamma + Cytokine Flow Cytometry Assay Correlate with Clinical Evidence of Protective Immunity in Patients with AIDS with CMV Retinitis. J Infect Dis 189:1362.  Link to PubMed abstract

Ruitenberg, J. J., and C. A. Waters. 2003. A rapid flow cytometric method for the detection of intracellular cyclooxygenases in human whole blood monocytes and a COX-2 inducible human cell line. J Immunol Methods 274:93.  Link to PubMed abstract

Maecker, H. T., and V. C. Maino. 2003. T cell immunity to HIV: defining parameters of protection. Current HIV Research 1:249.  Link to PubMed abstract

Tu, W., S. Chen, M. Sharp, C. Dekker, A. M. Manganello, E. C. Tongson, H. T. Maecker, T. H. Holmes, Z. Wang, G. Kemble, S. Adler, A. Arvin, and D. B. Lewis. 2004. Persistent and selective deficiency of CD4+ T cell immunity to cytomegalovirus in immunocompetent young children. J Immunol 172:3260.  Link to PubMed abstract

Maecker, H. T. 2004. Cytokine flow cytometry. In Flow Cytometry Protocols. T. S. Hawley, and R. G. Hawley, eds. Humana Press, Totowa, NJ, p. 95.  Link to PubMed abstract

Walker, E. B., D. Haley, W. Miller, K. Floyd, K. P. Wisner, N. Sanjuan, H. T. Maecker, P. Romero, H. M. Hu, W. G. Alvord, J. W. Smith, B. A. Fox, and W. J. Urba. 2004. gp100(209-2M) peptide immunization of HLA-A2+ stage I-III melanoma patients induces significant increase in antigen-specific effector and long-term memory CD8+ T cells. Clin Cancer Res 10:668.  Link to pdf

Deeks, S. G., J. N. Martin, E. Sinclair, J. Harris, T. B. Neilands, H. T. Maecker, E. Hagos, T. Wrin, C. J. Petropoulos, B. Bredt, and J. M. McCune. 2004. Strong cell-mediated immune responses are associated with the maintenance of low-level viremia in antiretroviral-treated individuals with drug-resistant human immunodeficiency virus type 1. J Infect Dis 189:312.  Link to PubMed abstract

Dunne, J. F., and H. T. Maecker. 2004. Flow cytometry. In Principles of Immunopharmacology. F. P. Nijkamp, ed. Verlag, Basel.  Link to publisher description

Shinn, A. H., N. C. Bravo, H. T. Maecker, and J. W. Smith. 2003. TNF-a detection using a flow cytometric assay to determine cellular responses to anthrax vaccine. J Immunol Methods 282:169.  Link to PubMed abstract

Suni, M. A., H. S. Dunn, P. L. Orr, R. deLaat, E. Sinclair, S. A. Ghanekar, B. M. Bredt, J. F. Dunne, V. C. Maino, and H. T. Maecker. 2003. Performance of plate-based cytokine flow cytometry with automated data analysis. BMC Immunology 4:9.  Link to pdf

Ghanekar, S. A., and H. T. Maecker. 2003. Cytokine flow cytometry: multiparametric approach to immune function analysis. Cytotherapy 5:1.  Link to PubMed abstract

Nomura, L. E., E. D. DeHaro, L. N. Martin, and H. T. Maecker. 2003. Optimal preparation of rhesus macaque blood for cytokine flow cytometric analysis. Cytometry 53A:28.  Link to PubMed abstract

Smith, J. W., 2nd, E. B. Walker, B. A. Fox, D. Haley, K. P. Wisner, T. Doran, B. Fisher, L. Justice, W. Wood, J. Vetto, H. Maecker, A. Dols, S. Meijer, H. M. Hu, P. Romero, W. G. Alvord, and W. J. Urba. 2003. Adjuvant immunization of HLA-A2-positive melanoma patients with a modified gp100 peptide induces peptide-specific CD8+ T-cell responses. J Clin Oncol 21:1562.  Link to PubMed abstract

He, X. S., K. Mahmood, H. T. Maecker, T. H. Holmes, G. W. Kemble, A. M. Arvin, and H. B. Greenberg. 2003. Analysis of the frequencies and of the memory T cell phenotypes of human CD8+ T cells specific for influenza A viruses. J Infect Dis 187:1075.  Link to PubMed abstract

Maecker, H. T. 2003. Human CD81 Directly Enhances TH1 and TH2 Cell Activation, but Preferentially Induces Proliferation of TH2 Cells upon Long-Term Stimulation. BMC Immunol 4:1.  Link to pdf

Maecker, H. L., Z. Yun, H. T. Maecker, and A. J. Giaccia. 2002. Epigenetic changes in tumor Fas levels determine immune escape and response to therapy. Cancer Cell 2:139.  Link to PubMed abstract

Dunn, H. S., D. J. Haney, S. A. Ghanekar, P. Stepick-Biek, D. B. Lewis, and H. T. Maecker. 2002. Dynamics of CD4 and CD8 T cell responses to cytomegalovirus in healthy human donors. J Infect Dis 186:15.  Link to PubMed abstract

Maino, V. C., H. T. Maecker, and L. J. Picker. 2002. Antigen-specific cytokine responses in HIV disease. In Cellular Aspects of HIV Infection. A. Cossarizza, and D. Kaplan, eds. Wiley-Liss, New York, p. 371.

