Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Human intestinal enteroids as a model of Clostridioides difficile -induced enteritis

doi: 10.1152/ajpgi.00045.2020

Figure Lengend Snippet: Clostridioides difficile strains secrete toxins, maintain shape and viability after incubation in Fluorobrite DMEM. A: toxin A and B ELISAs with C. difficile strains 37, M28, 630, and R20291 after 48 h in BHIS or 24-h incubation in BHIS and an additional 24 h incubation in Fluorobrite DMEM. ELISAs were examined on a plate reader at optical density (OD) of 450 nm. B: FM 4–64 membrane staining in C. difficile 37, M28, 630, and R20291 after 24-h incubation with BHIS and an additional 24 h incubation in Fluorobrite DMEM. Scale bar = 50 μm. C: cell viability as determined by BacLight live/dead cell staining. Cell staining was examined at excitation: 485 nm/emission: 530 nm (live) and excitation: 485 nm/emission: 630 nm (dead) on a Synergy H1 Microplate Reader, and viabilities were calculated by dividing the fluorescence intensity of 485/530 by the fluorescence intensity at 485/630 × 100%.

Article Snippet: For live-cell imaging, LifeActRuby-expressing cells were grown to confluence on 10-well CELLview chamber slides (GreinerBio) and then changed to an optically clear FluoroBrite DMEM media (Invitrogen) supplemented with 15 mM HEPES (Invitrogen), 1× sodium pyruvate (Invitrogen), 1× GlutaMax (Invitrogen), and 1× nonessential amino acids (Invitrogen) (Fluorobrite-Plus) C. difficile conditioned FluoroBrite DMEM was added in uninoculated FluoroBrite DMEM (50% final concentration).

Techniques: Incubation, Membrane, Staining, Fluorescence

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Human intestinal enteroids as a model of Clostridioides difficile -induced enteritis

doi: 10.1152/ajpgi.00045.2020

Figure Lengend Snippet: Human intestinal enteroids (HIEs) exhibited delayed cell rounding in response to toxigenic Clostridioides difficile strains. HIEs derived from the jejunum were transduced with the LifeAct-Ruby sensor, which labels F-actin with red fluorescent protein. Cell rounding was visualized by live cell microscopy on a Nikon TiE with a ×20 Plan Apo (NA 0.75) differential interference contrast objective, using a SPECTRA X LED light source and ORCA-Flash 4.0 sCMOS camera (scale bar, 50 µm). A: representative images of LifeAct-Ruby HIEs over time (1–16 h) after exposure to Fluorobrite DMEM medium alone or conditioned Fluorobrite from C. difficile strains CD37, M68, 630, or R20291. Insets indicate significant rounding occurs with toxigenic C. difficile strains (M68, 630, and R20291), but this cell rounding occurs at later time points. B: FIJI (formerly ImageJ) software was used to define cell membranes (as denoted by actin labeling) and cell diameter over time. C: resulting curves were assessed for the area under the curve (AUC), which demonstrates decreased cell diameter with M68, 630, and R20291. *P < 0.05, 1-way ANOVA; n = 3 independent experiments, n = 6/experiment. NS, not significant.

Article Snippet: For live-cell imaging, LifeActRuby-expressing cells were grown to confluence on 10-well CELLview chamber slides (GreinerBio) and then changed to an optically clear FluoroBrite DMEM media (Invitrogen) supplemented with 15 mM HEPES (Invitrogen), 1× sodium pyruvate (Invitrogen), 1× GlutaMax (Invitrogen), and 1× nonessential amino acids (Invitrogen) (Fluorobrite-Plus) C. difficile conditioned FluoroBrite DMEM was added in uninoculated FluoroBrite DMEM (50% final concentration).

Techniques: Derivative Assay, Transduction, Microscopy, Software, Labeling

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Human intestinal enteroids as a model of Clostridioides difficile -induced enteritis

