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Flow Cytometry–Based Single Cell Analyses of Bacterial Adaptation to Intracellular Environments

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Part of the Methods in Molecular Biology book series (MIMB,volume 2427)

Abstract

Since decades, flow cytometry (FC) is a powerful technique to perform single cell analyses with high accuracy and throughput. Moreover, FC is the method of choice to study bacterial cell heterogeneity and complements single-cell imaging techniques. The complex experimental approaches for FC sample preparation and the subsequent FC adjustment and gating strategy demand careful considerations to be successful when analyzing complex microbial populations, especially when liberated populations of intracellular bacterial pathogens, or bacterial pathogens inside intact host cells are analyzed. Here, we provide a set of experimental protocols for FC sample preparation of (1) in vitro cultured bacterial cells, (2) liberated intracellular bacteria from host cells, or (3) preparation of intact infected phagocytic or epithelial cells commonly used as host cells in infection biology. Since sample preparation, cytometer adjustment, and gating strategy are essential for experimental success, we aim to provide our expertise to support application of FC by other researchers.

Key words

  • Flow cytometry
  • Single cell analysis
  • Intracellular bacteria
  • Host cells
  • High-throughput analyses

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References

  1. Dittrich W, Göhde W (1969) Impulsfluorimetrie bei Einzelzellen in Suspensionen. Z Naturf 24b:360–361

    Google Scholar 

  2. Brown M, Wittwer C (2000) Flow cytometry: principles and clinical applications in hematology. Clin Chem 46(8):1221–1229. https://doi.org/10.1093/clinchem/46.8.1221

    CAS  CrossRef  PubMed  Google Scholar 

  3. Rieseberg M, Kasper C, Reardon KF, Scheper T (2001) Flow cytometry in biotechnology. Appl Microbiol Biotechnol 56(3–4):350–360. https://doi.org/10.1007/s002530100673

    CAS  CrossRef  PubMed  Google Scholar 

  4. Chow S, Hedley D (2001) Flow cytometric measurement of intracellular pH. Curr Protoc Cytom. Chapter 9:Unit 9 3. https://doi.org/10.1002/0471142956.cy0903s14

  5. Eruslanov E, Kusmartsev S (2010) Identification of ROS using oxidized DCFDA and flow-cytometry. Methods Mol Biol 594:57–72. https://doi.org/10.1007/978-1-60761-411-1_4

    CAS  CrossRef  PubMed  Google Scholar 

  6. Hedley DW, Chow S (1994) Evaluation of methods for measuring cellular glutathione content using flow cytometry. Cytometry 15(4):349–358. https://doi.org/10.1002/cyto.990150411

    CAS  CrossRef  PubMed  Google Scholar 

  7. June CH, Abe R, Rabinovitch PS (1997) Measurement of intracellular calcium ions by flow cytometry. Curr Protoc Cytom. Chapter 9:Unit 9 8. https://doi.org/10.1002/0471142956.cy0908s02

  8. Pozarowski P, Darzynkiewicz Z (2004) Analysis of cell cycle by flow cytometry. Methods Mol Biol 281:301–311. https://doi.org/10.1385/1-59259-811-0:301

    CAS  CrossRef  PubMed  Google Scholar 

  9. Riccardi C, Nicoletti I (2006) Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat Protoc 1(3):1458–1461. https://doi.org/10.1038/nprot.2006.238

    CAS  CrossRef  PubMed  Google Scholar 

  10. Shapiro HM (2000) Membrane potential estimation by flow cytometry. Methods 21(3):271–279. https://doi.org/10.1006/meth.2000.1007

    CAS  CrossRef  PubMed  Google Scholar 

  11. Vermes I, Haanen C, Reutelingsperger C (2000) Flow cytometry of apoptotic cell death. J Immunol Methods 243(1–2):167–190. https://doi.org/10.1016/S0022-1759(00)00233-7

    CAS  CrossRef  PubMed  Google Scholar 

  12. Hansmeier N, Miskiewicz K, Elpers L, Liss V, Hensel M, Sterzenbach T (2017) Functional expression of the entire adhesiome of Salmonella enterica serotype Typhimurium. Sci Rep 7(1):10326. https://doi.org/10.1038/s41598-017-10598-2

