Measurement of Intracellular Calcium Levels by Flow Cytometry Following Treatment of Murine Macrophage/Monocytes with Mistletoe Lectin
The purified mistletoe lectin, ML-I, with galactoside specificity has a dramatic antitumor effect and also causes secretion of cytokines such as tumor necrosis factor-α, interleukin-1 and interleukin-6 (Hajto et al. 1990; Joshi et al. 1991; Gabius et al. 1992). The precise mechanism(s) of action of ML-I at the cellular level is not known. Some of the cellular regulatory factors can lead to rapid changes in a variety of cellular functions, including changes in intracellular second messenger systems affected by the binding of lectin ML-I. These intracellular messengers may further mediate the induction of nuclear activities essential for the production of immune regulating/stimulating cytokines and other factors. One of the main factors involved in intracellular second messenger pathways is changes in kinetics of intracellular calcium mobilization/concentration. The effects of lectins on the levels of intracellular calcium levels can be measured using appropriate fluorescent dyes such as Indo-1 or Flura-2 or Fluo-3 and flow cytometry techniques (June and Rabinovitch 1991). The flow cytometer can be used to measure various functional parameters that are of interest to lectinologists. The flow cytometry can be used to measure the concentration of intracellular free ions such as calcium in single living cells using appropriate fluorescent probes. The commonly used fluorescent probes are Indo-1 and Fluo-3. Indo-1 requires a flow cytometer which is capable of UV illumination, whereas in the case of Fluo-3 dye, the UY source of illumination is not required. Fluo-3 requires regular argon ion laser. This chapter focuses on the determination of intracellular calcium levels in a murine macrophage/monocyte cell line J774A.1 following treatment with purified mistletoe lectin ML-I using flow cytometry techniques using Fluo-3 as fluorescent probe. Fluo-3 is a fluorescein-based calcium probe originally developed by Minta et al. (1989).
KeywordsFluorescent Probe Intracellular Calcium Level Monocytic Leukemia Acetoxymethyl Ester Mistletoe Lectin
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