Login processing...

Trial ends in Request Full Access Tell Your Colleague About Jove

Immunology and Infection


doi: 10.3791/55107 Published: April 5, 2017


Name Company Catalog Number Comments
Candida albicans ATCC 10231 Vitroids  80 CFU Sigma-Aldrich RQC14003-10EA Place directly on a spread agar plate according to the manufacturer's instructions
YPD broth Sigma-Aldrich Y1375 Follow manufacturer's instructions, various providers exist
Dextran Clinical grade MW = 200,000-300,000 MP Biomedicals 2101514 Use gloves 
Heparin Sodium solution for infusion (preservative free), 1,000 IU/mL  Fannin, Wockhardt UK Ltd FP1077 We purchased from UCH inpatient pharmacy
SNARF-1 Carboxylic Acid, Acetate, Succinimidyl Ester - Special Packaging Thermo Scientific/Invitrogen SS22801 Use gloves
5-(and-6)-Carboxy SNARF-1, Acetoxymethyl Ester, Acetate Thermo Scientific/Invitrogen C1272 Use gloves
Vivaglobin human IgG  160 mg/mL solution CSL Behring received as a gift Various providers exist
Normal mouse serum Abcam ab7486 Various providers exist
Lymphoprep (density gradient medium) Alere Ltd 1114547 Keep sterile and away from direct sunlight, can be substituted by Ficoll-Paque
Nigericin Sigma-Aldrich N7143 Toxic, use gloves, various providers exist
Saponin Sigma-Aldrich 47036 Toxic, use gloves, various providers exist
Dimethyl sulphoxide (DMSO) Sigma-Aldrich D8418 Toxic, use gloves, various providers exist
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) Sigma-Aldrich H3375 Various providers exist
Tris(hydroxymethyl)aminomethane (Tris) Sigma-Aldrich T1503  Various providers exist
Potassium chloride (KCl) Sigma-Aldrich P9333 Various providers exist
Sodium chloride (NaCl) Sigma-Aldrich S7653 Various providers exist
Magnesium chloride (MgCl2) Sigma-Aldrich M8266 Various providers exist
Magnesium sulphate (MgSO4) Sigma-Aldrich M7506 Various providers exist
Potassium dihydrogen phosphate (KH2PO4) Sigma-Aldrich P9791 Various providers exist
Calcium chloride (CaCl2) Sigma-Aldrich C1016 Various providers exist
Glucose Sigma-Aldrich D9434 Various providers exist
Absolute Ethanol  Sigma-Aldrich 2860 Various providers exist
Diphenylene iodonium (DPI) Sigma-Aldrich D2926 Various providers exist
Zinc chloride Sigma-Aldrich 208086 Various providers exist, do not dilute in phosphate based buffers as it will precipitate
Poly L lysine Sigma P4707 Various providers exist
Laser scanning confocal microscope (Zeiss LSM 700) Carl Zeiss The microscope must be able to excite at 555 nm and measure emission at two channels simultaneously (560-600 nm, >610 nm)
Ibidi µ slide 8 well ibiTreat Thistle Scientific IB-80826 Other appropriate imaging dishes can be substituted
Soniprep 150 MSE Any appropriate sonicator will suffice
TC20 Automated Cell Counter BioRad Various providers exist
1.5 mL Protein LoBind Tubes Sigma-Aldrich Z666505-100EA Specially used as Candida can stick to normal eppendorfs



