Flow cytometric measurement of [Ca2+]i and pHi in conjugated natural killer cells and K562 target cells during the cytotoxic process.
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 Ca(2+)-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.
Van Graft
M
M
Department of Applied Physics, University of Twente, Enschede, The Netherlands.
Kraan
Y M
YM
Segers
I M
IM
Radosevic
K
K
De Grooth
B G
BG
Greve
J
J
D000814:Aniline Compounds; D001578:Benzopyrans; D002118:Calcium / Q000032:analysis*; D002460:Cell Line / Q000737:chemistry*; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007694:Killer Cells, Natural / Q000737:chemistry*; D014966:Xanthenes
D000814:Aniline Compounds; D001578:Benzopyrans; D005456:Fluorescent Dyes; C063917:SNARF dye; D014966:Xanthenes; C059715:Fluo-3; D002118:Calcium