Journal: bioRxiv

Article Title: Resolving cell cycle speed in one snapshot with a live-cell fluorescent reporter

doi: 10.1101/494252

Figure Lengend Snippet: A . Schematic of inducible H2B-FT expression construct. B. Representative images of HeLa cells expressing the H2B-FT. C. Selected time series of H2B-FT fluorescence in HeLa cells from a representative field of view following doxycycline (Dox) induction. D-E . Quantification of average nuclear blue (D) and red (E) FT intensity over time following Dox treatment. F. Selected time series of HeLa cells after Dox washout at T=4.5 h. G . Data from (D-E) plotted as blue/red ratio. Error bars represent standard deviation across n = 3 culture wells. Nuclear intensity measurements were normalized to timepoint 0. Scale bars = 50µm.

Article Snippet: H2B-mCherry and H2B-BFP (BFP template from Addgene #52115) inserts were similarly cloned to serve as single-color controls for the color-changing H2B-FT.

Techniques: Expressing, Construct, Fluorescence, Standard Deviation

Journal: bioRxiv

Article Title: Resolving cell cycle speed in one snapshot with a live-cell fluorescent reporter

doi: 10.1101/494252

Figure Lengend Snippet: A. Changes in cell number over time in primary iH2B-FT MEFs following transduction with either c-Myc or empty vector control (EV). Error bars denote standard deviation across n=4 culture wells. B. The ratio of blue/red fluorescence intensity was determined for individual cells at 25h and 70h post transduction. Box plots represent the median and interquartile range and whiskers represent 5th-95th percentile. P=0.295 (25h) and P<0.0001 (70h), determined using Mann-Whitney test with a 99% confidence interval. C. The blue and red fluorescence level of individual cells at 70h post transduction. Each dot denotes a single cell. FACS-style gates were applied to representative scatter plots. D. The percentage of cells within each gate. Error bars show standard deviation across n=4 culture wells. P=0.0035 (Red Cells) and P=0.0498 (Blue Cells), determined using Student’s T-Test with Welch’s correction, 95% confidence interval. dF=4.057 (Red Cells) and dF=3.221 (Blue Cells). E. The cell cycle lengths of individual cells from each gate were determined by image tracking. Cell cycle length represents the time interval between two consecutive mitoses within the same cell lineage. Cell cycle length is heterogeneous and the distribution of cell cycle lengths for each condition are shown as histograms. n values refer to the number of cells tracked for each condition. F. The relationship between cell cycle length and H2B-FT blue/red ratio. Each dot denotes an individual cell. All trackable cells (n=189) from both conditions are plotted. Spearman correlation coefficient = −0.7808. P<0.0001 was calculated with a 95% confidence interval.

Article Snippet: H2B-mCherry and H2B-BFP (BFP template from Addgene #52115) inserts were similarly cloned to serve as single-color controls for the color-changing H2B-FT.

Techniques: Transduction, Plasmid Preparation, Standard Deviation, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Resolving cell cycle speed in one snapshot with a live-cell fluorescent reporter

doi: 10.1101/494252

Figure Lengend Snippet: A. BaF3 cells proliferate at different rates in varying IL-3 concentrations, as determined by cell counting. Error bars denote standard deviation across n=6 culture wells. B. Representative FACS plots of BaF3 cells expressing the H2B-FT reporter grown under different IL-3 concentrations. C. Histograms of blue/red fluorescence ratio derived from FACS data in (B). D. Representative colony morphology and FT fluorescence of H2B-FT-Medium knock-in mESCs maintained in feeder-free conditions. E. Confirmation of cell cycle change following 48h of RA treatment, as analyzed by EdU pulse-labeling and DNA content profiling. F. Representative FACS plots of red vs. blue fluorescence in pluripotent and RA-treated mESCs (top). These FACS data were re-plotted as histograms of blue/red ratio (bottom). G-H. Pluripotent and RA-treated mESCs stably transfected with H2B-FT via Sleeping Beauty transposon were FACS sorted according to their blue/red FT ratio. Representative gating strategy is shown. H2B-FT-negative gate was determined using non-transfected WT mESCs. I. DAPI/EdU cell cycle profiles of sub-populations sorted from mESCs and RA-treated cells. J. Frequency of cells in G1 vs. G2/M from the FACS-sorted populations shown in (I). K. The number of alkaline phosphatase positive colonies formed by the same number of cells sorted in (G) following 6 days of culture. Error bars denote standard deviation across n=3-4 culture wells. Significance determined using Student’s T-Test with a 95% confidence interval, df=6 (mESCs) and df=5-6 (RA 48h). Exact P values provided in Table S3. Scale bar in (D) = 80µm.