doi: 10.1152/ajpgi.00045.2020

Figure Lengend Snippet: Human intestinal enteroids (HIEs) are less responsive than Vero cells to purified Clostridioides difficile toxin A: HIEs derived from jejunum were transduced with the LifeAct-Ruby sensor, which labels F-actin with red fluorescent protein. Cell rounding was visualized by live-cell microscopy on a Nikon TiE with a ×20 Plan Apo (NA 0.75) differential interference contrast objective, using a SPECTRA X LED light source and ORCA-Flash 4.0 sCMOS camera (scale bar, 50 µm). A: representative images of LifeAct-Ruby HIEs over time (1–16 h) after exposure to Fluorobrite DMEM medium alone or purified TcdA (List Biologicals) in Fluorobrite DMEM. Insets indicate that significant rounding occurs with TcdA as low as 0.01 μg/mL (10 ng/mL). Similar to the C. difficile strains, cell rounding in response to purified toxins occurs at later time points. B: FIJI (formerly ImageJ) software was used to define cell membranes (as denoted by actin labeling) and cell diameter over time. C: resulting curves were assessed for the area under the curve (AUC), which demonstrates decreased cell diameter with TcdA. *P < 0.05, 1-way ANOVA; n = 3 independent experiments, n = 6/experiment.

Article Snippet: For live-cell imaging, LifeActRuby-expressing cells were grown to confluence on 10-well CELLview chamber slides (GreinerBio) and then changed to an optically clear FluoroBrite DMEM media (Invitrogen) supplemented with 15 mM HEPES (Invitrogen), 1× sodium pyruvate (Invitrogen), 1× GlutaMax (Invitrogen), and 1× nonessential amino acids (Invitrogen) (Fluorobrite-Plus) C. difficile conditioned FluoroBrite DMEM was added in uninoculated FluoroBrite DMEM (50% final concentration).

Techniques: Purification, Derivative Assay, Transduction, Microscopy, Software, Labeling

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Human intestinal enteroids as a model of Clostridioides difficile -induced enteritis

doi: 10.1152/ajpgi.00045.2020

Figure Lengend Snippet: Human intestinal enteroids (HIEs) are less responsive than Vero cells to purified Clostridium difficile toxin B. HIEs derived from jejunum were transduced with the LifeAct-Ruby sensor, which labels F-actin with red fluorescent protein. Cell rounding was visualized by live-cell microscopy on a Nikon TiE with a ×20 Plan Apo (NA 0.75) differential interference contrast objective, using a SPECTRA X LED light source and ORCA-Flash 4.0 sCMOS camera (scale bar, 50 µm). A: representative images of LifeAct-Ruby HIEs cell over time (1–16 h) after exposure to Fluorobrite DMEM medium alone or purified TcdB (List Biologicals) in Fluorobrite DMEM. Insets indicate significant rounding occurs with TcdB as low as 0.1 μg/mL (100 ng/mL). Similar to the C. difficile strains, cell rounding in response to purified toxins occurs at later time points. B: FIJI (formerly ImageJ) software was used to define cell membranes (as denoted by actin labeling) and cell diameter over time. C: resulting curves were assessed for the area under the curve (AUC), which demonstrates decreased cell diameter with TcdB treatment. *P < 0.05, 1-way ANOVA; n = 3 independent experiments, n = 6/experiment.

Article Snippet: For live-cell imaging, LifeActRuby-expressing cells were grown to confluence on 10-well CELLview chamber slides (GreinerBio) and then changed to an optically clear FluoroBrite DMEM media (Invitrogen) supplemented with 15 mM HEPES (Invitrogen), 1× sodium pyruvate (Invitrogen), 1× GlutaMax (Invitrogen), and 1× nonessential amino acids (Invitrogen) (Fluorobrite-Plus) C. difficile conditioned FluoroBrite DMEM was added in uninoculated FluoroBrite DMEM (50% final concentration).

Techniques: Purification, Derivative Assay, Transduction, Microscopy, Software, Labeling

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Human intestinal enteroids as a model of Clostridioides difficile -induced enteritis

doi: 10.1152/ajpgi.00045.2020

Figure Lengend Snippet: Toxigenic Clostridioides difficile strains cause rapid cell rounding in monkey kidney fibroblast Vero cells. Vero cells were transduced with the LifeAct-Ruby sensor, which labels F-actin with red fluorescent protein. Cell rounding was visualized by live cell microscopy on a Nikon TiE with a ×20 Plan Apo (NA 0.75) differential interference contrast objective, using a SPECTRA X LED light source and ORCA-Flash 4.0 sCMOS camera (scale bar, 50 µm). A: representative images of LifeAct-Ruby Vero cells over time (1–4 h) after exposure to Fluorobrite DMEM medium alone or conditioned Fluorobrite from C. difficile strains CD37, M68, 630, or R20291. Insets indicate significant rounding occurs with toxigenic C. difficile strains (M68, 630, and R20291). B: FIJI (formerly ImageJ) software was used to define cell membranes (as denoted by actin staining) and cell diameter over time. C: resulting curves were assessed for the area under the curve, which demonstrates decreased cell diameter with M68, 630, and R20291. *P < 0.05, 1-way ANOVA; n = 3 independent experiments, n = 6/experiment.