  13. Berney M, Hammes F, Bosshard F, Weilenmann HU, Egli T (2007) Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight kit in combination with flow cytometry. Appl Environ Microbiol 73(10):3283–3290. https://doi.org/10.1128/AEM.02750-06

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  14. Sasaki DT, Dumas SE, Engleman EG (1987) Discrimination of viable and non-viable cells using propidium iodide in two color immunofluorescence. Cytometry 8(4):413–420. https://doi.org/10.1002/cyto.990080411

    CAS  CrossRef  PubMed  Google Scholar 

  15. Stapels DAC, Hill PWS, Westermann AJ, Fisher RA, Thurston TL, Saliba AE, Blommestein I, Vogel J, Helaine S (2018) Salmonella persisters undermine host immune defenses during antibiotic treatment. Science 362 (6419):1156–1160. https://doi.org/10.1126/science.aat7148

  16. Röder J, Hensel M (2020) Presence of SopE and mode of infection result in increased Salmonella-containing vacuole damage and cytosolic release during host cell infection by Salmonella enterica. Cell Microbiol 22(5):e13155. https://doi.org/cmi.13155/cmi.13155

    Google Scholar 

  17. Helaine S, Thompson JA, Watson KG, Liu M, Boyle C, Holden DW (2010) Dynamics of intracellular bacterial replication at the single cell level. Proc Natl Acad Sci U S A 107(8):3746–3751. https://doi.org/10.1073/pnas.1000041107

    CrossRef  PubMed  PubMed Central  Google Scholar 

  18. Helaine S, Cheverton AM, Watson KG, Faure LM, Matthews SA, Holden DW (2014) Internalization of Salmonella by macrophages induces formation of nonreplicating persisters. Science 343(6167):204–204. https://doi.org/10.1126/science.1244705

  19. Schulte M, Olschewski K, Hensel M (2021) The protected physiological state of intracellular Salmonella enterica persisters reduces host cell-imposed stress. Commun Biol 4(1):520. https://doi.org/10.1038/s42003-021-02049-6

  20. Noster J, Chao TC, Sander N, Schulte M, Reuter T, Hansmeier N, Hensel M (2019) Proteomics of intracellular Salmonella enterica reveals roles of Salmonella pathogenicity island 2 in metabolism and antioxidant defense. PLoS Pathog 15(4):e1007741. https://doi.org/10.1371/journal.ppat.1007741

  21. Galeev A, Suwandi A, Bakker H, Oktiviyari A, Routier FH, Krone L, Hensel M, Grassl GA (2020) Proteoglycan-dependent endo-lysosomal fusion affects intracellular survival of Salmonella Typhimurium in epithelial cells. Front Immunol 11: 731. https://doi.org/10.3389/fimmu.2020.00731

  22. Schulte M, Olschewski K, Hensel M (2021) Fluorescent protein-based reporters reveal stress response of intracellular Salmonella enterica at level of single bacterial cells. Cell Microbiol 23(3):e13293. https://doi.org/10.1111/cmi.13293

  23. Reuter T, Scharte F, Franzkoch R, Liss V, Hensel M (2021). Single cell analyses reveal distinct adaptation of typhoidal and non-typhoidal Salmonella enterica serovars to intracellular lifestyle. PLoS Pathog 17: e1009319. https://doi.org/10.1371/journal.ppat.1009319

  24. Röder J, Felgner P, Hensel M (2021) Single cell analyses reveal phosphate availability as critical factor for nutrition of Salmonella enterica within mammalian host cells. Cell Microbiol: e13374. https://doi.org/10.1111/cmi.13374

  25. Röder J, Felgner P, Hensel M (2021) Comprehensive single cell analyses of the nutritional environment of intracellular Salmonella enterica. Front Cell Infect Microbiol 11:624650. https://doi.org/10.3389/fcimb.2021.624650

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Acknowledgments

This work was supported by the DFG by project P15 in SFB 944.

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Correspondence to Michael Hensel .

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Schulte, M., Hensel, M. (2022). Flow Cytometry–Based Single Cell Analyses of Bacterial Adaptation to Intracellular Environments. In: Gal-Mor, O. (eds) Bacterial Virulence. Methods in Molecular Biology, vol 2427. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1971-1_10

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  • DOI: https://doi.org/10.1007/978-1-0716-1971-1_10

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1970-4

  • Online ISBN: 978-1-0716-1971-1

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