HUN-REN BRC CF.

Magyar

Flow cytometry

Main research areas

  • Disturbances in immunehomeaostasis in human chronic disease
  • Multiparametric immunophenotyping of systemic autoimmune disorders (RA, SLE, SSC)
  • Investigation of thrombotic events in anti-phospholipid syndrome and COVID-19
  • Single cell mass cytometric characterization of the inflammatory complications in type II diabetes mellitus
  • Investigation of plasma proteins and single cell PBMCs in stable vs exacerbating COPD
  • High multiplex investigation of the intratumor heterogeneity in human primary adenocarcinoma
  • Analysis of the effect of drug candidate molecules using single cell mass cytometry

About us

The cooperation of highly specialized cell types maintains the homeostasis of multicellular organisms. The disturbance of that harmony contributes to the development of several diseases. Most of the cellular functions are executed by proteins so it is essential to investigate biological processes at the protein level. One of the routinely used methods to study cellular proteins is flow cytometry which detects cell surface or intracellular proteins by fluorescently labeled antibodies at single cell resolution. Currently, we use BC Cytoflex S (4 lasers), one BD FACSCalibur (2 lasers) analyzators and two BD Jazz (2 lasers; 3 lasers) sorters, and the state of the art spectral Cytek Aurora sorter (4 lasers). Overlap among the fluorescence spectra of different dyes limits the possibility of high multiplexicity in one single tube. To overcome the limitations of flow cytometry, antibodies are labeled by stable heavy metal isotopes (min 96% monoisotopic) in mass cytometry. Mass cytometer detects the distinct atomic mass of heavy metal isotopes which offers the possibility to acquire up to 42 markers in one sample. Mass cytometry is unique technology in our laboratory within Hungary. The characterization of cellular heterogeneity is achieved at the protein level with single cell resolution from homogenous cell suspensions of different biological samples (blood, solid tumors, different tissues) followed by antibody staining for mass cytometry. High content data analysis (Cytobank, FlowJo, CATALYTES, Astrolabe, Maxpar Pathsetter) is also integrated in our portfolio
We focus on the disturbances of the activation and regulation of immune system; therefore, we study human patient-derived blood or tissue samples at single cell resolution. The high content immunophenotyping is performed on PBMCs or different tissues such as primary adenocarcinoma.
The comparative bioinformatic analysis of biological specimens (patients vs healthy controls) reveals differences in the expression pattern of proteins of interest at single cell resolution. To complement the investigation of PBMCs we quantify the cytokine/chemokine content of the corresponding liquid biopsies by Luminex (MagPix) or Legendplex (Biolegend) technologies. We describe expression patterns of proteins associated with a given pathology such as rheumatoid arthritis, adenocarcinoma, COPD to find diagnostic, prognostic markers or novel therapeutic targets. We develop in vitro/ex vivo functional immune assays (i) to screen drug candidate molecules, (ii) to better understand the pathomechanism and reveal signal transduction pathways behind the given pathologies.
The laboratory is run on service and collaborative basis. We have built wide national and international connections to research groups and biotech companies.

Technology

Expertise and Equipment

1. Single cell mass cytometer (Helios, Fluidigm)

A special cytometer with 135 channels, antibodies are labeled with stable heavy metal isotopes. Currently 42 cell surface or intracellular markers can be studied. Complex bioinformatic analysis (Cytobank, Astrolabe, Maxper Pathsetter) is available.

2. Beckman Coulter Cytoflex S fluorescent flow cytometer

Beckman Coulter Cytoflex S fluorescent flow cytometer analyzer equipped with 4 lasers (405 nm, 488 nm, 561 nm, 638 nm) and 96-well plate microtiter plate adapter. Thirteen proteins, markers can be measured simultaneously, plus the SSC and FSC channels. The Kaluza and Cytexpert software are available for data analysis.

3. Becton Dickinson FACSCalibur fluorescent flow cytometer

Fluorescent flow cytometer analyser equipped with 2 lasers (488 nm, 633 nm). The Cellquest and Modfit software is available for data analysis.

4. Beckton Dickinson Jazz flow cytometer sorter

3 lasers (405 nm, 488 nm, 633 nm), Two-way single cell sorting is possible.

5. Beckton Dickinson Jazz flow cytometer sorter

2 lasers (405 nm, 488 nm), Two-way single cell sorting is possible.

6. Cytek Aurora flow cytometer

Spectral sorter equipped with 4 lasers (405, 488, 561 and 633 nm). It has 48 channels and capable of 6-way sorting.

7. Luminex MagPix

Multiplex, magnetic bead based technology for quantitative analysis of proteins. Max. 50 proteins and 76 (Merck) or 80 (Thermo) samples can be measured in one assay. The Millipplex software is available for data analysis.