Suni, M. A., S. A. Ghanekar, D. W. Houck, H. T. Maecker, S. B. Wormsley, L. J. Picker, R. B. Moss, and V. C. Maino. 2001. CD4(+)CD8(dim) T lymphocytes exhibit enhanced cytokine expression, proliferation and cytotoxic activity in response to HCMV and HIV-1 antigens. Eur J Immunol 31:2512.  Link to PubMed abstract

Maecker, H. T., H. S. Dunn, M. A. Suni, E. Khatamzas, C. J. Pitcher, T. Bunde, N. Persaud, W. Trigona, T. M. Fu, E. Sinclair, B. M. Bredt, J. M. McCune, V. C. Maino, F. Kern, and L. J. Picker. 2001. Use of overlapping peptide mixtures as antigens for cytokine flow cytometry. J Immunol Methods 255:27.  Link to PubMed abstract

Maecker, H. T., S. A. Ghanekar, M. A. Suni, X. S. He, L. J. Picker, and V. C. Maino. 2001. Factors affecting the efficiency of CD8+ T cell cross-priming with exogenous antigens. J Immunol 166:7268.  Link to pdf

Ghanekar, S. A., L. E. Nomura, M. A. Suni, L. J. Picker, H. T. Maecker, and V. C. Maino. 2001. Gamma interferon expression in CD8(+) T cells is a marker for circulating cytotoxic T lymphocytes that recognize an HLA A2-restricted epitope of human cytomegalovirus phosphoprotein pp65. Clin Diagn Lab Immunol 8:628.  Link to pdf

Maecker, H. T., S. Auffermann-Gretzinger, L. E. Nomura, A. Liso, D. K. Czerwinski, and R. Levy. 2001. Detection of CD4 T-cell responses to a tumor vaccine by cytokine flow cytometry. Clin Cancer Res 7:902s.  Link to PubMed abstract

He, X. S., B. Rehermann, J. Boisvert, J. Mumm, H. T. Maecker, M. Roederer, T. L. Wright, V. C. Maino, M. M. Davis, and H. B. Greenberg. 2001. Direct functional analysis of epitope-specific CD8+ T cells in peripheral blood. Viral Immunol 14:59.  Link to PubMed abstract

Maecker, H. T., V. C. Maino, and L. J. Picker. 2000. Immunofluorescence analysis of T-cell responses in health and disease. J Clin Immunol 20:391.  Link to PubMed abstract

Nomura, L. E., J. M. Walker, and H. T. Maecker. 2000. Optimization of whole blood antigen-specific cytokine assays for CD4(+) T cells. Cytometry 40:60.  Link to PubMed abstract

Asanuma, H., M. Sharp, H. T. Maecker, V. C. Maino, and A. M. Arvin. 2000. Frequencies of memory T cells specific for varicella-zoster virus, herpes simplex virus, and cytomegalovirus by intracellular detection of cytokine expression. J Infect Dis 181:859.  Link to PubMed abstract

Tools for Immune Function Assays

The following are some useful tools for immune function assays:

To calculate sample size required for statistical significance in intracellular cytokine assays:  Download sample_size_calc.xls

To determine a confidence interval around an intracellular cytokine result:  Download conf_interval_calc.xls

To create a list of 15 aa peptides, overlapping by 11 aa each, for a sequence of your choice:  Download Peptidechopper2.xls

Immune Function Literature from BD

Finding technical literature on the BD website can be difficult.  Here I've gathered the BD links to technical documents related to cellular immune function assays and related topics.

BD Application Notes:

Selecting Reagents for Multicolor Flow Cytometry:  Link to pdf  (guide to development of multicolor reagent panels)

Detecting Cytokines in Antigen-Activated CD4+ and CD8+ Lymphocytes:  Link to pdf  (For 4-color FastImmune kits, including peptide stimulation)

Detecting Cytokines in Antigen-Activated Lymphocytes:  Link to pdf  (For 3-color FastImmune kits, whole antigen stimulation)

Simultaneous Detection of Proliferation and Cytokine Expression in Peripheral Blood Mononuclear Cells:  Link to pdf  (For use with anti-BrdU FITC+DNAse reagent from BD, Link to product description)

Detecting Intracellular Cytokines in Activated Lymphocytes:  Link to pdf  (For polyclonal activation of lymphocytes)

Detecting Intracellular Cytokines in Activated Monocytes:  Link to pdf  (For polyclonal activation of monocytes)

BD Manuals:

Performance Characteristics of Cytokine Flow Cytometry Assays:  Link to pdf  (Covers relevant parameters for intracellular cytokine assays, from choosing antigens to interpreting results)

Tools and Solutions for Immune Function Analysis:  Link to pdf  (Overviews of CBA, FastImmune Cytokine, ELISPOT, DimerX, and ELISA assays from BD)

A Setup System for Compensation: BD CompBeads plus BD FACS Diva Software:  Link to pdf  (describes use of CompBeads as single-color compensation controls, and autocompensation in FACS Diva software)

BD Posters:

Mouse CD chart:  Link to pdf

Signaling pathways of immune function:  Link to pdf

Cytokine research web page

I was recently alerted to the following resource for cytokine researchers:  http://www.cytok.com.  There are job postings, recent literature, and a directory of laboratories with citation rankings (http://www.cytok.com/labs.php).  Yes, I'm on the list, at #906!  I never knew there were so many labs doing cytokine research....

Hints for non-human primate CFC

We've published a techniques paper that addresses sample handling and other issues for macaque CFC (Link to PubMed abstract).  Here are answers to a few common questions:

How should samples best be handled, if they can't be stimulated on the day of harvest?  We suggest isolating PBMC on the day of harvest (BD CPT tubes work well for this), shipping the PBMC overnight, and stimulating the next day.  Stimulations can be done in a timed water bath, cooling to <18 C after the desired stimulation time of 6-10 hours, with processing done the next day.

What about plates versus tubes?  For human PBMC, we routinely use 96-well plates and the results are just as good as in tubes (see Download suniplate_cfcbmci.pdf).  We haven't yet done the comparison with non-human primate cells.

What are the best cytokines to monitor?  In general, in rhesus:  TNFa > IFNg > IL-2.  We haven't tried Th2 cytokines much.  The clones are important, especially for IFNg, where the B27 clone from BD Pharmingen is much superior to any others we've tested.

Peptide Mix Antigens

Overlapping peptide mixes are commercially available from vendors like Jerini Ag in Germany (http://www.jpt.com/index.htm) or BD Pharmingen (see Download peptidemix_hotlines.pdf).  These are commonly designed as 15 aa in length, with 11 aa overlaps, a paradigm pioneered by Florian Kern and colleagues (see for example Kern et al., 1999).  The utility of this approach, and comparison with mixes of 9 and 20 aa peptides, can be found in our JIM paper from 2001 (Link to PubMed abstract).  Here are some frequently asked questions:

How many peptides can be mixed together in a single stimulation?  The answer seems to be "many", maybe up to 300 or so, without significant competition for MHC sites.  However, as a practical matter, peptides are usually solubilized in DMSO, and there is a limit to their solubility (determined largely by sequence).  More than 0.5% final concentration of DMSO can cause toxicity to lymphocytes in a 6 hour assay.  So you may be limited on how many peptides you can mix together based simply on DMSO concentration.

What purity of peptides is required?  We use 80% purity as a standard, but this is more to ensure roughly equal representation of different peptides in a mix, rather than a requirement for performance.  We have not been able to see a difference in stimulation capability between purified and crude versions of a single epitope peptide for CMV.

Do you order them pre-mixed?  We have been able to get manufacturers to pre-mix and aliquot lyophilized mixes, and to QC these mixes for us.  This is much more convenient than solubilizing individual peptides and then mixing them, and allows for higher per-peptide concentrations when dissolving the final mix.  Vendors that offer pre-mixing, QC, and aliquoting include CPC Scientific (http://www.cpcscientific.com/) and American Peptide Company (http://www.americanpeptide.com/).

How do you stimulate with peptides?  Brefeldin A can be added at time 0 without detriment, because internal processing of peptides is not required.  6 hour stimulation is standard, but this can be lengthened up to 12 hours or more with increasing intensity of cytokine staining, before toxicity of the brefeldin A begins to interfere.

How do 15-mers stimulate CD8 responses?  We don't know for sure, but it seems likely that cell-surface and/or serum-derived proteases play a role in clipping longer peptides to generate species that can bind efficiently to class I MHC molecules.  Internalization does not seem to be required, as stimulation is not inhibited by chloroquine or inhibitors of TAP processing.

What concentration of peptide is required?  We typically use 1-2 ug/mL of each peptide in the mix.  The optimal concentration will actually be donor-dependent, since some donors will respond to high-affinity epitopes and/or epitopes that are easily presented, while other donors will not...so this is just a compromise recommendation.