gelatin-treated microfluidic channel ibidi μ-slide i0.4  (ibidi GmbH)

Journal: Journal of the Royal Society Interface

Article Title: Enhanced wall shear stress prevents obstruction by astrocytes in ventricular catheters

doi: 10.1098/rsif.2019.0884

Figure Lengend Snippet: (a) Transparent microfluidic channel used in this study (left) [18] and illustration of channel cross-section (right), with a bottom plate that is gas-permeable so that pH remains controlled with sufficient gas exchange. The bottom surface is chemically treated for adherence of cells. (b) Schematic of microfluidic experiment set-up embedded in controlled CO2 environmental chamber.

Article Snippet: We cultured a mouse astrocyte cell line (C8D1A, ATCC) in a commercial transparent microfluidic channel ( μ -Slide I Luer, ibidi GmbH) to investigate the response of astrocytes under fluid shear stress ( ).

Techniques:

Journal: PLoS Biology

Article Title: Cilia-based peptidergic signaling

doi: 10.1371/journal.pbio.3000566

Figure Lengend Snippet: A. Agarose block assay. CC124 minus gametes were uniformly spread on the surface of a Petri dish containing 1% agar in M-N medium. Agarose blocks containing 10 μM GATI-amide, 10 μM GATI-OH, M-N medium, or M-N medium containing 0.1% DMSO were placed onto the Petri dish; after 30 minutes in the dark, minus gametes had accumulated around the agarose block containing GATI-amide (red arrow). Similar results were obtained in three independent experiments. B. Microfluidic chemotaxis assay. Microfluidic channel slides were used for chemoattractant gradient formation. An image of a gradient of red food dye is shown. For each treatment, the number of cells in regions 0, I, and 2 were counted over time. C. Chemotaxis assay of minus gametes in microchannel slide . The chemotaxis index (CI) was measured over a period of 4 hours in response to 10 μM GATI-amide, GATI-OH, GATI-Gly, M-N medium, and vehicle control. The CI in the presence of GATI-amide was significantly higher at 3 hours (** P < 0.01) and 4 hours (* P < 0.05) compared to 0.1% DMSO. D. Dose dependence of the response of minus gametes to GATI-amide . A 4-hour incubation period was used; 0.2% DMSO was used as the vehicle control to accommodate the use of 20 μM peptide. The CI of 5 μM GATI-amide was significantly higher than the control at 3 hours (** P < 0.01); for 10 μM GATI-amide, the CI was significantly higher at 3 hours (** P < 0.01) and 4 hours (* P < 0.05), while for 20 μM GATI-amide it was significantly higher at 2 hours (* P < 0.05), 3 hours (*** P < 0.01), and 4 hours (* P < 0.05). The only CI to ever exceed 1.00 was for GATI-amide. E. Chemotactic response . The response of minus and plus gametes to 10 μM GATI-amide and 0.1% DMSO. The number of cells for each treatment was counted along the entire channel length, and the ratio of the number of cells in each slice/number of cells in slice 0 (n slice X /n slice 0 ) was plotted for the 4-hour time point. The response of gametic CC124 cells to GATI-amide was significantly (** P < 0.001) different from gametic CC125 cells. Data are the average of three independent experiments ± SEM. F. Trajectory plots . Plots are shown for minus and plus gametes. Data are representative of three independent experiments; all plots shown are from one experiment. G. Population response. The center of mass was calculated for each population from trajectory plots like those shown in F . Results are the average of three independent experiments ± SEM. H. Motile behavior. The swimming velocities of minus and plus gametes were not altered by the presence of the amidated peptide. Results are the average of three independent experiments ± SEM. One-way and two-way ANOVAs were used as appropriate. The underlying numeric data for this figure can be found in . GATI-amide, VLYPNDPAAYAAYAPGTGGGATI-amide; M-N medium, gametic medium.

Article Snippet: Microfluidic channel slides (μ slide I 0.2 Luer; ibidi GmbH, Gräfelfing, Germany) were used for gradient formation.

Techniques: Blocking Assay, Chemotaxis Assay, Control, Incubation

Journal: mSphere

Article Title: Intracellular Signaling by the comRS System in Streptococcus mutans Genetic Competence

doi: 10.1128/mSphere.00444-18

Figure Lengend Snippet: comS deletion is not fully complemented by synthetic XIP. (A) Comparison of levels of P comX-gfp activity in S. mutans cells of the UA159 (wild-type) background (magenta) and Δ comS mutant background (cyan). The median GFP fluorescence is shown for cells that were supplied with a continuous flow of exogenous synthetic XIP in FMC medium in microfluidic chambers. Data are shown at 30 min (circles, dashed-dotted lines) and 90 min (squares, dashed lines) of flow. The smooth curves represent spline fits to the data. (B) Median GFP levels in the two strains at the 90-min time point of the flow experiment. (C and D) Also shown are the histograms of the individual cell P comX-gfp reporter activity versus exogenous XIP concentration for (C) the UA159 background and (D) the Δ comS background. Solid black curves in panels C and D show the best-fit gamma probability distribution for each histogram. A cutoff of 40 units of P comX-gfp fluorescence has been applied to exclude background autofluorescence. (E) Parameter a of the (two-parameter) gamma probability distribution, obtained from the fits in panels C and D, reflecting the ratio of transcription rate to protein degradation rate. (F) Distribution parameter b , reflecting the ratio of the rate of translation to the rate of mRNA degradation. In panels E and F, cyan indicates the Δ comS mutant and magenta indicates the UA159 background.