Our best

Services

I. Measurement of antigen specific T-cell response

After vaccination, a major issue in vaccine efficacy studies is the quantitative measurement of the humoral immune response, i.e. T-cell immunity in addition to antibody production. T cells are capable of killing virus-infected cells and develop a long-term T cell memory that leads to the accumulation of reactive clones in the event of a subsequent viral infection. In immunosuppressed, i.e. immune-compromised patients (elderly, cancer patients, autoimmune patients) this is a particularly important parameter to monitor.
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II. Cytotoxic assessment of drug- candidate molecules

For candidate antitumour agents, it is questionable whether they exert cytostatic and/or cytotoxic effects. A further question for bioactive antitumour agents is whether they have an apoptotic or necrotic effect, and whether, in the case of an apoptotic effect, programmed cell death occurs in mammary cancer cells via an extrinsic or intrinsic pathway.
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III. Measurement of plant genome poliploidity

A fundamental parameter in plant breeding is the monitoring of DNA stocks, the so-called polyploidy test. This characteristic changes dynamically during plant breeding and provides information on the chromosome set.
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IV. Multiplex measurement of cytokine/chemokine concentrations

Quantification of cytokines, chemokines, hormones is often not possible from a limited amount of biological samples (CSF, newborn blood, tears).
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V. Multiplex immunophenotyping

The plasticity, dynamic changes and polarization of cells in the immune system are very important parameters that may underlie a therapeutic response, drug resistance. Multiparametric immunophenotyping and isolation of specific immune cell subclasses would greatly facilitate a better understanding of diseases resulting from dysregulation of the immune system, such as cancer or chronic autoimmune diseases.
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Grants awarded to the Flowcytometry Unit

Awarded Hungarian grants

Million HUF in total

List of grants
University of Ferrara
University of Szeged
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National Institute of Oncology
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Articles