Article Snippet: H2B-mCherry and H2B-BFP (BFP template from Addgene #52115) inserts were similarly cloned to serve as single-color controls for the color-changing H2B-FT.

Techniques: Cell Counting, Standard Deviation, Expressing, Fluorescence, Derivative Assay, Knock-In, Labeling, Stable Transfection, Transfection

Journal: bioRxiv

Article Title: Resolving cell cycle speed in one snapshot with a live-cell fluorescent reporter

doi: 10.1101/494252

Figure Lengend Snippet: A. Representative FACS plots of blue/red profile in LKS and GMP cells from reconstituted mice 2 months after transplantation with HSPCs virally expressing the H2B-FT reporter. B. Distribution of blue/red ratio in H2B-FT-expressing LKS and GMP populations, overlaid on that of whole bone marrow. C. FACS plots of myeloid (Mac1+) and erythroid (Ter119+) cells from reconstituted mouse bone marrow. D. Data from (C) plotted as histograms of blue/red H2B-FT ratio. Gates in (A) and (C) were used to exclude the non-transduced (H2B-FT-negative) cells. E. Targeting strategy for the HPRT:: TetO-H2B-FT mouse allele. F. DAPI/EdU cell cycle profiles of GMPs vs. early erythroid cells following 2 hours of in vivo EdU labeling. G. Distributions of H2B-FT blue/red ratio in LKS, GMPs, and early erythroid cells. H. Frequency of myeloid (Mac1+), B-cell (B220+), and T-cell (CD3+) lineages in peripheral blood of healthy H2B-FT knock-in mice vs. those crossed with MLL-ENL (n=3 mice per group). All mice were treated with Dox for at least 8 days prior to analysis. Error bars represent standard deviation. P=0.0090 (Mac1+), P=0.0072 (B220+), and P=0.7629 (CD3+) determined using Student’s T-Test with a 95% confidence interval, dF=4. I. Representative H2B-FT blue/red profiles within defined peripheral blood subsets as in (H).

Article Snippet: H2B-mCherry and H2B-BFP (BFP template from Addgene #52115) inserts were similarly cloned to serve as single-color controls for the color-changing H2B-FT.

Techniques: Transplantation Assay, Expressing, In Vivo, Labeling, Knock-In, Standard Deviation

Journal: bioRxiv

Article Title: Resolving cell cycle speed in one snapshot with a live-cell fluorescent reporter

doi: 10.1101/494252

Figure Lengend Snippet: A. 5µm frozen sections from kidney and pylorus in two orientations of an adult iH2B-FT reporter mouse were scanned by microscopy to capture blue and red fluorescence. Gastric glands are shown in elongated orientation (pylorus I) and oblique/cross-sectional orientation (pylorus II). Bottom, the neocortex of a representative E17.5 iH2B-FT mouse embryo. L= pyloric lumen; S=submucosa. Dotted line in bottom left image follows the line of a cerebral lateral ventricle. B. The blue/red ratio for each image shown in (A) was quantified and plotted as a histogram, from which five populations of increasing blue/red ratio (P1-P5) were determined. C. Left, a heatmap corresponding to populations P1-P5 as shown in (B) was applied onto the original images (A) to indicate the relative locations occupied by cells of different blue/red ratios. Right, the same regions as imaged after fixation and DAPI/EdU labeling. EdU was injected 35 minutes before harvesting mice/embryos. D. Magnified detail of white boxed pyloric regions shown in (C). White arrow indicates the base of a gastric gland. E. Tiled images showing a zoomed-out view of an E17.5 embryonic brain hemisphere. Left, H2B-FT blue/red merged image. Middle, heatmap of blue/red ratio binned from histograms as in (B). Right, same region imaged after DAPI/EdU labeling. Dotted line indicates cerebral lateral ventricle; IZ=intermediate zone, CP=cortical plate, MZ=marginal zone, Th=thalamus. F. Blue/red ratio histograms from all three tissues overlaid on the same plot. “Pylorus” histogram includes combined data from both images of the pylorus (middle images, A). All images contained within a single panel are shown at the same scale. Scale bar in (A,C) = 200µm. Scale bar in (D) = 80µm. Scale bar in (E) = 500µm.

