Flow Cytometric Measurement of [Ca2+]i and pHi in Conjugated Natural Killer Cells and K562 Target Cells during the Cytotoxic Process1,2

Marja van Graft, M. van Graft, Yvonne M. Kraan, Gezina M.J. Segers-Nolten, K. Radosevic, Katarina Radosevic, B.G. de Grooth, Jan Greve

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We describe a flow cytometric assay that enables one to follow conjugate formation between cytotoxic cells and their target cells during the cytotoxic process. In addition, the internal calcium concentration ([Ca2+]i) and internal pH (pHi) of the conjugated cells can be monitored and directly compared to the nonconjugated cells. This is achieved by labeling one cell type with the Ca2+-specific dye Fluo-3, while the other cell type is labeled with the pH-sensitive dye SNARF-1. As these fluorochromes have different emission spectra, events positive for both fluorochromes are identified as conjugates. The results show that the conjugates can be clearly distinguished from single cytotoxic cells [natural killer (NK) cells] and target cells [K562 cells, (TC)]. Upon binding, [Ca2+]i is increased in the NK cells as well as in the TC. In conjugated NK cells this increase of [Ca2+]i is temperature dependent and is followed by a decrease to a normal [Ca2+]i value later on. The [Ca2+]i in NK cells increases in 2 steps, which may be related to the binding-and lethal hit phase. Upon conjugate formation, NK cells show a slight increase in pHi (0.2-0.3 pH units). TC do not reveal a significant change in pHi.
Original languageUndefined
Pages (from-to)257-264
Number of pages8
Issue number3
Publication statusPublished - 1993


  • Cytotoxic interaction
  • IR-60718
  • SNARF-1
  • METIS-129387
  • intracellular [Ca2+]
  • intracellular pH
  • Fluo-3

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