Key publications

  1. Zsófia Ruppert, Patrícia Neuperger, Bettina Rákóczi, Nikolett Gémes, Brigitta Dukay, Petra Hajdu, Mária Péter, Gábor Balogh, László Tiszlavicz, László Vígh, Zsolt Török, László G. Puskás, Gábor J. Szebeni, Melinda E. Tóth
    Characterization of obesity-related diseases and inflammation using single cell immunophenotyping in two different diet-induced obesity models, International Journal of Obesity, Volume 48, Pages 1568–1576, 2024.
    https://www.nature.com/articles/s41366-024-01584-6
  2. Eniko Szabó, Anna Faragó, Gergely Bodor, Laszlo G. Puskas, László Kovács, Gabor J. Szebeni
    Identification of Immune Subsets with Distinct Lectin Binding Signatures using Multi-Parameter Flow Cytometry: Correlations with Disease Activity in Systemic Lupus Erythematosus, Frontiers in Immunology, Section: Autoimmune and Autoinflammatory Disorders: Autoimmune Disorders, May 7, 2024, Article 1380481.
    https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2024.1380481/full
  3. József Ágoston Balog, Klaudia Horti-Oravecz, Dorottya Kövesdi, Anikó Bozsik, János Papp, Henriett Butz, Attila Patócs, Gábor János Szebeni, Vince Kornél Grolmusz
    Peripheral immunophenotyping reveals lymphocyte stimulation in healthy women living with hereditary breast and ovarian cancer syndrome, iScience, Volume 27, Issue 6, 21 June 2024, Article 109882.
    https://www.sciencedirect.com/science/article/pii/S2589004224011040
  4. József Á Balog, Ágnes Zvara, Vivien Bukovinszki, László G. Puskás, Attila Balog, Gábor János Szebeni
    Comparative single-cell multiplex immunophenotyping of therapy-naive patients with rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus shed light on disease-specific composition of the peripheral immune system, Frontiers in Immunology, Section: Autoimmune and Autoinflammatory Disorders: Autoimmune Disorders, April 25, 2024, Article 1376933.
    https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2024.1376933/full
  5. Anna Faragó, Ágnes Zvara, László Tiszlavicz, Éva Hunyadi-Gulyas, Zsuzsanna Darula, Zoltán Hegedűs, Enikő Szabó, Sára Eszter Surguta, József Tóvári, Laszló G. Puskás, Gábor J. Szebeni
    Lectin-based immunophenotyping and whole proteomic profiling of CT-26 colon carcinoma murine model, International Journal of Molecular Sciences, April 4, 2024, Volume 25, Issue 7, Article 4022.
    https://www.mdpi.com/1422-0067/25/7/4022
  6. Nikolett Gémes, József Á Balog, Patrícia Neuperger, Erzsébet Schlegl, Imre Barta, János Fillinger, Balazs Antus, Ágnes Zvara, Zoltán Hegedűs, Zsolt Czimmerer, Máté Manczinger, Gergő Balogh, József Tóvári, László G. Puskás, Gabor Janos Szebeni
    Single cell immunophenotyping revealed the association of CD4+ central-and CD4+ effector memory T-cells linking exacerbating chronic obstructive pulmonary disease and NSCLC, Frontiers in Immunology, December 20, 2023, Article 1297577.
    https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2023.1297577/full
  7. Patrícia Neuperger, Klára Szalontai, Nikolett Gémes, József Á Balog, László Tiszlavicz, József Furák, György Lázár, Laszlo G Puskas, Gábor J. Szebeni
    Single-Cell Mass Cytometric Analysis of Peripheral Immunity and Multiplex Plasma Marker Profiling of Non-Small Cell Lung Cancer Patients Receiving PD-1 Targeting Immune Checkpoint Inhibitors in Comparison with Platinum-Based Chemotherapy, Frontiers in Immunology, October 13, 2023, Article 1243233.
    https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2023.1243233/full
  8. Gábor J. Szebeni, Róbert Alföldi, Lajos I. Nagy, Patrícia Neuperger, Nikolett Gémes, József A. Balog, László Tiszlavicz, László G. Puskás
    Introduction of an ultraviolet C irradiated 4T1 murine breast cancer whole cell vaccine model, Vaccines, 2023, Volume 11, Issue 7, Article 1254.
    https://www.mdpi.com/2076-393X/11/7/1254
  9. Enikő Szabó, Szabolcs Modok, Benedek Rónaszéki, Anna Faragó, Nikolett Gémes, Lajos I. Nagy, László Hackler Jr., Katalin Farkas, Patrícia Neuperger, József Á. Balog, Attila Balog, László G. Puskás, Gabor J. Szebeni
    Comparison of humoral and cellular immune responses in hematologic diseases following completed vaccination protocol with BBIBP-CorV, or AZD1222, or BNT162b2 vaccines against SARS-CoV-2, Frontiers in Medicine, July 17, 2023, Article 1176168.
    https://www.frontiersin.org/journals/medicine/articles/10.3389/fmed.2023.1176168/full
  10. Nikolett Gémes, Zsófia Makra, Patrícia Neuperger, Enikő Szabó, József Á. Balog, Lili Borbála Flink, Beáta Kari, László Hackler Jr., László G. Puskás, Iván Kanizsai, Gábor J. Szebeni
    A Cytotoxic Survey On 2-Amino-1H-Imidazol Based Synthetic Marine Sponge Alkaloid Analogues, Drug Development Research, December 2022, Volume 83, Issue 8, Pages 1906-1922.
    https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/full/10.1002/ddr.22006
  11. Dániel Honfi, Nikolett Gémes, Enikő Szabó, Patrícia Neuperger, József Á. Balog, Lajos I. Nagy, Gergely Toldi, László G. Puskás, Gábor J. Szebeni, Attila Balog
    Comparison of homologous and heterologous booster SARS-CoV-2 vaccination in autoimmune rheumatic and musculoskeletal patients, International Journal of Molecular Sciences, 2022, Volume 23, Issue 19, Article 11411.