Article Snippet: H2B-mCherry and H2B-BFP (BFP template from Addgene #52115) inserts were similarly cloned to serve as single-color controls for the color-changing H2B-FT.

Techniques: Microscopy, Fluorescence, Labeling, Injection

Journal: bioRxiv

Article Title: Resolving cell cycle speed in one snapshot with a live-cell fluorescent reporter

doi: 10.1101/494252

Figure Lengend Snippet: A. Cell cycle length distribution of cultured GMPs. Individual cell cycle length was determined by time-lapse microscopy. Dotted lines show the indicated percentiles. B. H2B-FT blue/red ratio distribution of the same GMPs as analyzed by flow cytometry after 24 hours culture. C. Calculated cell cycle length distributions of designated bone marrow populations from a representative H2B-FT mouse. D. Cell cycle length distribution of normal GMPs and L-GMPs from a representative mouse induced to express MLL-ENL. E. Median cell cycle length variation among three H2B-FT mice, shown by percent coefficient of variation (CV%). F. Cell cycle length distributions of LKS and GMP populations from the three mice. Boxes in panels (E), (F), and (H) represent the median and interquartile range of each group; whiskers represent 5th-95th percentile.

Article Snippet: H2B-mCherry and H2B-BFP (BFP template from Addgene #52115) inserts were similarly cloned to serve as single-color controls for the color-changing H2B-FT.

Techniques: Cell Culture, Time-lapse Microscopy, Flow Cytometry

Journal: Virology Journal

Article Title: Development of simple and rapid assay to detect viral RNA of tick-borne encephalitis virus by reverse transcription-loop-mediated isothermal amplification

doi: 10.1186/1743-422X-10-68

Figure Lengend Snippet: Gene amplification and detection of the TBEV by TBEV-specific RT-LAMP assay. ( A ) Real-time kinetics of the RT-LAMP amplification was monitored by real-time turbidimeter. In vitro transcribed RNA of TBEV Oshima strain was serially diluted to make 10 -1 to 10 4 copies. The reaction was repeated three times and a representative result is shown. ( B ) Agarose gel electrophoresis profile of the RT-LAMP products. M indicates 100-bp DNA marker (Sigma). N indicates a sample containing no viral RNA. ( C and D ) Visual detection of the RT-LAMP products amplified in a reaction tube. Positive amplification is indicated by change of color to cloudy yellow ( C ) or by fluorescent green under UV irradiation ( D ). ( E ) Real-time kinetics of TaqMan rRT-PCR with TBEV-specific primer/probe set. In vitro transcribed RNA of Oshima was serially diluted to make 10 -1 to 10 6 copies. The reaction was repeated three times and a representative result is shown.

Article Snippet: For visualization of the positive reaction, fluorescent detection reagent (FD; Eiken Chemical Co., Ltd. Tokyo, Japan) was added to the reaction mixture and a change of color was recognized directly (transparent to cloudy yellow color) or under UV irradiation (fluorescent green).

Techniques: Amplification, RT Lamp Assay, In Vitro, Agarose Gel Electrophoresis, Marker, Irradiation, Quantitative RT-PCR

Journal: Virology Journal

Article Title: Development of simple and rapid assay to detect viral RNA of tick-borne encephalitis virus by reverse transcription-loop-mediated isothermal amplification

doi: 10.1186/1743-422X-10-68

Figure Lengend Snippet: Detection of TBEV gene from mouse tissue samples by TBEV RT-LAMP assay. Visual detection of RT-LAMP products using 10 ng of RNA samples extracted from blood, plasma, liver, spleen and brain samples collected from laboratory mice on the 5th and 9th day following peripheral infections with TBEV Oshima strain. Three mice in each group and one mouse for mock infected mice were used. Positive amplification was indicated by the change of the color to cloudy yellow ( A ) or fluorescent green under UV irradiation ( B ). The reaction was repeated three times for each sample and a representative result is shown.

Article Snippet: For visualization of the positive reaction, fluorescent detection reagent (FD; Eiken Chemical Co., Ltd. Tokyo, Japan) was added to the reaction mixture and a change of color was recognized directly (transparent to cloudy yellow color) or under UV irradiation (fluorescent green).

