cell membrane impermeable dyes  (New England Biolabs)

Journal: ACS Synthetic Biology

Article Title: Bioprinting of Synthetic Cell-like Lipid Vesicles to Augment the Functionality of Tissues after Manufacturing

doi: 10.1021/acssynbio.4c00137

Figure Lengend Snippet: Bioprinting of giant unilamellar vesicles (GUVs). A) Production of GUVs with defined filling (Alexa Fluor 488 phalloidin, inset: scale bar of 10 μm) by electroformation. B) Filtration of GUVs by size with a 10 μm filter membrane to collect a stock solution with an average GUV diameter similar to human cells. C) Bioprinting GUVs by DOD or extrusion.

Article Snippet: PEG5-GUVs were produced with a buffer consisting of sucrose (300 mM), Ce6 (125 μM), and the cell-membrane impermeable dye Alexa Fluor 488 phalloidin (2 μM, A12379, Thermo Fisher Scientific, Waltham, USA).

Techniques: Filtration, Membrane

Journal: ACS Synthetic Biology

Article Title: Bioprinting of Synthetic Cell-like Lipid Vesicles to Augment the Functionality of Tissues after Manufacturing

doi: 10.1021/acssynbio.4c00137

Figure Lengend Snippet: Studying the release of a fluorescent dye from bioprinted Ce6-PEG5-GUVs upon illumination. A) Fluorescence image of Ce6-PEG5-GUVs. B) Ce6-PEG5-GUVs loaded with sucrose and cultured in glucose solution before and after illumination with an LED set at a wavelength of 357 nm for 5 min. C) Fluorescence images showing bioprinted 1% w/v agarose constructs encapsulated with Alexa Fluor 488-loaded Ce6-PEG-GUVs before (upper images) and after (lower images) the release of the dye by illumination with an LED set at a wavelength of 357 nm for 5 min. Scale bars represent 50 μm; in insets, they are 10 μm; in macroscopic images, they are 2 mm.

Article Snippet: PEG5-GUVs were produced with a buffer consisting of sucrose (300 mM), Ce6 (125 μM), and the cell-membrane impermeable dye Alexa Fluor 488 phalloidin (2 μM, A12379, Thermo Fisher Scientific, Waltham, USA).

Techniques: Fluorescence, Cell Culture, Construct

Journal: PLoS ONE

Article Title: Dissecting the Molecular Properties of Prokaryotic Flotillins

doi: 10.1371/journal.pone.0116750

Figure Lengend Snippet: (A) A cartoon of the two putative topologies of SNAP-YuaG is shown. YuaG has either a hairpin loop tethering it to the membrane or a trans-membrane helix as predicted by bioinformatics tools. SNAP dye TMR-Star is membrane permeable and hence sufficient to label in- and outside the cell, labelling with SNAP dye BG-488 (SNAP-surface 488), which is membrane impermeable, is only possible with an extracellular SNAP tag. (B) Cells expressing SNAP-YuaG or free SNAP were labelled with the cell impermeable SNAP dye BG-488 and the cell permeable SNAP dye TMR-Star. SNAP dyes are all false coloured in green. Zoomed in regions are indicated with a red frame. Scale bar 2 µm. (C) A representative Proteinase K (PK) sensitivity assay is shown for protoplasted B. subtilis cells. Control experiments using wild type cells incubated with TMR-Star and unlabeled cells expressing SNAP-YuaG confirmed that TMR-Star does not unspecifically label B. subtilis proteins. As a positive control protoplasts were resuspended in water instead of MSMNB and TritonX-100 was added to a final concentration of 1%. (D) The in gel-fluorescence of the PK assay was quantified. Fluorescence of 0 minutes PK was always defined as 100%. Standard deviation is shown; n = 4. (E) Alignment of the hydrophobic helix of B. subtilis YuaG with different flotillins from various bacteria. Note the conserved glycine residue, highlighted in red. The alignment was performed using Kalign with default settings .

Article Snippet: We incubated cells expressing SNAP-YuaG with the membrane impermeable SNAP dye BG-488 (NEB) and the membrane permeable dye SNAP-CellCell TMR-Star (NEB).

Techniques: Expressing, Sensitive Assay, Incubation, Positive Control, Concentration Assay, Fluorescence, PK Assay, Standard Deviation

Journal: ChemRxiv

Article Title: Synthetic Siglec-9 agonists inhibit neutrophil activation associated with COVID-19

doi: 10.26434/chemrxiv.13378148

Figure Lengend Snippet: A cis-binding Siglec-9 agonist (pS9L) inhibits R848-induced NETosis via Siglec-9 and SHP-1. ( a - c ) Primary neutrophils were cotreated with R848 (10 μM) and glycopolypeptide (500 nM) in IMDM supplemented 0.5% hiFBS containing the membrane impermeable DNA intercalators Cytotox Green or Red (250 nM). Images were acquired by fluorescence microscopy every 15 min for 12 h. The area of all green fluorescent objects >300 μm 2 was quantified and the total area was averaged across three images per well. Relative NETosis was determined by normalizing to the maximal NET area from R848 treatment alone (t = 8 h). ( a ) Representative phase contrast and fluorescence images from t = 8 h. Scale bars indicate 40 μm. ( b ) Quantitation of NETosis over time as area under the curve in ( c ). Error bars represent SD. ( c ) NET formation and degradation as a function of time. Error bands represent SEM. ( d ) Treatment of R848-stimulated neutrophils with various glycopolypeptides. Error bars represent SD. ( e ) pS9L is a mucin-like glycopolypeptide that bears high affinity and specific ligands for Siglec-9 and is functionalized with a membrane-tethering lipid tail. ( f ) HL-60 cells were transfected with siRNAs against SIGLEC9 (encoding Siglec-9), PTPN6 (encoding SHP-1), or a scrambled control and then grown for two days. Cells were then cotreated with R848 (10 μM) and vehicle or pS9L (500 nM). Relative NETosis is determined as in ( b ), except all objects >200 μm 2 were quantified and the R848 maximum in dHL-60’s was observed at 2.5 h post induction. Error bars represent SD. Statistics were determined by two-way ANOVA ( b ) or one-way ANOVA ( c , d , f ). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: DNA staining was performed using membrane impermeable fluorogenic DNA intercalators Cytotox Green (Essen Biosciences, 4633) or Cytotox Red (Essen Biosciences, 4632).

Techniques: Binding Assay, Membrane, Fluorescence, Microscopy, Quantitation Assay, Transfection

Journal: ChemRxiv

Article Title: Synthetic Siglec-9 agonists inhibit neutrophil activation associated with COVID-19

doi: 10.26434/chemrxiv.13378148

Figure Lengend Snippet: Reagent Table and Usage.

Article Snippet: DNA staining was performed using membrane impermeable fluorogenic DNA intercalators Cytotox Green (Essen Biosciences, 4633) or Cytotox Red (Essen Biosciences, 4632).

Techniques: Negative Control