Journal: eLife

Article Title: Spatial quality control bypasses cell-based limitations on proteostasis to promote prion curing

doi: 10.7554/eLife.04288

Figure Lengend Snippet: ( A ) A [ PSI + ] Weak HSP104GFP culture (SY2126) was imaged over time in a microfluidics chamber at 30°C after a 30 min incubation at 30°C, 37°C, 40°C, or 37°C before 40°C. Fluorescence intensity in daughter and mother cells was quantified at the first cell division in cells that were budded (gray) or unbudded (orange) after thermal stress. Lines represent medians; boxes represent upper and lower quartiles, and whiskers represent maximum and minimum. All pairwise comparisons are significantly distinct, with a p < 0.015, except where indicated (N.S.), by unpaired t-test; n ≥ 10. ( B ) A [ PSI + ] Weak HSP104GFP WT (SY2126, gray) or BNI1 deletion strain (Δ bni1 ) (SY2486, green) was imaged over time in a microfluidics chamber at 30°C after a 30 min incubation at 40°C. Fluorescence intensity in daughter and mother cells was quantified at the first cell division. Lines represent medians; boxes represent upper and lower quartiles; and whiskers represent maximum and minimum; n ≥ 14; p = 0.0075 by unpaired t-test. ( C ) [ PSI + ] Weak WT (SLL2600) or Δ bni1 strains (SY1888), treated as described in ( B ), were plated on YPD to analyze curing by colony color phenotype. Data represent means; error bars represent standard deviations; n = 3; p < 0.0001 by unpaired t-test. ( D ) A [ PSI + ] Weak HSP104GFP strain (SY2126) was imaged over time in a microfluidics chamber at 30°C after a 30 min incubation at 40°C and with GdnHCl added before or after the 40°C incubation. Fluorescence intensity in daughter and mother cells was quantified at the first cell division. Lines represent medians; boxes represent upper and lower quartiles; and whiskers represent maximum and minimum; n > 11; *p = 0.0003, **p = 0.0026 by unpaired t-test. ( E ) A [ PSI + ] Weak strain (SLL2600) was incubated at 40°C for 30 min and plated on rich medium. Mother and daughter pairs were separated by micromanipulation and allowed to form colonies, which were then dispersed to YPD for analysis of curing by colony color phenotype. n = 15. ( F ) A [ PSI + ] Weak HSP104GFP culture (SY2126) was incubated at 30°C (dotted) or at 40°C for 30 min and allowed to recover for 30 min at 30°C (solid) before analysis of GFP fluorescence intensity by flow cytometry. Based on these intensities, cells were sorted into four fractions (orange, blue, purple, red) by FACS. ( G ) Cells collected in ( F ) were plated on YPD to analyze curing by colony color phenotype. Data represent means; error bars represent standard deviations; n = 2; *p = 0.02 by paired t-test. DOI: http://dx.doi.org/10.7554/eLife.04288.010

Article Snippet: Microfluidics experiments were performed on a Zeiss Axio Observer Z1 using a CellAsics microfluidics plate with temperature controls and media flow of 2 psi on a Y0C4 yeast perfusion plate (channel size 3.5–5 μm).

Techniques: Incubation, Fluorescence, Micromanipulation, Flow Cytometry

Journal: eLife

Article Title: Spatial quality control bypasses cell-based limitations on proteostasis to promote prion curing

doi: 10.7554/eLife.04288

Figure Lengend Snippet: ( A ) A [ psi − ] strain expressing heat-inducible untagged GFP (SY2091) was imaged over time in a microfluidics chamber at 30°C after 30 min incubation at 40°C (red) or 30°C (gray). Fluorescence intensity in daughter and mother cells was quantified at the first cell division in budded cells. Lines represent medians, boxes represent upper and lower quartiles, and whiskers represent maximum and minimum; n ≥ 11. ( B ) A [ PSI + ] Weak strain expressing a GFP-tagged endogenous Ssa1 and DsRedNLS (SY2659) was imaged after a 90 min incubation at 30°C, 37°C, 40°C, or 37°C before 40°C. Scale bar = 2 μm. ( C ) A [ PSI + ] Weak strain expressing a GFP-tagged endogenous Sis1 and DsRedNLS (SY2485) was imaged after a 90-min incubation at 30°C, 37°C, 40°C, or 37°C before 40°C. Scale bar = 2 μm. ( D ) A [ PSI + ] Weak SSA1GFP culture (SY2658) was imaged over time in a microfluidics chamber at 30°C after a 30 min incubation at 40°C (red) or 30°C (gray). Fluorescence intensity in daughter and mother cells was quantified at the first cell division in budded cells. Lines represent medians, boxes represent upper and lower quartiles, and whiskers represent maximum and minimum; n > 15. ( E ) A [ PSI + ] Weak SIS1GFP culture (SY2447) was imaged over time in a microfluidics chamber at 30°C after a 30 min incubation at 40°C (red) or 30°C (gray). Fluorescence intensity in daughter and mother cells was quantified at the first cell division in budded cells. Lines represent medians, boxes represent upper and lower quartiles, and whiskers represent maximum and minimum; n ≥ 7. ( F ) Quantitative immunoblotting for Hsp104 was performed on lysates from WT (SLL2600) or Δbni1 (SY1888) [ PSI + ] Weak cultures treated at 30°C (black) and 40°C (white) for 30 min following SDS-PAGE. Data represent means; error bars represent standard deviations; n = 3. ( G ) Lysates were isolated from WT (SLL2600) or Δbni1 (SY1888) [ PSI + ] Weak strains that were incubated at 30°C or 40°C for 30 min, and heat-induced protein aggregates were analyzed by differential centrifugation and Bradford assay. Data represent means; error bars represent standard error; n = 3. DOI: http://dx.doi.org/10.7554/eLife.04288.011

