UBE2NL protein in virus-infected or mock-infected MDCK cells at 6 h p.i..

<p>MDCK cells were infected with the viruses at MOI of 0.1 in the presence of 1 µg/ml TPCK-trypsin. After adsorption for 1 h at 37°C, the inocula were removed and the cultures were incubated for 6 h at 37°C in the maintenance media. Then, the cells were processed for indirect immunofluorescence assay, and the infected cells were detected with polyclonal antisera to UBE2NL protein and NP protein. (A) The fluorescence images (10×) of the infected and mock-infected cells at 6 h p.i. The FITC-fluorescence signal was expressed as UBE2NL protein and TRITC-fluorescence signal was expressed as the infected cells. (B) The fluorescence images (60×) of the cells infected by rPR8-H9N2NA or rPR8-H5N1NA viruses.</p

List of differentially expressed protein spots in influenza virus-infected and mock-infected MDCKs identified by MALDI-TOF/TOF.

a<p>The arrow“↑” represents the identified proteins were upregulated and the arrow “↓”represents the identified proteins were downregulated.</p

Protein expression profiles of the influenza- and mock-infected MDCK cells.

<p>Cell lysates (120 µg) were separated on 13-cm (isoelectric point [pI] 4–7) linear gradient IPG strips using 12.5% SDS-PAGE. Differentially expressed protein spots are indicated with green squares. (A) Representative 2-DE gels of influenza- and mock-infected MDCK cells. T1/C: PR8-wt infected/mock infected, T3/C: rH1N1NA infected/mock infected, T4/C: rH9N2NA infected/Mock infected, T5/C: rH5N1NA infected/mock infected, T3/T1: rH1N1NA infected/PR8-wt infected, T4/T1: rH9N2NA infected/PR8-wt infected, T5/T1: rH5N1NA infected/PR8-wt infected. (B) Numbers of differentially expressed protein spots detected by 2-DE in virus-infected MDCK cells compared with mock-infected MDCK cells. The number of spots ≥0 indicated the proteins were upregulated, and the number <0 indicated the proteins were downregulated. (C) Numbers of differentially expressed protein spots detected by 2-DE in recombinant viruses compared with wild-type virus (wt-PR8)-infected MDCK cells.</p

List of differentially expressed protein spots in MDCK cells infected with recombinant viruses and PR8-wt virus identified by MALDI-TOF/TOF.

a<p>The arrow“↑” represents the identified proteins were upregulated and the arrow “↓”represents the identified proteins were downregulated.</p

Western blots of representative proteins in influenza virus-infected MDCKs.

<p>The samples were prepared from MDCK cells that were virus-infected or mock-infected cells at 6 h p.i.. The β-actin protein was used as a control. (A) Western blot confirmation of differentially expressed proteins for PSMC2 (C08) and UBE2NL (C26). (B) ImageJ software analysis of the ratios of proteins changes according to Fig. 4A.</p

Classification of the identified proteins based on their functional annotations using Gene Ontology (GO) categories.

<p>The proteins were annotated into three main categories: cellular component, biological process, or molecular function. The Y-axis indicates the number and percentages of genes, the X-axis indicates the GO category.</p

Transcriptional profiles of differentially expressed proteins in influenza virus-infected MDCKs.

<p>Total cellular RNA from MDCKs with or without influenza virus infection was subjected to real-time RT-PCR. Samples were normalized to mock-infected MDCKs using β-actin as the reference gene.</p

Rapid and Accurate Quantification of Viable <i>Lactobacillus</i> Cells in Infant Formula by Flow Cytometry Combined with Propidium Monoazide and Signal-Enhanced Fluorescence In Situ Hybridization

Lactobacillus is an important member of the probiotic bacterial family for regulating human intestinal microflora and preserving its normalcy, and it has been widely used in infant formula. An appropriate and feasible method to quantify viable Lactobacilli cells is urgently required to evaluate the quality of probiotic-fortified infant formula. This study presents a rapid and accurate method to count viable Lactobacilli cells in infant formula using flow cytometry (FCM). First, Lactobacillus cells were specifically and rapidly stained by oligonucleotide probes based on a signal-enhanced fluorescence in situ hybridization (SEFISH) technique. A DNA-binding fluorescent probe, propidium monoazide (PMA), was then used to accurately recognize viable Lactobacillus cells. The entire process of this newly developed PMA-SEFISH-FCM method was accomplished within 2.5 h, which included pretreatment, dual staining, and FCM analysis; thus, this method showed considerably shorter time-to-results than other rapid methods. This method also demonstrated a good linear correlation (R2 = 0.9994) with the traditional plate-based method with a bacterial recovery rate of 91.24%. To the best of our knowledge, the present study is the first report of FCM combined with PMA and FISH for the specific detection of viable bacterial cells

MOESM1 of Quantification of plasmid DNA reference materials for Shiga toxin-producing Escherichia coli based on UV, HR-ICP-MS and digital PCR

Additional file 1. Supplementary information

International Comparison of Enumeration-Based Quantification of DNA Copy-Concentration Using Flow Cytometric Counting and Digital Polymerase Chain Reaction

Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to “absolute quantification”, which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration-based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter. In addition, 2 orthogonal chemical-analysis methods based on nucleotide quantification, isotope-dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) were applied to quantify a more concentrated solution of the plasmid. Although 9 dPCR results from 8 laboratories showed some dispersion (relative standard deviation [RSD] = 11.8%), their means were closely aligned with those of the FCM-based counting method and the orthogonal chemical-analysis methods, corrected for gravimetric dilution factors. Using the means of dPCR results, the RSD of all 4 methods was 1.8%, which strongly supported the validity of the recent enumeration approaches. Despite a good overall agreement, the individual dPCR results were not sufficiently covered by the reported measurement uncertainties. These findings suggest that some laboratories may not have considered all factors contributing to the measurement uncertainty of dPCR, and further investigation of this possibility is warranted