The Alamar Blue® assay to determine drug sensitivity of African trypanosomes (T.b. rhodesiense and T.b. gambiense) in vitro

There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

Abstract

Alamar Blue, an indicator for metabolic cell function, was evaluated as a fluorescent and as a colorimetric dye in drug sensitivity assays for human pathogenic African trypanosomes, Trypanosoma brucei rhodesiense and T.b. gambiense. The experimental conditions were adjusted to find those where the relationship between trypanosome number and Alamar Blue signal was linear over the widest possible range. Fluorescent signals correlated to trypanosome numbers from 10(4) trypanosomes/ml (T.b. rhodesiense) and 10(5) trypanosomes/ml (T.b. gambiense) up to 2-3 x 10(6) trypanosomes/ml when trypanosomes were incubated for 2 h with 10% Alamar Blue. Trypanocidal activity of common drugs (melarsoprol, DFMO, suramin, pentamidine and diminazene aceturate) was determined employing this assay. The IC50 values obtained were comparable to those obtained with another fluorochrome, BCECF-AM. The Alamar Blue assay can be applied for drug screening, since it is simple, reproducible and economical. The assay can also be used in field sites with less equipped laboratories, because in addition to fluorometric endpoint determination, a colorimetric reading is possible.

Related collections

Most cited references 10

Record: found
Abstract: found
Article: not found

A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay.

S. Ansar Ahmed, Robert M. Gogal, Jane E. Walsh (1994)

A one-step non-radioactive assay to determine the proliferation of murine lymphocytes, lymphoid tumor cells and hybridoma cells is described. This assay requires the addition of Alamar Blue dye to cell cultures and the degree of change in its color, which is reflective of the extent of cellular proliferation, can be determined by an ELISA plate reader. Alamar Blue must be added during the initial phase of cell culture. The pattern of concanavalin A (ConA) or anti-CD3 antibody-induced proliferative response of murine lymphocytes as assessed by Alamar Blue was similar to that of a [3H]thymidine assay. Similarly, the spontaneous proliferation curve of anti-CD3 antibody secreting cell line (YCD3-1), monocytic macrophage cell lines (PU5-1.8, P388D1, J774.1) and myeloma cells (Sp2/0) as determined by Alamar Blue closely resembled that of the [3H]thymidine assay. The minimum detectable number of proliferating cells was comparable in Alamar Blue and [3H]thymidine assays. Since cell lysis/extraction and washing procedures are not involved in the Alamar Blue assay, this approach has several distinct advantages over currently available assays (eg. [3H]thymidine). First, it allows daily monitoring of proliferation without compromising the sterility of cultures. An indication of proliferation can be evaluated (spectrophotometrically or visually) as early as 24 h after ConA stimulation. Second, unlike previously reported assays, Alamar Blue permits further analysis of proliferating cells by other methods. Analysis of cells in culture with Alamar Blue for various surface antigens (CD44, CD45RB, CD4, heat stable antigen) by flow cytometry revealed that the fluorescent profile and relative percentage of cells in cultures with the Alamar Blue were comparable to those without this reagent. The salient advantages of Alamar Blue assay over the [3H]thymidine assay include: (i) non-radioactivity; (ii) simplicity; (iii) less costly; (iv) non-labor intensive; (v) rapidity of assessment of proliferation of large number of samples; (vi) non-toxicity; (vii) usefulness in determining the kinetics of cell growth of hybridomas; and (viii) non-interference of secretion of antibodies by a hybridoma cell line.

0 comments Cited 213 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: not found
Article: not found

A comparison of three methods of estimating EC50 in studies of drug resistance of malaria parasites.

W Huber, Jacob Koella (1993)

0 comments Cited 94 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

Current situation of African trypanosomiasis.

F.A.S. Kuzoe (1993)

African trypanosomiasis (sleeping sickness) is fatal, if untreated, and occurs in 36 African countries, south of the Sahara, where some 50 million people are at risk of acquiring infection. In the absence of adequate control measures epidemics occur, which are costly and difficult to control. The history of sleeping sickness has been characterized by waves of epidemics, resurgences and outbreaks. Nevertheless, sleeping sickness has been brought practically under control in the early 1950s, in West and Central Africa, through systematic surveillance of the population at risk and in East Africa, mainly by vector control. Following the attainment of independence from colonial rule in subsequent years, failure by national health authorities to give due attention to sleeping sickness control, due to civil and political unrest, lack of adequate resources and competing national health priorities, has resulted in epidemics and the recrudescence of many old foci and the appearance of new ones. Thus, sleeping sickness is currently a major concern among many countries, particularly in East and Central Africa. During the past decade, progress has been achieved through research in the development of new tools for diagnosis, which are simple to use by national health personnel and for vector control, which can be used at the community level. Eflornithine, a new drug, has been registered for the treatment of gambiense sleeping sickness, and although it is expensive, it is relatively safe and provides an alternative therapy to the existing treatment, which may cause severe adverse effects. These tools have raised hopes for improved control, but their integration into health care systems, which could improve surveillance of the population at risk, has been slow. In view of the worsening economic situation of endemic countries, and the focus of attention and resources on the AIDS pandemic, prospects of any significant improvement in the sleeping sickness situation would largely depend on the successful mobilization of external resources.