Article Snippet: Microfluidics experiments were performed on a Zeiss Axio Observer Z1 using a CellAsics microfluidics plate with temperature controls and media flow of 2 psi on a Y0C4 yeast perfusion plate (channel size 3.5–5 μm).

Techniques: Expressing, Incubation, Fluorescence, Western Blot, SDS Page, Isolation, Centrifugation, Bradford Assay

Journal: eLife

Article Title: Spatial quality control bypasses cell-based limitations on proteostasis to promote prion curing

doi: 10.7554/eLife.04288

Figure Lengend Snippet: ( A ) The number of [ PSI + ] Weak HSP104GFP (SY2126) cells containing fluorescent foci was quantified in cultures recovering at 30°C over time following a 90 min incubation at 40°C (white). Colony forming units in these cultures were quantified by plating (black). Data represent means; error bars represent standard deviations; n = 3. ( B ) [ PSI + ] Weak HSP104GFP cells (SY2126) treated for 30 min at 40°C and imaged over time in a microfluidics chamber are shown. Cells that were budded at the time of thermal stress are outlined in white, while unbudded cells are outlined in orange. Solid lines mark mothers, and dotted lines mark daughters. Scale bar = 1 µm. ( C ) A [ PSI + ] Weak HSP104GFP strain (SY2126) was imaged over time in a microfluidics at 30°C after a 30 min incubation at 40°C chamber. Budded or unbudded cells were scored at the first cell division for the presence or absence of fluorescent aggregates. Data represent means; error bars represent standard deviations; n = 3; p = 0.0005 by unpaired t-test. DOI: http://dx.doi.org/10.7554/eLife.04288.015

Article Snippet: Microfluidics experiments were performed on a Zeiss Axio Observer Z1 using a CellAsics microfluidics plate with temperature controls and media flow of 2 psi on a Y0C4 yeast perfusion plate (channel size 3.5–5 μm).

Techniques: Incubation

Journal: NPJ Biofilms and Microbiomes

Article Title: Bacterial biofilm functionalization through Bap amyloid engineering

doi: 10.1038/s41522-022-00324-w

Figure Lengend Snippet: Time-lapse fluorescence microscopy was performed to monitor mCherry expression at single-cell level in S. aureus Bap-mCherry. In order to visually compare image patterns, the histogram range of each image was modified accordingly. Bacteria were grown at 37 °C in CellAsic microfluidic plates with a continuous flow of LB for 330 min. Next, bacteria were challenged with LB (neutral pH) a or with acidified LB b for 90 min. Arrows show the point in which the media was changed. Images were taken in 30 min intervals. The mCherry fluorescence and the differential interference contrast (DIC) images are shown. Right panels show the magnification of images taken 30 min before and 60 min after media challenge. Scale bar of panels represents 5 μm.

Article Snippet: S. aureus Bap-Spy was grown in CellAsic microfluidic plates at 37 °C with a continuous flow for 300 min. Then, rCatcher-GFP (left column) and rGFP (right column) were added and the flow was maintained during 2 h with the recombinant proteins.

