Flow cytometric measurement of [Ca2+]i and pHi in conjugated natural killer cells and K562 target cells during the cytotoxic process

Abstract

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 (pH_i) 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 pH_i (0.2–0.3 pH units). TC do not reveal a significant change in pH_i.