dq collagen type iv  (Thermo Fisher)

Journal: NPJ Biofilms and Microbiomes

Article Title: Protease activities of vaginal Porphyromonas species disrupt coagulation and extracellular matrix in the cervicovaginal niche

doi: 10.1038/s41522-022-00270-7

Figure Lengend Snippet: a Cell suspensions of P. asaccharolytica and P. uenonis at 10 7 or 10 8 CFU/reaction were incubated with fluorophore-conjugated type I collagen. Results are presented as mean ± standard error from three independent experiments performed in technical triplicate or quadruplicate. Collagen degradation was measured every 3 min by detecting the increase in fluorescence over a 2-h time course. b , c Cell-free supernatants of P. asaccharolytica and P. uenonis were incubated with fluorophore-conjugated b type I or c type IV collagen over an 18-h time course. Results are presented as mean ± standard error from seven independent experiments (type I collagen) and four independent experiments (type IV collagen) performed in technical triplicate. d Cell-free supernatants of P. asaccharolytica and P. uenonis were incubated with fluorescein (FITC)-conjugated casein over a 5-h time course. Results are presented as mean ± standard error from five independent experiments performed in technical triplicate. The mean fluorescence readings of the negative control were subtracted from experimental wells and relative fluorescence units (RFU) were plotted over time, with negative values adjusted to zero.

Article Snippet: Cell suspensions or cell-free supernatants were tested for collagenase activity with the EnzChek Gelatinase/Collagenase assay kit (Invitrogen, Carlsbad, CA) using fluorophore-labeled DQ TM gelatin conjugate (predominantly type I collagen, Invitrogen) or a fluorophore-labeled DQ TM type IV collagen conjugate from the human placenta (Invitrogen).

Techniques: Incubation, Fluorescence, Negative Control

Journal: NPJ Biofilms and Microbiomes

Article Title: Protease activities of vaginal Porphyromonas species disrupt coagulation and extracellular matrix in the cervicovaginal niche

doi: 10.1038/s41522-022-00270-7

Figure Lengend Snippet: a , b Cell-free supernatants of a P. asaccharolytica and b P. uenonis were incubated with fluorophore-conjugated casein in the presence of the metalloprotease inhibitor 1,10-phenanthroline (0.2 mM), the cysteine protease inhibitor iodoacetamide (0.4 mM), or the serine protease inhibitor aprotinin (0.01 mM). Casein degradation was measured every 10 min by detecting the increase in fluorescence over a 5-h time course. Results are presented as a ratio normalized to the no inhibitor control and presented as mean ± standard error from five independent experiments performed in technical triplicate. The mean fluorescence readings of the negative control were subtracted from experimental wells and relative fluorescence units (RFU) was plotted over time, with negative values adjusted to zero. c Maximum fluorescence of P. asaccharolytica supernatant caseinase activity in the presence of inhibitors. Results are presented as mean ± standard error from five independent experiments performed in technical triplicate. Statistical significance was assessed with a one-way ANOVA and Tukey’s post hoc comparison, where inhibitor combination vs. no inhibitor * p = 0.0413. d Maximum fluorescence of P. uenonis supernatant caseinase activity in the presence of inhibitors. Results are presented as mean ± standard error from five independent experiments performed in technical triplicate. Statistical significance was assessed with a one-way ANOVA and Tukey’s post hoc comparison, where 1,10-phenanthroline 0.2 mM vs. no inhibitor p = 0.0145, inhibitor combination vs. no inhibitor p = 0.0180.

Article Snippet: Cell suspensions or cell-free supernatants were tested for collagenase activity with the EnzChek Gelatinase/Collagenase assay kit (Invitrogen, Carlsbad, CA) using fluorophore-labeled DQ TM gelatin conjugate (predominantly type I collagen, Invitrogen) or a fluorophore-labeled DQ TM type IV collagen conjugate from the human placenta (Invitrogen).

Techniques: Incubation, Protease Inhibitor, Fluorescence, Negative Control, Activity Assay

Journal: NPJ Biofilms and Microbiomes

Article Title: Protease activities of vaginal Porphyromonas species disrupt coagulation and extracellular matrix in the cervicovaginal niche

doi: 10.1038/s41522-022-00270-7

Figure Lengend Snippet: a SDS-PAGE of control in vitro transcription/translation (myTXTL ® ) reaction (RNA polymerase only) and PepO myTXTL ® reaction. b – d PepO myTXTL ® reactions were incubated with b fluorescein (FITC)-casein or c fluorophore-conjugated type I collagen in the presence of 0.5 mM 1,10-phenanthroline. Casein degradation was measured by detecting the increase in fluorescence over a 24-h time course. Results are presented as mean ± standard error from three independent experiments. Collagen degradation was measured by detecting the increase in fluorescence over an 18-h time course. Results are presented as mean ± standard error from five independent experiments. d PepO myTXTL ® reactions were incubated with fluorophore-conjugated type IV collagen over an 18-h time course and results are presented as mean ± standard deviation from one independent experiment. The mean fluorescence readings of the negative control were subtracted from experimental wells and relative fluorescence units (RFU) was plotted over time, with negative values adjusted to zero.

Article Snippet: Cell suspensions or cell-free supernatants were tested for collagenase activity with the EnzChek Gelatinase/Collagenase assay kit (Invitrogen, Carlsbad, CA) using fluorophore-labeled DQ TM gelatin conjugate (predominantly type I collagen, Invitrogen) or a fluorophore-labeled DQ TM type IV collagen conjugate from the human placenta (Invitrogen).

Techniques: SDS Page, In Vitro, Incubation, Fluorescence, Standard Deviation, Negative Control