Techniques: Fluorescence, Microscopy, Expressing, Modification

Journal: NPJ Biofilms and Microbiomes

Article Title: Bacterial biofilm functionalization through Bap amyloid engineering

doi: 10.1038/s41522-022-00324-w

Figure Lengend Snippet: a Schematic diagram of the experiment. S. aureus Bap-Spy was grown in CellAsic microfluidic plates at 37 °C with a continuous flow for 300 min. Then, rCatcher-GFP (left column) and rGFP (right column) were added and the flow was maintained during 2 h with the recombinant proteins. Next, channels were washed with PBS during 90 min to remove the background fluorescence. b Images of GFP fluorescence and the differential interference contrast (DIC) at different times are shown. S. aureus Bap-Spy was grown in microfluidic plates and were incubated with rCatcher-GFP (left column) and rGFP (right column). Images were recorded every 30 min in separate fields to avoid photobleaching. Scale bar of panels represents 5 μm.

Article Snippet: S. aureus Bap-Spy was grown in CellAsic microfluidic plates at 37 °C with a continuous flow for 300 min. Then, rCatcher-GFP (left column) and rGFP (right column) were added and the flow was maintained during 2 h with the recombinant proteins.

Techniques: Recombinant, Fluorescence, Incubation

Journal: Cell systems

Article Title: tRNA Methylation Is a Global Determinant of Bacterial Multi-Drug Resistance

doi: 10.1016/j.cels.2019.03.008

Figure Lengend Snippet: Key Resources Table

Article Snippet: Microfluidic perfusion plates , CellASIC , Cat. #B04a.

Techniques: Virus, Recombinant, Plasmid Preparation, Purification, Software, Mass Spectrometry, Targeted Proteomics, Membrane, Imaging, Fluorescence, Microscopy

Journal: Blood

Article Title: Dynamic intercellular redistribution of HIT antigen modulates heparin-induced thrombocytopenia

doi: 10.1182/blood-2018-02-830737

Figure Lengend Snippet: Effect of endothelial cells on binding of PF4 to platelets and WBCs. Whole blood from PF4null mice was flowed through a microfluidic channel coated with HUVECs that had been either previously unexposed to PF4 (A) or previously incubated with 100 µg/mL of hPF4 (B). (A-B) The infused whole blood was also preincubated with 0, 10, or 100 µg/mL of PF4, as indicated. Shown is relative cell-surface HIT antigenicity detected using KKO on platelets (red circles) or WBCs (yellow squares) after traversing the channel compared with results before entering the channel. N = 6 separate studies per arm, each in duplicate. *P < .01, **P < .001 with values determined using analysis of variance of calculated values against a hypothetical value 1 = no change (dashed horizontal line).

Article Snippet: A well-plate microfluidic device (Fluxion Biosciences) was used to analyze surface distribution of PF4 on various blood cell types under flow in endothelial-lined channels.

Techniques: Binding Assay, Incubation

Journal: Blood

Article Title: Dynamic intercellular redistribution of HIT antigen modulates heparin-induced thrombocytopenia

doi: 10.1182/blood-2018-02-830737

Figure Lengend Snippet: Endothelial activation enhances PF4 binding. HUVECs lining microfluidic wells were injured with hematoporphyrin and perfused with PF4null murine blood supplemented with 10 or 100 µg/mL of hPF4 as indicated. (A) VWF exposure on the surface of endothelial cells showing the effects of the photochemical injury using binding of a labeled anti-VWF antibody. —, no injury; +++, photochemical injury. N = 4 separate experiments, each performed in duplicate; each experimental result is shown as well as the mean. P values were determined using analysis of variance comparing MFI of injured with noninjured endothelium. (B) Same as panel A, but for PF4 binding to endothelial cells detected by labeled KKO.

Article Snippet: A well-plate microfluidic device (Fluxion Biosciences) was used to analyze surface distribution of PF4 on various blood cell types under flow in endothelial-lined channels.

Techniques: Activation Assay, Binding Assay, Labeling

Journal: Journal of Dental Research

Article Title: Biochemical Indicators of Implantation Success of Tissue-Engineered Oral Mucosa

doi: 10.1177/0022034514554225

Figure Lengend Snippet: Box plots of the distribution of interleukin-8 (IL-8), human β-defensin 1 (hBD-1), and vascular endothelial growth factor (VEGF) (A), and tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and TIMP-2) (B) enzyme-linked immunosorbent assay data by control and thermally stressed groups of ex vivo–produced oral mucosa equivalents. The P values from the test for differences in means were 0.04 for IL-8, 0.03 for hBD-1, 0.07 for VEGF, 0.15 for TIMP-1, and 0.02 for TIMP-2. Statistical analysis strategy is detailed in the Materials and Methods section.

Article Snippet: For microchannel ELISA, OptimserTM microfluidic ELISA plate assembly, buffers, and reagents were from Siloam Biosciences (Cincinnati, OH).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Ex Vivo, Produced