  1. Segal, A. W. NADPH oxidases as electrochemical generators to produce ion fluxes and turgor in fungi, plants and humans. Open Biol. 6, (5), 515-517 (2016).
  2. Segal, A. W. How neutrophils kill microbes. Annu Rev Immunol. 23, 197-223 (2005).
  3. Cross, A. R., Segal, A. W. The NADPH oxidase of professional phagocytes - Prototype of the NOX electron transport chain systems. BBA Bioenergetics. 1657, (1), 1-22 (2004).
  4. Demaurex, N., El Chemaly, A. Physiological roles of voltage-gated proton channels in leukocytes. J Physiol. 588, (23), 4659-4665 (2010).
  5. Levine, A. P., Duchen, M. R., de Villiers, S., Rich, P. R., Segal, A. W. Alkalinity of neutrophil phagocytic vacuoles is modulated by HVCN1 and has consequences for myeloperoxidase activity. PLoS One. 10, (4), 0125906 (2015).
  6. Reeves, E. P., Lu, H., et al. Killing activity of neutrophils is mediated through activation of proteases by K+ flux. Nature. 416, (6878), 291-297 (2002).
  7. Menegazzi, R., Busetto, S., Dri, P., Cramer, R., Patriarca, P. Chloride ion efflux regulates adherence, spreading, and respiratory burst of neutrophils stimulated by tumor necrosis factor-alpha (TNF) on biologic surfaces. J Cell Biol. 135, (2), 511-522 (1996).
  8. Ohkuma, S., Poole, B. Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Cell Biol. 75, (7), 3327-3331 (1978).
  9. Jankowski, A., Scott, C. C., Grinstein, S. Determinants of the phagosomal pH in neutrophils. J Biol Chem. 277, (8), 6059-6066 (2002).
  10. Tsien, R. Y. Fluorescent Indicators of Ion Concentrations. Methods in Cell Biol. 30, 127-156 (1989).
  11. Lavis, L. D., Rutkoski, T. J., Raines, R. T. Tuning the pK a of Fluorescein to Optimize Binding Assays. Anal Chem. 79, (17), 6775-6782 (2007).
  12. Hurst, J. K., Albrich, J. M., Green, T. R., Rosen, H., Klebanoff, S. Myeloperoxidase-dependent fluorescein chlorination by stimulated neutrophils. J Biol Chem. 259, (8), 4812-4821 (1984).
  13. Nunes, P., Guido, D., Demaurex, N. Measuring Phagosome pH by Ratiometric Fluorescence Microscopy. J Vis Exp. (106), e53402 (2015).
  14. Canton, J., Khezri, R., Glogauer, M., Grinstein, S. Contrasting phagosome pH regulation and maturation in human M1 and M2 macrophages. Mol Biol Cell. 25, (21), 3330-3341 (2014).
  15. Schlafer, S., Dige, I. Ratiometric Imaging of Extracellular pH in Dental Biofilms. J Vis Exp. (109), e53622 (2016).
  16. Buckler, K. J., Vaughan-Jones, R. D. Application of a new pH-sensitive fluoroprobe (carboxy-SNARF-1) for intracellular pH measurement in small, isolated cells. Pfl ügers Archiv EJP. 417, (2), 234-239 (1990).
  17. El Chemaly, A., Nunes, P., Jimaja, W., Castelbou, C., Demaurex, N. Hv1 proton channels differentially regulate the pH of neutrophil and macrophage phagosomes by sustaining the production of phagosomal ROS that inhibit the delivery of vacuolar ATPases. J Leuko Biol. 95, (5), 827-839 (2014).
  18. Morgan, D., Capasso, M., et al. Voltage-gated proton channels maintain pH in human neutrophils during phagocytosis. Proc Natl Acad Sci USA. 106, (42), 18022-18027 (2009).
  19. Thomas, J. A., Buchsbaum, R. N., Zimniak, A., Racker, E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry. 18, (11), 2210-2218 (1979).
  20. Levine, A. P., Segal, A. W. The NADPH Oxidase and Microbial Killing by Neutrophils, With a Particular Emphasis on the Proposed Antimicrobial Role of Myeloperoxidase within the Phagocytic Vacuole. Microbiol Spectr. 4, (4), (2016).
  21. Segal, A. W., Geisow, M., Garcia, R., Harper, A., Miller, R. The respiratory burst of phagocytic cells is associated with a rise in vacuolar pH. Nature. 290, (5805), 406-409 (1981).
  22. Hunter, R. C., Beveridge, T. J. Application of a pH-sensitive fluoroprobe (C-SNARF-4) for pH microenvironment analysis in Pseudomonas aeruginosa biofilms. Appl Environ Microbiol. 71, (5), 2501-2510 (2005).
  23. Van Erp, P. E. J., Jansen, M. J. J. M., De Jongh, G. J., Boezeman, J. B. M., Schalkwijk, J. Ratiometric measurement of intracellular pH in cultured human keratinocytes using carboxy-SNARF-1 and flow cytometry. Cytometry. 12, (2), 127-132 (1991).
  24. Minamikawa, T., Takamatsu, T., Kashima, S., Fushiki, S., Fujita, S. Confocal calcium imaging with ultraviolet laser- scanning microscopy and indo-1. Micron. 24, (6), 551-556 (1993).
  25. Takahashi, A., Camacho, P., Lechleiter, J. D., Herman, B. Measurement of Intracellular Calcium. Physiol Rev. 79, (4), (1999).
  26. Hillson, E. J., Dewitt, S., Hallett, M. B. Optical methods for the measurement and manipulation of cytosolic free calcium in neutrophils. Methods Mol Biol. 412, Clifton, N.J. 125-137 (2007).
  27. Bernardo, J., Long, H. J., Simons, E. R. Initial cytoplasmic and phagosomal consequences of human neutrophil exposure to Staphylococcus epidermidis. Cytometry Part A. 77, (3), 243-252 (2010).
  28. Riazanski, V., Gabdoulkhakova, A. G., et al. TRPC6 channel translocation into phagosomal membrane augments phagosomal function. Proc Natl Acad Sci USA. 112, (47), 6486-6495 (2015).
  29. Martínez-Zaguilán, R., Martínez, G. M., Lattanzio, F., Gillies, R. J. Simultaneous measurement of intracellular pH and Ca2+ using the fluorescence of SNARF-1 and fura-2. Am J Physiol Cell Physiol. 260, (2), (1991).
Play Video

Cite this Article

Foote, J. R., Levine, A. P., Behe, P., Duchen, M. R., Segal, A. W. Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator. J. Vis. Exp. (122), e55107, doi:10.3791/55107 (2017).More

Foote, J. R., Levine, A. P., Behe, P., Duchen, M. R., Segal, A. W. Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator. J. Vis. Exp. (122), e55107, doi:10.3791/55107 (2017).

Copy Citation Download Citation Reprints and Permissions
View Video

Get cutting-edge science videos from JoVE sent straight to your inbox every month.

Waiting X
simple hit counter