Dye exclusion tests are used to determine the number of live and dead cells. These
assays are based on the principle that intact plasma membranes in live cells exclude
specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB)
exclusion assay has limitations. The dye can be incorporated by live cells after a
short exposure time, and personal reliability, related to the expertise of the
analyst, can affect the results. We propose an alternative assay for evaluating cell
viability that combines the TB exclusion test and the high sensitivity of the flow
cytometry technique. Previous studies have demonstrated the ability of TB to emit
fluorescence when complexed with proteins. According to our results, TB/bovine serum
albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is
detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v)
was defined as the optimum concentration for distinguishing unstained living cells
from fluorescent dead cells, and fluorescence emission was stable for 30 min after
cell treatment. Although previous studies have shown that TB promotes green
fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in
human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal
antibody. We observed a high correlation between the percentage of propidium
iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar
double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the
results indicate that a TB exclusion assay by flow cytometry can be employed as an
alternative tool for quick and reliable cell viability analysis.
HTLV-I/II infection is present in all regions of Brazil, but its prevalence varies according to the geographical area, being higher in Bahia, Pernambuco and Pará. It has been estimated that Brazil has the highest absolute number of infected individuals in the world. Blood donors screening and research conducted with special groups (indigenous population of Brazil, IV drug users and pregnant women) are the major sources of information about these viruses in our Country. HTLV-I causes adult T cell leukemia/lymphoma (ATLL), HTLV associated myelopathy/tropical spastic paraparesis (HAM/TSP), HTLV associated uveitis (HAU), dermatological and immunological abnormalities. HTLV-II is not consistently associated with any disease. Diagnosis is established using screening (enzymatic assays, agglutination) and confirmatory (Western blot, PCR) tests. The viruses are transmitted by blood and contaminated needles, by sexual relations and from mother to child, especially by breast feeding. Prevention efforts should focus on education of positive blood donors, infected mothers and IV drug users.
Dimethylsulfoxide (DMSO) is an amphipathic molecule composed of a polar domain characterized by the sulfinyl and two nonpolar methyl groups, for this reason it is able to solubilize polar and nonpolar substances and transpose hydrophobic barriers. DMSO is widely used to solubilize drugs of therapeutic applications and studies indicated that 10% v/v concentration did not modify culture viability when used to treat human peripheral blood mononuclear cells (PBMC). However, some DMSO concentrations could influence lymphocyte activation and present anti-inflammatory effects. Therefore, the objective of this study was to evaluate the effect of DMSO on lymphocyte activation parameters. Cell viability analysis, proliferation, and cytokine production were performed on PBMC from six healthy subjects by flow cytometry. The results indicated that 2.5% v/v DMSO concentrations did not modify lymphocytes viability. DMSO at 1% and 2% v/v concentrations reduced the relative proliferation index of lymphocytes and at 5% and 10% v/v concentrations reduced the percentage of total lymphocytes, cluster of differentiation 4+ (CD4+) T lymphocytes and CD8+ T lymphocytes interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2) producers. Thus, it was concluded that DMSO has an in vitro anti-inflammatory effect by reducing lymphocyte activation demonstrated with proliferation reduction and the decrease of cytokine production.
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