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It is well established that the immune response to alloantigens is characterized by the formation of effector cells which specifically destroy target cells carrying the sensitizing alloantigens in the absence of antibody and/or complement (for references, see 1). These effector cells belong to the thymus-derived (T) lymphocyte series and are generally referred to as cytotoxic T lymphocytes (CTL)1With the development of a precise and reproducible cytotoxic assay system (2), it has been possible to follow the appearance of CTL during the primary and secondary response of mice to normal or tumor allografts. In certain instances, such studies have shown an earlier appearance and a higher peak of cytotoxic activity in spleens of mice injected twice with allogeneic tumor cells as compared to that observed after primary immunization, suggesting the existence of an anamnestic response in T-cell-mediated immunity (3-4).Recently, it has been found that mouse CTL can be generated in vitro using mixed leukocyte cultures (MLC). Following the work of H~yry and Defendi (5), several studies have established the usefulness of the MLC system in providing an in vitro experimental model to analyze the series of events resulting in the appearance of CTL (6-10).The present study was undertaken to investigate the effect of in vivo immunization on CTL generation in MLC. Using improved tissue culture conditions, we found that cell-mediated cytotoxic responses were significantly higher when the responding cells were obtained from immune (MLC-Imm) rather than normal spleens. At the peak of the response, MLC-Imm populations were five times more cytotoxic than MLC populations. Physical and immunological characterization of the effector cells generated in MLC-Imm gave results simi-

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BackgroundThe effect that traditional and modern DNA extraction methods have on applications to study the role of gut microbiota in health and disease is a topic of current interest. Genomic DNA was extracted from three faecal samples and one probiotic capsule using three popular methods; chaotropic (CHAO) method, phenol/chloroform (PHEC) extraction, proprietary kit (QIAG). The performance of each of these methods on DNA yield and quality, microbiota composition using quantitative PCR, deep sequencing of the 16S rRNA gene, and sequencing analysis pipeline was evaluated.ResultsThe CHAO yielded the highest and the QIAG kit the lowest amount of double-stranded DNA, but the purity of isolated nucleic acids was better for the latter method. The CHAO method yielded a higher concentration of bacterial taxa per mass (g) of faeces. Sequencing coverage was higher in CHAO method but a higher proportion of the initial sequencing reads were retained for assignments to operational taxonomic unit (OTU) in the QIAG kit compared to the other methods. The QIAG kit appeared to have longer trimmed reads and shorter regions of worse quality than the other two methods. A distinct separation of α-diversity indices between different DNA extraction methods was not observed. When compositional dissimilarities between samples were explored, a strong separation was observed according to sample type. The effect of the extraction method was either marginal (Bray–Curtis distance) or none (unweighted Unifrac distance). Taxon membership and abundance in each sample was independent of the DNA extraction method used.ConclusionsWe have benchmarked several DNA extraction methods commonly used in gut microbiota research and their differences depended on the downstream applications intended for use. Caution should be paid when the intention is to pool and analyse samples or data from studies which have used different DNA extraction methods.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-2171-7) contains supplementary material, which is available to authorized users.

Cell-mediated cytotoxic responses in vitro to surface antigens associated with murine sarcoma virus (MSV)-induced tumors were investigated using mixed leukocyte-tumor cell cultures (MLTC). The source of responding cells was either spleens from normal C57BL/6 mice (primary MLTC) or spleens of C57BL/6 mice carrying or having rejected a MSV-induced tumor (secondary MLTC). Graffi virus-induced GiL-4 leukemia cells, Rauscher virus-induced RB1-5 leukemia cells, and MSV-induced MSV-B16 sarcoma cells were used as stimulating syngeneic tumor cells and/or target cells. Under appropriate culture conditions, cytolytic T lymphocytes (CTL) were generated in both primary and secondary MLTC. As assessed by a quantitative short-term 51Cr release assay system, CTL activity in secondary MLTC populations was at least 10-fold higher than that in primary MLTC populations, and 100-fold higher than that in spleen cells taken at the peak of the in vivo response of MSV-infected mice. The ability of spleen cells to mount a secondary CTL response in vitro could be observed as early as 5 days after virus injection, increased up to the time of maximum tumor size and persisted long after tumor regression. This suggests the development of increased numbers of CTL progenitors and/or the formation of "memory" CTL in spleens of MSV-injected mice.

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