Article Snippet: For live-cell imaging, LifeActRuby-expressing cells were grown to confluence on 10-well CELLview chamber slides (GreinerBio) and then changed to an optically clear FluoroBrite DMEM media (Invitrogen) supplemented with 15 mM HEPES (Invitrogen), 1× sodium pyruvate (Invitrogen), 1× GlutaMax (Invitrogen), and 1× nonessential amino acids (Invitrogen) (Fluorobrite-Plus) C. difficile conditioned FluoroBrite DMEM was added in uninoculated FluoroBrite DMEM (50% final concentration).

Techniques: Transduction, Microscopy, Software, Staining

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Human intestinal enteroids as a model of Clostridioides difficile -induced enteritis

doi: 10.1152/ajpgi.00045.2020

Figure Lengend Snippet: Purified toxin A (TcdA) and B (TcdB) cause rapid cell rounding in monkey kidney fibroblast Vero cells. LifeAct-Ruby-transduced Vero cells were incubated with varying concentrations of purified TcdA or TcdB (List Biologicals). Cell rounding was visualized by live-cell microscopy on a Nikon TiE with ×20 Plan Apo (NA 0.75) differential interference contrast objective, using a SPECTRA X LED light source and ORCA-Flash 4.0 sCMOS camera (scale bar = 50 µm). A: representative images of LifeAct-Ruby Vero cells 4 h after exposure to Fluorobrite DMEM medium alone or purified toxins in Fluorobrite DMEM. FIJI (formerly ImageJ) software was used to define cell membranes (as denoted by actin staining) and cell diameter over time for TcdA (B) and TcdB (C). Area under the curve (AUC) analysis of TcdA (D) and TcdB (E). One-way ANOVA; n = 3 independent experiments, n = 6/experiment.

Article Snippet: For live-cell imaging, LifeActRuby-expressing cells were grown to confluence on 10-well CELLview chamber slides (GreinerBio) and then changed to an optically clear FluoroBrite DMEM media (Invitrogen) supplemented with 15 mM HEPES (Invitrogen), 1× sodium pyruvate (Invitrogen), 1× GlutaMax (Invitrogen), and 1× nonessential amino acids (Invitrogen) (Fluorobrite-Plus) C. difficile conditioned FluoroBrite DMEM was added in uninoculated FluoroBrite DMEM (50% final concentration).

Techniques: Purification, Incubation, Microscopy, Software, Staining

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Human intestinal enteroids as a model of Clostridioides difficile -induced enteritis

doi: 10.1152/ajpgi.00045.2020

Figure Lengend Snippet: Vero cell and human intestinal enteroids (HIEs) exhibit distinct morphologies following Clostridioides difficile R20291 toxin exposure by scanning electron microscopy (SEM). SEM was performed on Vero and HIE monolayers exposed to C. difficile R20291 Fluorobrite DMEM for 16 h. Images depict increasing magnification for both cell types, ending with a 10-μm scale.

Article Snippet: For live-cell imaging, LifeActRuby-expressing cells were grown to confluence on 10-well CELLview chamber slides (GreinerBio) and then changed to an optically clear FluoroBrite DMEM media (Invitrogen) supplemented with 15 mM HEPES (Invitrogen), 1× sodium pyruvate (Invitrogen), 1× GlutaMax (Invitrogen), and 1× nonessential amino acids (Invitrogen) (Fluorobrite-Plus) C. difficile conditioned FluoroBrite DMEM was added in uninoculated FluoroBrite DMEM (50% final concentration).

Techniques: Electron Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Multiple Sclerosis-Associated hnRNPA1 Mutations Alter hnRNPA1 Dynamics and Influence Stress Granule Formation

doi: 10.3390/ijms22062909

Figure Lengend Snippet: Key resources table. Listed are the reagents/resources utilized in this study. Their purchase source and catalogue identifiers are included.

Article Snippet: FluoroBrite DMEM Media , Fisher Scientific , Cat# A1896701.

Techniques: Modification, Membrane, Western Blot, Extraction, Recombinant, Software, Microscopy