    https://www.mdpi.com/1422-0067/23/19/11411
  12. Kupai, K.; Várkonyi, T.; Török, S.; Gáti, V.; Czimmerer, Z.; Puskás, L.G.; Szebeni, G.J.
    Recent Progress in the Diagnosis and Management of Type 2 Diabetes Mellitus in the Era of COVID-19 and Single Cell Multi-Omics Technologies, Life, 2022, Volume 12, Article 1205.
    https://www.mdpi.com/2075-1729/12/8/1205
  13. Gábor J. Szebeni, Nikolett Gémes, Dániel Honfi, Enikő Szabó, Patrícia Neuperger, József Á. Balog, Lajos I. Nagy, Zoltan Szekanecz, Laszlo G Puskas, Gergely Toldi, Attila Balog
    Humoral and Cellular Immunogenicity and Safety of Five Different SARS-CoV-2 Vaccines in Patients With Autoimmune Rheumatic and Musculoskeletal Diseases in Remission or With Low Disease Activity and in Healthy Controls: A Single Center Study, Frontiers in Immunology, 2022, Article 846248.
    https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2022.846248/full
  14. Neuperger, P.; Balog, J.Á.; Tiszlavicz, L.; Furák, J.; Gémes, N.; Kotogány, E.; Szalontai, K.; Puskás, L.G.; Szebeni, G.J.
    Analysis of the Single-Cell Heterogeneity of Adenocarcinoma Cell Lines and the Investigation of Intratumor Heterogeneity Reveals the Expression of Transmembrane Protein 45A (TMEM45A) in Lung Adenocarcinoma Cancer Patients, Cancers, 2022, Volume 14, Article 144.
    https://www.mdpi.com/2072-6694/14/1/144
  15. Klára Szalontai, Nikolett Gémes, József Furák, Tünde Varga, Patrícia Neuperger, József Á. Balog, László G. Puskás, Gábor J. Szebeni
    Chronic Obstructive Pulmonary Disease: Epidemiology, Biomarkers and Paving the Way to Lung Cancer, Journal of Clinical Medicine, 2021, Special Issue: Living with Chronic Obstructive Pulmonary Disease, Volume 10, Issue 13, Article 2889.
    https://www.mdpi.com/2077-0383/10/13/2889
  16. Jozsef A. Balog, Agnes Kemeny, Laszlo G. Puskas, Szilard Burcsar, Attila Balog, Gabor J. Szebeni
    Investigation of newly diagnosed drug-naive patients with systemic autoimmune diseases revealed the cleaved peptide tyrosine tyrosine (PYY 3-36) as a specific plasma biomarker of rheumatoid arthritis, Mediators of Inflammation, 2021, Article 5523582.
    https://onlinelibrary.wiley.com/doi/10.1155/2021/5523582
  17. József Á. Balog, Viktor Honti, Éva Kurucz, Beáta Kari, László G. Puskás, István Andó, Gábor J. Szebeni
    Immunoprofiling of Drosophila Hemocytes by Single-cell Mass Cytometry, Genomics Proteomics & Bioinformatics, 2021, March 10, Article S1672-0229.
    https://academic.oup.com/gpb/article/19/2/243/7229852
  18. Kotogány, E.; Balog, J.Á.; Nagy, L.I.; Alföldi, R.; Bertagnolo, V.; Brugnoli, F.; Demjén, A.; Kovács, A.K.; Batár, P.; Mezei, G.; Szabó, R.; Kanizsai, I.; Varga, C.; Puskás, L.G.; Szebeni, G.J
    Imidazo[1,2-b]pyrazole-7-Carboxamide Derivative Induces Differentiation-Coupled Apoptosis of Immature Myeloid Cells Such as Acute Myeloid Leukemia and Myeloid-Derived Suppressor Cells, International Journal of Molecular Sciences, 2020, Volume 21, Article 5135.
    https://www.mdpi.com/1422-0067/21/14/5135
  19. József Á. Balog, László Hackler Jr., Anita K. Kovács, Patrícia Neuperger, Róbert Alföldi, Lajos I. Nagy, László G. Puskás, Gábor J. Szebeni
    Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model, International Journal of Molecular Sciences, 2020, Volume 21, Issue 1, Article 170.
    https://www.mdpi.com/1422-0067/21/1/170
  20. Alföldi R, Balog JÁ, Faragó N, Halmai M, Kotogány E, Neuperger P, Nagy LI, Fehér LZ, Szebeni GJ, Puskás LG
    Single Cell Mass Cytometry of Non-Small Cell Lung Cancer Cells Reveals Complexity of In vivo And Three-Dimensional Models over the Petri-dish, Cells, 2019, Volume 8, Issue 9, Article 1093.
    https://www.mdpi.com/2073-4409/8/9/1093
  21. Gábor J. Szebeni, Lajos I. Nagy, Anikó Berkó, Alexandra Hoffmann, Liliána Z. Fehér, Mária Bagyánszki, Beáta Kari, József A. Balog, László Hackler Jr., Iván Kanizsai, Anikó Pósa, Csaba Varga, László G. Puskás
    The Anti-Inflammatory Role of Mannich Curcuminoids; Special Focus on Colitis, Molecules, 2019, Volume 24, Issue 8, Article 1546.
    https://www.mdpi.com/1420-3049/24/8/1546
  22. Gabor J. Szebeni, Csaba Vizler, Klara Kitajka, Laszlo G. Puskas
    Inflammation and Cancer: extra- and intracellular determinants of tumor-associated macrophages as tumor promoters, Mediators of Inflammation, 2017, Article 9294018.
    https://onlinelibrary.wiley.com/doi/10.1155/2017/9294018
  23. Gabor J. Szebeni, Csaba Vizler, Lajos I. Nagy, Klara Kitajka, Laszlo G. Puskas
    Pro-tumoral inflammatory myeloid cells as emerging therapeutic targets, International Journal of Molecular Sciences, 2016, Volume 17, Issue 11, Article 1958.
    https://www.mdpi.com/1422-0067/17/11/1958

Team

Check our Team

László PUSKÁS

scientific adviser

Gábor SZEBENI

senior research associate

Ágnes ZVARA

senior research associate

Fanni BALOGH

research associate

Nóra Katalin FARAGÓ

research associate

Beáta KARI

research associate

Nikolett GÉMES

research associate

Patrícia NEUPERGER

research associate

Edit, KOTOGÁNY

junior research associate

Beáta MANDÁKNÉ MILITÁR

laboratory assistant/administrator expert

Cynthia SZABÓ

laboratory assistant/administrator expert