Flow cytometric measurement of cytoplasmic pH: a critical evaluation of available fluorochromes

Cytometry. 1986 Jul;7(4):347-55. doi: 10.1002/cyto.990070409.


Three pH-sensitive fluorochromes-4-methyl-umbelliferone(4MU),2, 3-dicyano-hydroquinone (DCH), and 2',7'-bis(carboxyethyl)-5,6-carboxy fluorescein (BCECF)--were evaluated for their resolution, range, and stability of cellular fluorescence. Flow cytometric techniques for determining cytoplasmic pH (pHi) have been fully described for 4MU and DCH; BCECF has previously been used for fluorimetric estimation of pHi, and was adapted to flow cytometry. For each fluorochrome, the ratio of fluorescence intensity at two wavelengths gives a measure of pHi, which may be calibrated by obtaining the fluorescence ratios for cells suspended in buffers of varying pH in the presence of a proton ionophore. Reliable calibration proved difficult using 4MU, partly because of poor retention within cells. Both DCH and BCECF could be calibrated using a fluorescence ratio and had resolutions of 0.2 and 0.4 pH units, respectively. The fluorescence of DCH is so strongly pH dependent that there were practical difficulties in its use over a wide pH range; however, pHi measurements are possible between pH 6.0 and pH 7.5 using either DCH or BCECF. Substantial dye leakage was found for 4MU and, to a lesser extent, DCH, while BCECF was retained by cells for up to 2 hours. Despite its lower resolution BCECF had a usable range of more than 1.5 pH units and this coupled with its stable fluorescence and excitation at 488 nm rather than UV suggests a wide application.

MeSH terms

  • Cell Line
  • Cytoplasm / metabolism*
  • Flow Cytometry*
  • Fluoresceins
  • Fluorescent Dyes*
  • Humans
  • Hydrogen-Ion Concentration
  • Hydroquinones
  • Hymecromone
  • Leukemia / metabolism
  • T-Lymphocytes / metabolism


  • Fluoresceins
  • Fluorescent Dyes
  • Hydroquinones
  • Hymecromone
  • 2,3-dicyanohydroquinone
  • 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein