Skip to main content
SpringerLink
Log in

Detection and Enumeration of Cytokine-Secreting Cells by FluoroSpot

  • Protocol
  • First Online:
Single-Cell Protein Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2386))

Abstract

The FluoroSpot assay is a development of the highly sensitive enzyme-linked immunospot (ELISpot) assay which enables functional measurement of immunity at the single-cell level. Both assays are performed in a 96-well format and measures the frequency of analyte-secreting cells, in ELISpot usually limited to one analyte per well due to the use of enzymes and precipitating substrates for detection. FluoroSpot, performed in a similar way as ELISpot, overcomes this limitation by detecting each analyte with an assigned fluorophore instead of an enzyme. By using readers equipped with fluorophore-specific filters, cells producing single or multiple cytokines can be identified simultaneously in the same well. This greatly facilitates the analysis of functionally distinct subpopulations in heterogenous cell samples, for example, the frequency of polyfunctional T cells, suggested to be of importance in various disease states. FluoroSpot maintains the simplicity and sensitivity of the ELISpot while taking the assay a step further towards a multiplex analysis and an in-depth understanding of the quality of an immune response. We describe here a 96-well plate method to analyze cells that have secreted up to four different cytokines simultaneously (Four-color Fluorospot).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Van de Pol MA, Lutter R, van Ree R, van der Zee JS (2012) Increase in allergen-specific IgE and ex vivo Th2 responses after a single bronchial challenge with house dust mite in allergic asthmatics. Allergy 67(1):67–73

    Article  Google Scholar 

  2. Oseroff C, Sidney J, Tripple V, Grey H, Wood R, Broide DH, Greenbaum J, Kolla R, Peters B, Pomés A, Sette A (2012) Analysis of T cell responses to the major allergens from German cockroach: epitope specificity and relationship to IgE production. J Immunol 189(2):679–688

    Article  CAS  Google Scholar 

  3. Hill PC, Brookes RH, Fox A, Jackson-Sillah D, Jeffries DJ, Lugos MD, Donkor SA, Adetifa IM, de Jong BC, Aiken AM, Adegbola RA, McAdam KP (2007) Longitudinal assessment of an ELISpot test for Mycobacterium tuberculosis infection. PLoS Med 4(6):e192

    Article  Google Scholar 

  4. Ewer KJ, O’Hara GA, CJA D, Collins KA, Sheehy SH, Reyes-Sandoval A, Goodman AL, Edwards NJ, Elias SC, Halstead FD, Longley RJ, Rowland R, Poulton ID, Draper SJ, Blagborough AM, Berrie E, Moyle S, Williams N, Siani L, Folgori A, Colloca S, Sinden RE, Lawrie AM, Cortese R, Gilbert SC, Nicosia A, Hill AVS (2013) Protective CD8+ T-cell immunity to human malaria induced by chimpanzee adenovirus-MVA immunisation. Nat Commun 4:2836

    Article  Google Scholar 

  5. Streeck H, Frahm N, Walker BD (2009) The role of IFN-γ Elispot assay in HIV vaccine research. Nat Protoc 4(4):461–468

    Article  CAS  Google Scholar 

  6. Higashide T, Kawamura T, Nagata M, Kotani R, Kimura K, Hirose M, Inada H, Niihira S, Yamano T (2006) T cell epitope mapping study with insulin overlapping peptides using ELISPOT assay in Japanese children and adolescents with type 1 diabetes. Pediatr Res 59:445–450

    Article  CAS  Google Scholar 

  7. Powell WE, Hanna SJ, Hocter CN, Robinson E, Lewis M, Dunseath G, Luzio S, Howell A, Dayan CM, Wong FS (2019) Detecting autoreactive B cells in the peripheral blood of people with type 1 diabetes using ELISpot. J Immunol Methods 471:61–65

    Article  CAS  Google Scholar 

  8. Greiner J, Ono Y, Hofmann S, Schmitt A, Mehring E, Götz M, Guillaume P, Döhner K, Mytilineos J, Döhner H, Schmitt M (2012) Mutated regions of nucleophosmin 1 elicit both CD4(+) and CD8(+) T-cell responses in patients with acute myeloid leukemia. Blood 120(6):1282–1289

