The Agarose Overlay Method as a Screening Approach for Ocular Irritancy: Application to Cosmetic Products

The in vitro agarose overlay method was investigated as an alternative to the Draize eye irritation test for the evaluation of the ocular irritancy of cosmetic products. Modifications to the original protocol include the definition of an area of lysis (expressed as a weighty)/toxicity relationship for each product by a planimetric method and the creation of an agarose classification. 56 different cosmetic formulations, including emulsions, gels, lotions and tonics, were evaluated using this modified test, and the results were correlated with in vivo Draize data. Four different agarose classes were delimited: non-irritant; minimally irritant; mildly irritant; and irritant; corresponding respectively to the following scoring scales (x 10 4g): 0; 0 &lt;x&lt;200; 200≤ x &lt;450 and ≥ 450. A high correlation between the agarose overlay method and the Draize test was observed when taking into account two classes: non-irritant (non-irritant, minimally irritant) and irritant (mildly irritant, moderately irritant [Draize] or irritant [agarose]) as shown by an 86% concordance value. In this study, the sensitivity was 92% (11–12 irritants were predicted by the agarose diffusion method) and the specificity was 84%. The overestimation error of 16% suggests that the agarose overlay method might be slightly more sensitive than the Draize eye test. When applying the kappa test, the agarose overlay method reached a 64% coefficient, attesting that the good correlation with the Draize test was not due to chance. In relation to the Landis &amp; Koch classification, this percentage places the agarose overlay method in the “good concordance” class. Based on these results, the use of the agarose overlay method for screening the ocular irritation potential for the cosmetics industry can be considered to be a very interesting alternative to be included in a battery of tests. </jats:p

High resolution analysis of cell cycle-correlated vimentin expression in asynchronously grown, TPA-treated MPC-11 cells by the novel flow cytometric multiparameter BrdU-hoechst/PI and immunolabeling technique

High resolution, multiparameter analysis using the flow cytometric BrdU/Hoechst quenching technique has been applied to study cell cycle kinetics and vimentin expression in individual cells of asynchronously grown MPC−11 mouse plasmacytoma cell cultures treated with 12−O−tetradecanoylphorbol−13−acetate (TPA) to induce in vitro differentiation. BrdU treatment up to 16 h in the absence or presence of TPA did not affect either cell cycle progression or the kinetics or quantity of vimentin expression. TPA−treated cells became arrested in G1 phase of the second cell cycle; however, this G1 phase arrest was transient only. In addition, G1 phase cells located prior to a putative transition point at the beginning of TPA treatment were completely blocked in cell cycle progression. There is also evidence that cells located in G1 or G2/M phase at the beginning of TPA treatment finally expressed low levels of vimentin. On the contrary, cells located in S phase at TPA exposure showed high vimentin levels after treatment. The results presented here show that, with the flow cytometric BrdU/Hoechst quenching technique, one can correlate time−dependent protein expression at the single cell level in asynchronously grown cultures not only with the actual cell cycle state, but also with the history of cell replicatio

Guidelines for the use of flow cytometry and cell sorting in immunological studies

International audienceThe classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127(-) and CD127(+) early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127(-) and CD127(+) ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127(-) ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127(+) ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis