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 (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. © 1993 Wiley‐Liss, Inc. Copyright © 1993 Wiley‐Liss, Inc.