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Measurement of Intracellular Calcium Levels by Flow Cytometry Following Treatment of Murine Macrophage/Monocytes with Mistletoe Lectin

  • S. S. Joshi
  • H.-J. Gabius
Part of the Springer Laboratory book series (SLM)

Abstract

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).

Keywords

Fluorescent Probe Intracellular Calcium Level Monocytic Leukemia Acetoxymethyl Ester Mistletoe Lectin 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. Gabius HJ, Walzel H, Joshi SS, Kruip J, Kojima S, Goke V, Kratziu H, Gabius S (1992) The immunomodulatory galactoside-specific lectin from mistletoe: partial sequence analysis, cell and tissue binding, and impact on intracellular biosignaling of monocytic leukemia cells. Anticancer Res 12: 669–676PubMedGoogle Scholar
  2. Hajto T, Hostanska K, Frei K, Rordorf C, Gabius HJ (1990) Increased secretion of tumor necrosis factor-α, interleukin-1 and interleukin-6 by human mononuclear cells exposed to β-galactoside specific lectin from clinically applied mistletoe extract. Cancer Res 50: 3322–3326PubMedGoogle Scholar
  3. Joshi SS, Komanduri KC, Gabius S, Gabius HJ (1991) Immunotherapeutic effects of purified mistletoe lectin, ML-I, on murine large cell lymphoma. In: Lectins and Cancer. HJ Gabius and S Gabius (Eds.), Springer-Verlag, Heidelberg, pp. 207–217CrossRefGoogle Scholar
  4. June CH, Rabinovitch PS (1991) In: Current protocols in immunology. JE Coligan, AM Kruisbeek, EM Shevaeh and W Strober (Eds.), John Wiley & Sons, New York, pp. 5.5.2Google Scholar
  5. Minta A, Kao JPY, Tsien RY (1989) Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J Biol Chem 264: 8171–8178PubMedGoogle Scholar
  6. Rijkers GT, Justement LB, Grifferen AW, Cambier JC (1990) Improved method for measuring intracellular Ca++ with Fluo-3. Cytometry 11: 923–927PubMedCrossRefGoogle Scholar
  7. Vandenberghe PA, Ceuppens JL (1990) Flow cytometric measurement of cytoplasmic free calcium in human peripheral blood T-lymphocytes with Fluo-3, a new fluorescent indicator. J Immunol Methods 127: 197–205PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1993

Authors and Affiliations

  • S. S. Joshi
  • H.-J. Gabius

There are no affiliations available

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