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  • Fig demonstrates cell damage induced by ultra sonication and

    2018-11-14

    Fig. 2 demonstrates cell damage induced by ultra-sonication and evaluated by estimation of the total protein content of the cell suspensions. The data demonstrates that ultra-sonication of Rosiglitazone manufacturer induces varying degrees of cell damage directly proportional to the duration of ultra-sonication representing increasing cell lysis and release of protein. However, when the same samples were totally lysed by incubation at 65°C (1h), the total protein was not significantly different from each other. Addition of cellulose significantly increased the OD600 of cell suspensions and this increase (noise) was not cleared even when suspensions were allowed to stand for up to 15min (Fig. 4). This observation was the same for all the different strains of bacteria used. Furthermore, a similar observation was made for suspensions with higher or lower numbers of cells. To test whether the presence of an insoluble substrate will only add proportional increase to the OD600 or not, cellulose was added to cell suspensions. There was no correlation between the original OD600 and the OD600 when insoluble substrates are present (Fig. 5). The correlation was not improved even when the cell suspensions were left to stand for 5min or 15min.
    Experimental design, materials and methods Human genomic DNA (200ngµl-1) was obtained from Bioline, London, UK, diluted serially in nuclease free water and used as a calibration standard. The diluted standards were stained with SYBR-I and/or PI and the fluorescence was measured using the Modulus™ Single Tube multimode reader. The blue (P/N 9200-040, λex = 460nm, λem = 515-570nm) or green (P/N 9200-042, λex = 525nm, λem = 580–640) Modulus™ fluorescence kits (Turner BioSystems, Sunnyvale, CA, USA) were used for the measurement of green and red fluorescence respectively. Cell density was determined by measuring absorbance of cell suspensions at 600nm using the absorbance module (Model E6076, GLOMAX MultiJR, Promega, Southampton, UK) on the Modulus reader. To induce cell damage and evaluate how useful dual staining with SYBR-I and PI is for quantification of live bacterial cells demonstrated in flow cytometry [4–10] and demonstrated for fluorimetry in ref [1], a cell suspension was prepared and aliquoted into different tubes numbered 1 to 6. The tubes (1 to 6) were ultra-sonicated for one pulse at 10µm (amplitude) for 0, 3, 7, 10, 15 and 20s each respectively on ice using the MSE Soniprep 150 Ultrasonic Disintegrator. The degree of damage was accessed by the quantity of extracellular protein using the Pierce Coomassie plus (Bradford) assay kit (ThermoScientific, Rockford, lL, USA) following the manufacturer׳s instructions. Dual staining of the ultra-sonicated cells with SYBR-I and PI was performed to determine whether damage to the cells correlated with dead cells as estimated from fluorescence measurements. To determine the effect of the presence of an insoluble substrate in the cell suspension, the suspensions were spiked with equal amounts of microcrystalline cellulose (avicel). The OD600 was determined. Suspensions were also left to stand for 5 and 15minutes to check whether OD600 values or the live or dead cell quantification will be improved. Dual staining with SYBR-I and PI for fluorimetry has been described in the research article related to this data [1].
    Acknowledgements The authors thank the Darwin Trust of Edinburgh for the award of a scholarship for KOD.
    Data Data in the following Tables present the reference values for 38 distinct human T, B or NK lymphocyte subpopulations. When sex or age has a significant impact on Rosiglitazone manufacturer these subpopulations, separate reference values are given for male, female, younger (19–44) or older (45–67) individuals. Data are expressed either as absolute numbers of cells in G/L (a), or as the percentage of cells relative to total CD3+ (b), CD3+ CD4+ (c), CD3+ CD8+ (d) T lymphocytes, Tregs (e), total B (f) or NK cells (g); DN: CD4-CD8- double-negative T cells; DP: CD4+CD8+ double-positive T cells.