    Article  CAS  Google Scholar 

  9. Robbins PF, Lu Y-C, El-Gamil M, Li YF, Gross C, Gartner J, Lin JC, Teer JK, Cliften P, Tycksen E, Samuels Y, Rosenberg SA (2013) Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells. Nat Med 19(6):747–752

    Article  CAS  Google Scholar 

  10. Varnaité R, García M, Glans H, Maleki KT, Sandberg JT, Tynell J, Christ W, Lagerqvist N, Asgeirsson H, Ljunggren HG, Ahlén G, Frelin L, Sällberg M, Blom K, Klingström J, Gredmark-Russ S (2020) Expansion of SARS-CoV-2-specific antibody secreting cells and generation of neutralizing antibodies in hospitalized COVID-19 patients. J Immunol. https://doi.org/10.4049/jimmunol.2000717

  11. Slota M, Lim JB, Dang Y, Disis ML (2011) ELISpot for measuring human immune responses to vaccines. Expert Rev Vaccines 10(3):299–306

    Article  CAS  Google Scholar 

  12. Fiore-Gartland A, Manso BA, Friedrich DP, Gabriel EE, Finak G, Moodie Z, Hertz T, De Rosa SC, Frahm N, Gilbert PG, McElrath MJ (2016) Pooled-peptide epitope mapping strategies are efficient and highly sensitive: an evaluation of methods for identifying human T cell epitope specificities in large-scale HIV vaccine efficacy trials. PLoS One 11(2):e0147812

    Article  Google Scholar 

  13. De Rosa SC, Lu FX, Yu J, Perfetto SP, Falloon J, Moser S, Evans TG, Koup R, Miller CJ, Roederer M (2004) Vaccination in humans generates broad T cell cytokine responses. J Immunol 173(9):5372–5380

    Article  Google Scholar 

  14. Minton K (2013) Mechanisms of T cell polyfunctionality. Nat Rev Immunol 14:7

    Article  Google Scholar 

  15. Lin L, Finak G, Ushey K, Seshadri C, Hawn TR, Frahm N, Scriba TJ, Mahomed H, Hanekom W, Bart PA, Pantaleo G, Tomaras GD, Rerks-Ngarm S, Kaewkungwal J, Nitayaphan S, Pitisuttithum P, Michael NL, Kim JH, Robb ML, O’Conell RJ, Karasavvas N, Gilbert P, DeRosa S, McElrath MJ, Gottardo R (2015) COMPASS identifies T-cell subsets correlated with clinical outcomes. Nat Biotechnol 33(6):610–616

    Article  CAS  Google Scholar 

  16. Aagaard C, Hoang TTKT, Izzo A, Billeskov R, Troudt J, Arnett K, Keyser A, Elvang T, Andersen P, Dietrich J (2009) Protection and polyfunctional T cells induced by Ag85B-TB10.4/IC31® against Mycobacterium tuberculosis is highly dependent on the antigen dose. PLoS One 4(6):e5930

    Article  Google Scholar 

  17. Kannanganat S, Ibegbu C, Chennareddi L, Robinson HL, Amara RR (2007) Multiple-cytokine-producing antiviral CD4 T cells are functionally superior to single-cytokine-producing cells. J Virol 81:8468–8476

    Article  CAS  Google Scholar 

  18. Ciuffreda D, Comte D, Cavassini M, Giostra E, Bühler L, Perruchoud M, Heim MH, Battegay M, Genné D, Mulhaupt B, Malinverni R, Oneta C, Bernasconi E, Monnat M, Cerny A, Chuard C, Borovicka J, Mentha G, Pascual M, Gonvers JJ, Pantaleo G, Dutoit V (2008) Polyfunctional HCV-specific T-cell responses are associated with effective control of HCV replication. Eur J Immunol 38(10):2665–2677

    Article  CAS  Google Scholar 

  19. Darrah PA, Patel DT, De Luca PM, Lindsay RWB, Davey DF, Flynn BJ, Hoff ST, Andersen P, Reed SG, Morris SL, Roederer M, Seder RA (2007) Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major. Nat Med 13(7):843–850

    Article  CAS  Google Scholar 

  20. Attig S, Hennenlotter J, Pawelec G, Klein G, Koch SD, Pircher H, Feyerabend S, Wernet D, Stenzl A, Rammensee HG, Gouttefangeas C (2009) Simultaneous infiltration of polyfunctional effector and suppressor T cells into renal cell carcinomas. Cancer Res 69(21):8412–8419