Article Snippet: In order to measure the flow rate dependence of XIP signaling, we loaded cells into a commercial six-channel microfluidic slide (IBIDI μ-slide VI; IBIDI GmbH).

Techniques: Comparison, Activity Assay, Mutagenesis, Fluorescence, Concentration Assay

Journal: mSphere

Article Title: Intracellular Signaling by the comRS System in Streptococcus mutans Genetic Competence

doi: 10.1128/mSphere.00444-18

Figure Lengend Snippet: Activation of ComX in a ComS-overexpressing strain is independent of the rate of medium replacement. P comX-rfp reporter activity is shown in cells growing in microfluidic chambers supplied with continuously flowing fresh complex medium (BHI). (A) Histograms of individual cell P comX-rfp reporter fluorescence. (First column) wild-type background (negative control) with flow at 0.1 ml h − 1 . (Second column) ComS-overexpressing 184 comS Δ comS background at 0.1 ml h − 1 . (Columns 3 to 5) ComS-overexpressing (184 comS ) background at 0.02 ml h − 1 , 0.1 ml h − 1 , 0.5 ml h − 1 , and 1 ml h − 1 . (At a flow rate of 1 ml h − 1 , the medium in each flow chamber is replaced every 6 s.) (B) RFP fluorescence of cells that exceeded the wild type (negative-control) red fluorescence in column 1 of panel A. The black bar indicates the median of data in each channel. (Leftmost column) 184 comS Δ comS background. (Columns 2 to 5) ComS-overexpressing (184 comS ) background. All RFP measurements were made 4 h after addition of chloramphenicol to the cultures.

Article Snippet: In order to measure the flow rate dependence of XIP signaling, we loaded cells into a commercial six-channel microfluidic slide (IBIDI μ-slide VI; IBIDI GmbH).

Techniques: Activation Assay, Activity Assay, Fluorescence, Negative Control

Journal: mSphere

Article Title: Intracellular Signaling by the comRS System in Streptococcus mutans Genetic Competence

doi: 10.1128/mSphere.00444-18

Figure Lengend Snippet: ComS overexpresser does not induce expression of comX of strain Δ comS in coculture. (A and B) Histograms of (A) GFP and (B) RFP fluorescence of individual cells in cocultures of sender (184 comS P comX-rfp ) and receiver (P comX-gfp Δ comS ) strains in FMC medium. Strains were grown to equal levels of optical density, mixed in various proportions, and then incubated in microfluidic chambers containing stationary defined medium. Blue lines indicate population medians. Panels A and B show fluorescence of the UA159 background strain containing P comX rfp reporter, without added XIP (negative control) (first column); of strain P comX-gfp Δ comS (receiver) with added 50 nM XIP (positive control) (second column); and of cocultures of sender and receiver, with columns labeled by percentage by volume of 184 comS (sender) culture in the initial preparation of the coculture. (C to H) Phase-contrast images of cocultures, overlaid with red and green fluorescence images. (C) P comX-rfp reporter in UA159 background, with no added XIP (negative control). (D) P comX-gfp Δ comS cells with 50 nM added XIP (positive control). (E) P comX-gfp Δ comS (receiver) alone, with 0% sender. (F to H) Cocultures containing 30%, 80%, and 100% sender, respectively.

Article Snippet: In order to measure the flow rate dependence of XIP signaling, we loaded cells into a commercial six-channel microfluidic slide (IBIDI μ-slide VI; IBIDI GmbH).

Techniques: Expressing, Fluorescence, Incubation, Negative Control, Positive Control, Labeling

Journal: mSphere

Article Title: Intracellular Signaling by the comRS System in Streptococcus mutans Genetic Competence

doi: 10.1128/mSphere.00444-18

Figure Lengend Snippet: Proposed model for comRS feedback regulation of comX . The model represents comS feedback-enhanced activation of P comX-gfp reporter by exogenous synthetic XIP. (A) Illustration of the feedback model and the role of comS and extracellular XIP. ComS and XIP both interact with ComR to activate transcription of comS and comX. At low concentrations of extracellular XIP, comS expression is very low and comX is not expressed. At high concentrations of extracellular XIP, XIP is imported efficiently by Opp and interacts with ComR to drive expression of both comS and comR . Endogenously produced ComS is not readily exported in the absence of lysis, and so intracellular accumulation of ComS drives elevated comS and comX expression. Consequently, comX expression at any given XIP concentration is boosted by comS feedback. Cells lacking native comS can respond to synthetic XIP but cannot activate comX to the same level as the wild type. The figure presents the behavior seen in defined medium; in complex medium, extracellular XIP is not imported . (B) Integration of this intracellular feedback system into the wider com regulon, involving upstream activator ComE and its response to exogenous CSP through the ComD transmembrane kinase. ComS is allowed to be processed to XIP intracellularly by an unidentified protease. (C) Comparison of model fit with data. Red circles indicate median P comX-gfp fluorescence of the UA159 background strain supplied with synthetic XIP in microfluidic flow; blue circles indicate the median P comX-gfp fluorescence of the Δ comS background. Solid curves represent calculated values from a fit in which 11 parameters were fitted to the microfluidic data, as described in Materials and Methods. The model relates the predicted ComX concentration to the median GFP fluorescence by an offset and scale factor.

Article Snippet: In order to measure the flow rate dependence of XIP signaling, we loaded cells into a commercial six-channel microfluidic slide (IBIDI μ-slide VI; IBIDI GmbH).

Techniques: Activation Assay, Expressing, Produced, Lysis, Concentration Assay, Comparison, Fluorescence