Techniques: RT Lamp Assay, Clinical Proteomics, Infection, Amplification, Irradiation

Journal: PLoS ONE

Article Title: Detection of melon necrotic spot virus by one-step reverse transcription loop-mediated isothermal amplification assay

doi: 10.1371/journal.pone.0230023

Figure Lengend Snippet: (A) Specificity analysis of RT-LAMP by 1.5% agarose gel electrophoresis. Lane M, DL 2000 DNA marker; lane 1, MNSV-infected melon leaf from Shouguang, China; lane 2, MNSV-infected melon leaf from Changle, China; lane 3, CGMMV-infected cucumber leaf; lane 4, CMV-infected cucumber leaf; lane 5, CCYV-infected melon leaf; and lanes 6–7, negative control and blank control. (B) Visualization of the RT-LAMP following staining with SYBR Green I under UV light. (C) Visualization of the RT-LAMP by the naked eye with the color change of hydroxynaphthol blue from violet (negative) to sky blue (positive).

Article Snippet: Positive results could be translated into a fluorescent color change which could be observed under UV light at 365 nm by adding 0.2 μL SYBR Green I (10×, Invitrogen) to the post-reaction solution, or into a visual color change by adding 2 μL of 60 μM hydroxynaphthol blue (HNB; Sigma–Aldrich) to the pre-reaction solution, the latter change in color (from violet to sky blue) being clearly observable with the naked eye.

Techniques: Agarose Gel Electrophoresis, Marker, Infection, Negative Control, Control, Staining, SYBR Green Assay

Journal: PLoS ONE

Article Title: Detection of melon necrotic spot virus by one-step reverse transcription loop-mediated isothermal amplification assay

doi: 10.1371/journal.pone.0230023

Figure Lengend Snippet: (A) Sensitivity analysis of the RT-LAMP by agarose gel electrophoresis using a serial dilution of total RNA. Lane M, DL 2000 DNA marker (100–2000 bp); lanes 1–8, from 7.5×10 −1 to 7.5×10 −8 μg/μL of total RNA extracted from MNSV-infected melon leaves per assay; and lanes 9–10, negative control and blank control. Visualization of the RT-LAMP assay according to (A) with addition of SYBR Green I under UV light (B) or the color change of hydroxynaphthol blue (C). (D) Agarose gel illustrating the RT-PCR products from the same dilution series of total RNA as in lanes 1–10 in (A).

Article Snippet: Positive results could be translated into a fluorescent color change which could be observed under UV light at 365 nm by adding 0.2 μL SYBR Green I (10×, Invitrogen) to the post-reaction solution, or into a visual color change by adding 2 μL of 60 μM hydroxynaphthol blue (HNB; Sigma–Aldrich) to the pre-reaction solution, the latter change in color (from violet to sky blue) being clearly observable with the naked eye.

Techniques: Agarose Gel Electrophoresis, Serial Dilution, Marker, Infection, Negative Control, Control, RT Lamp Assay, SYBR Green Assay, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Detection of melon necrotic spot virus by one-step reverse transcription loop-mediated isothermal amplification assay

doi: 10.1371/journal.pone.0230023

Figure Lengend Snippet: (A) Agarose gel illustrating the RT-LAMP products from 24 melon leaf samples in lanes 1–24; lane 25, positive control; and lanes 26–27, negative control and blank control. (B) Visualization of the RT-LAMP assay according to 24 samples with the color change of adding hydroxynaphthol blue. (C) Agarose gel illustrating the RT-PCR products from 24 melon leaf samples in lanes 1–24; lane 25, positive control (MNSV samples that had been detected above); and lanes 26–27, negative control and blank control.

Article Snippet: Positive results could be translated into a fluorescent color change which could be observed under UV light at 365 nm by adding 0.2 μL SYBR Green I (10×, Invitrogen) to the post-reaction solution, or into a visual color change by adding 2 μL of 60 μM hydroxynaphthol blue (HNB; Sigma–Aldrich) to the pre-reaction solution, the latter change in color (from violet to sky blue) being clearly observable with the naked eye.

Techniques: Agarose Gel Electrophoresis, Positive Control, Negative Control, Control, RT Lamp Assay, Reverse Transcription Polymerase Chain Reaction