    Article  CAS  Google Scholar 

  21. Chauvat A, Benhamouda N, Gey A, Lemoine FM, Paulie S, Carrat F, Gougeon ML, Rozenberg F, Krivine A, Cherai M, Lehmann PV, Quintin-Colonna F, Launay O, Tartour E (2014) Clinical validation of IFNγ/IL-10 and IFNγ/IL-2 FluoroSpot assays for the detection of Tr1 T cells and influenza vaccine monitoring in humans. Hum Vaccine Immunother 10(1):104–113

    Article  CAS  Google Scholar 

  22. Gazagne A, Claret E, Wijdenes J, Yssel H, Bousquet F, Levy E, Vielh P, Scotte F, Goupil TL, Fridman WH, Tartour E (2003) A Fluorospot assay to detect single T lymphocytes simultaneously producing multiple cytokines. J Immunol Methods 283:91–98

    Article  CAS  Google Scholar 

  23. Jackson SE, Sedikides GX, Mason GM, Okecha G, Wills MR (2017) Human Cytomegalovirus (HCMV)-specific CD4+ T cells are polyfunctional and can respond to HCMV-infected dendritic cells in vitro. J Virol 91(6):e02128–e02116

    Article  CAS  Google Scholar 

  24. Birrueta G, Frazier A, Pomés A, Glesner J, Filep S, Schal C, Jeong KY, McMurtrey C, Schans TV, Hildebrand WH, Busse P, Beigelman A, Bacharier LB, Peters B, Sette A, Schulten V (2019) Variability in German cockroach extract composition greatly impacts T cell potency in cockroach-allergic donors. Front Immunol 10:313. https://doi.org/10.3389/fimmu.2019.00313

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Mullins CS, Gock M, Krohn M, Linnebacher M (2019) Human colorectal carcinoma infiltrating B lymphocytes are active secretors of the immunoglobulin isotypes A, G, and M. Cancers 11(6):776. https://doi.org/10.3390/cancers11060776

    Article  CAS  PubMed Central  Google Scholar 

  26. Körber N, Behrends U, Hapfelmeier A, Protzer U, Bauer T (2016) Validation of an IFNγ/IL2 FluoroSpot assay for clinical trial monitoring. J Transl Med 14(1):175. https://doi.org/10.1186/s12967-016-0932-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Levin MJ, Kroehl ME, Johnson MJ, Hammes A, Reinhold D, Lang N, Weinberg A (2018) Th1 memory differentiates recombinant from live herpes zoster vaccines. J Clin Invest 128(10):4429–4440

    Article  Google Scholar 

  28. Jahnmatz P, Bengtsson T, Zuber B, Färnert A, Ahlborg N (2016) An antigen-specific, four-color, B-cell FluoroSpot assay utilizing tagged antigens for detection. J Immunol Methods 433:23–30

    Article  CAS  Google Scholar 

  29. Jahnmatz P, Sundling C, Makower B, Sondén K, Färnert A, Ahlborg N (2020) Multiplex analysis of antigen-specific memory B cells in humans using reversed B-cell FluoroSpot. J Immunol Methods 478:112715. https://doi.org/10.1016/j.jim.2019.112715

    Article  CAS  PubMed  Google Scholar 

  30. Bittel P, Mayor D, Iseli P, Bodmer T, Suter-Riniker F (2014) IGRA-positive patients and interferon-gamma/interleukin-2 signatures: can the Fluorospot assay provide further information? Infection 42(3):539–543

    Article  CAS  Google Scholar 

  31. Bronge M, Ruhrman S, Carvalho-Queiroz C, Nilsson OB, Kaiser A, Holmgren E, Macrini C, Winklmeier S, Meinl E, Brundin L, Khademi M, Olsson T, Gafvelin G, Grönlund H (2019) Myelin oligodendrocyte glycoprotein revisited-sensitive detection of MOG-specific T-cells in multiple sclerosis. J Autoimmun 102:38–49

    Article  CAS  Google Scholar 

  32. Kesa G, Larsson PH, Ahlborg N, Axelsson B (2012) Comparison of ELISpot and FluoroSpot in the analysis of swine flu-specific IgG and IgA secretion by in vivo activated human B cells. Cell 1(2):27–34

    Article  CAS  Google Scholar 

  33. Romer PS, Berr S, Avota E, Na SY, Battaglia M, ten Berge I, Einsele H, Hunig T (2011) Preculture of PBMCs at high cell density increases sensitivity of T-cell responses, revealing cytokine release by CD28 superagonist TGN1412. Blood 118(26):6772–6782

    Article  Google Scholar 

  34. Janetzki S, Panageas KS, Ben-Porat L, Noyer J, Britten CM, Clay TM, Kalos M, Maecker HT, Romero P, Yuan J, Kast WM, Hoos A (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

    Article  Google Scholar 

  35. Janetzki S, Price L, Britten CM, van der Burg SH, Caterini J, Currier JR, Ferrari G, Gouttefangeas C, Hayes P, Kaempgen E, Lennerz V, Nihlmark K, Souza V, Hoos A (2010) Performance of serum-supplemented and serum-free media in IFN-γ Elispot assays for human T cells. Cancer Immunol Immunother 59:609–618

    Article  CAS  Google Scholar 

  36. Kutscher S, Dembek CJ, Deckert S, Russo C, Korber N, Bogner JR, Geisler F, Umgelter A, Neuenhahn M, Albrecht J, Cosma A, Protzer U, Bauer T (2013) Overnight resting of PBMC changes functional signatures of antigen specific T-cell responses: impact for immune monitoring within clinical trials. PLoS One 8(10):e76215

    Article  CAS  Google Scholar 

  37. Currier JR, Kuta EG, Turk E, Earhart LB, Loomis-Price L, Janetzki S, Ferrari G, Birx DL, Cox JH (2002) A panel of MHC class I restricted viral peptides for use as a quality control for vaccine trial ELISPOT assays. J Immunol Methods 260:157–172

    Article  CAS  Google Scholar 

  38. Quast S, Zhang W, Shive C, Kovalovski D, Ott PA, Herzog BA, Boehm BO, Tary-Lehmann M, Karulin AY, Lehmann PV (2005) IL-2 absorption affects IFN-gamma and IL-5, but not IL-4 producing memory T cells in double color cytokine ELISPOT assays. Cell Immunol 237:28–36

    Article  CAS  Google Scholar 

  39. Crotty S, Felgner P, Davies H, Glidewell J, Villarreal L, Ahmed R (2003) Cutting edge: long-term B cell memory in humans after smallpox vaccination. J Immunol 171(10):4969–4973

    Article  CAS  Google Scholar 

  40. Walsh PN, Friedrich DP, Williams JA, Smith RJ, Stewart TL, Carter DK, Liao HX, McElrath MJ, Frahm N (2013) Optimization and qualification of a memory B-cell ELISpot for the detection of vaccine-induced memory responses in HIV vaccine trials. J Immunol Methods 394(1–2):84–93

    Article  CAS  Google Scholar 

  41. Jahnmatz M, Kesa G, Netterlid E, Buisman A-M, Thorstensson R, Ahlborg N (2013) Optimization of a human IgG B-cell ELISpot assay for the analysis of vaccine-induced B-cell responses. J Immunol Methods 391(1–2):50–59

    Article  CAS  Google Scholar 

  42. Jahnmatz P (2020) Methods for studying memory-B-cell immunity against malaria. Dissertation, Karolinska University Hospital

    Google Scholar 

  43. Moodie Z, Price L, Gouttefangas C, Mander A, Janetzki S, Löwer M, Welters MJP, Ottensmeier C, van der Burg SH, Britten CM (2010) Response definition criteria for ELISPOT assays revisited. Cancer Immunol Immunother 59:1489–1501

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Karin Cavallin, Jens Gertow, and Kopek Nihlmark for critical reading of the manuscript, Peter Jahnmatz and Karin Cavallin for images and Tanja Zamani/Mabtech for illustrations.

Author information

Authors and Affiliations

  1. Mabtech AB, Nacka Strand, Stockholm, Sweden

    Bernt Axelsson

Authors
  1. Bernt Axelsson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bernt Axelsson .

Editor information

Editors and Affiliations

  1. Mission Bio, Inc., South San Francisco, CA, USA

    Aik T. Ooi

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Axelsson, B. (2022). Detection and Enumeration of Cytokine-Secreting Cells by FluoroSpot. In: Ooi, A.T. (eds) Single-Cell Protein Analysis. Methods in Molecular Biology, vol 2386. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1771-7_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1771-7_6

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1770-0

  • Online ISBN: 978-1-0716-1771-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation