A novel sulfhydryl-sensing fluorescent probe to monitor the redox status of intracellular compartments

Background: Sensing of intracellular redox state is important for detecting the effect of disease and therapeutics agents. However, a reliable assay that can simultaneously provide information about the redox state of intracellular compartments is currently not available. Such an assay will improve the detection of abnormal metabolic state and evaluate the impact of therapeutics. Purpose: To test the responsiveness of DSSQ1 (fluorescein- Donor tethered via a disulfide [S-S] to a para-methyl red Quencher), a novel sulfhydryl-sensing fluorescence probe, and monitor its intracellular distribution under oxidative and reduced conditions. Methods: Fibroblasts grown in culture were treated with redox sensor DSSQ1, and its intracellular distribution and localization was assessed using confocal fluorescent microscopy. Localization of DSSQ1 within mitochondria, lysosomes and nuclei was confirmed using specific fluorescent dyes – TMRM for mitochondria, LysoTracker® Red for lysosomes and Hoechst 33342 for nuclei. Results: Under the normal conditions, the green fluorescence of DSSQ1 was localized to the cytosol, lysosomes, nuclear membrane and within mitochondria. Oxidative stress (extracellular H2O2, 100 μM) significantly decreased the loading efficiency of the redox sensor DSSQ1 into the fibroblasts, while reducing agent (extracellular N-acetyl cysteine, 10 mM), which is known to increase intracellular levels of glutathione and cytoplasmic redox state, enhanced the uptake of DSSQ1. Conclusion: The chemical structure of DSSQ1 allows permeability of compound without losing viability of cells. The compound is distributed within the cytoplasm, and localizes to lysosomes, mitochondria and nuclear membrane, but excluded from the nuclei. DSSQ1 accumulation is affected by redox status of cells and could be used to monitor the redox status of the cell

A novel high throughput approach for quantification of cell density

Background: Current approach to cell counting using hemacytometer is limited by requirement for high cell concentration and is prone to error. In biological experiments using cells from human cardiac tissues with limited number of cells, this approach results in large variation in cell counts. Here, we demonstrate the utility of a novel approach using a 96-well microplate that accurately provides the density of cells as low as 15,000 cells/cm2, which fulfills an unmet need in experiments with limited cell availability. Purpose: To develop and test the accuracy of a high-throughput 96-well microplate assay in assessing the cell density in comparison to existing methods. Methods: NIH/3T3 fibroblasts were cultured and differentiated and grown to different cell density. Cell number obtained using hemacytometer was compared to the total fluorescence of propidium iodide, binding to the nuclei of cells permeabilized with Triton X-100 (0.25%), and assessed using multiplate reader. In addition, the total activity of lactate dehydrogenase, an intracellular enzyme, was used to assess the total volume of cytoplasm released from permeabilized cells. Furthermore, the ratio of live/ dead cells was determined by propidium iodide-positive cells and lactate dehydrogenase activity before and after permeabilization in each well of the 96-well plate. Results:There was a linear relationship between increasing intensity of propidium iodide fluorescence with the density of the cells in the 96-well microplate (ranging from 5,000 to 100,000 cells/cm2). Similarly, linear relationship was observed between the intensity of propidium iodide fluorescence and cellular lactate dehydrogenase activity in corresponding wells. At low cell density ( Conclusion: Proposed propidium iodide and lactate dehydrogenase assays are useful tools for quantification of cell number in high-throughput manner with greater accuracy at low cell density, higher reproducibility and overall time saving. This assay is especially useful in experiments using limited cell number such as cells isolated from the human heart

TGF-β1-mediated differentiation of fibroblasts is associated with increased mitochondrial content and cellular respiration

OBJECTIVES: Cytokine-dependent activation of fibroblasts to myofibroblasts, a key event in fibrosis, is accompanied by phenotypic changes with increased secretory and contractile properties dependent on increased energy utilization, yet changes in the energetic profile of these cells are not fully described. We hypothesize that the TGF-β1-mediated transformation of myofibroblasts is associated with an increase in mitochondrial content and function when compared to naive fibroblasts. METHODS: Cultured NIH/3T3 mouse fibroblasts treated with TGF-β1, a profibrotic cytokine, or vehicle were assessed for transformation to myofibroblasts (appearance of α-smooth muscle actin [α-SMA] stress fibers) and associated changes in mitochondrial content and functions using laser confocal microscopy, Seahorse respirometry, multi-well plate reader and biochemical protocols. Expression of mitochondrial-specific proteins was determined using western blotting, and the mitochondrial DNA quantified using Mitochondrial DNA isolation kit. RESULTS: Treatment with TGF-β1 (5 ng/mL) induced transformation of naive fibroblasts into myofibroblasts with a threefold increase in the expression of α-SMA (6.85 ± 0.27 RU) compared to cells not treated with TGF-β1 (2.52 ± 0.11 RU). TGF-β1 exposure increased the number of mitochondria in the cells, as monitored by membrane potential sensitive dye tetramethylrhodamine, and expression of mitochondria-specific proteins; voltage-dependent anion channels (0.54 ± 0.05 vs. 0.23 ± 0.05 RU) and adenine nucleotide transporter (0.61 ± 0.11 vs. 0.22 ± 0.05 RU), as well as mitochondrial DNA content (530 ± 12 μg DNA/106 cells vs. 307 ± 9 μg DNA/106 cells in control). TGF-β1 treatment was associated with an increase in mitochondrial function with a twofold increase in baseline oxygen consumption rate (2.25 ± 0.03 vs. 1.13 ± 0.1 nmol O2/min/106 cells) and FCCP-induced mitochondrial respiration (2.87 ± 0.03 vs. 1.46 ± 0.15 nmol O2/min/106 cells). CONCLUSIONS: TGF-β1 induced differentiation of fibroblasts is accompanied by energetic remodeling of myofibroblasts with an increase in mitochondrial respiration and mitochondrial content

TGF-β1-mediated enhancement of mitochondrial respiration in NIH/3T3 cells.

<p><b>A</b>, Endogenous oxygen consumption rate (OCR) of naive (TGF-β1-untreated) and differentiated (TGF-β1-treated) NIH/3T3 cells measured using Seahorse XF-96. Basal respiration was inhibited with oligomycin (inhibitor of oxidative phosphorylation), followed by treatment with FCCP (uncoupler of mitochondrial oxidative phosphorylation) and antimycin A (inhibitor of mitochondrial respiratory chain). <b>B</b>, Quantification of OCR in naive and TGF-β1-treated NIH/3T3 cells at baseline and following treatment with oligomycin, FCCP and antimycin A. Shown are averages of at least three independent measurements ± standard error of mean. White circles and bars for naive, TGF-β1-untreated cells and black circles and bars for TGF-β1-treated cells.</p

TGF-β1-mediated differentiation of NIH/3T3 fibroblasts into myofibroblasts.

<p><b>A and B</b>, Representative fluorescent images of naive (control) and differentiated (TGF-β1-treated) cells. Cell nuclei are stained blue (1 μg/mL in mounting media); intracellular vimentin, a characteristic protein of fibroblasts, and α-smooth muscle actin (α-SMA), a marker of myofibroblasts (green and red, respectively), were labeled using immunocytochemistry as described in the Materials and Methods section. Superimposed images of naive and differentiated NIH/3T3 cells (overlay) demonstrate increased expression of α-SMA following TGF-β1 treatment. <b>C</b>, Relative increase in expression of α-SMA in NIH/3T3 cells following differentiation (average of at least 3 experiments). Densitometry of western blot bands was performed using Image<i>J</i> software as described in the Materials and Methods sections.</p

TGF-β1 treatment increased the content of mitochondria and expression of mitochondria-specific proteins and mitochondrial DNA in differentiated NIH/3T3 fibroblasts.

<p><b>A</b>, Confocal images of NIH/3T3 cells loaded with nuclear (Hoechst 33342, blue) and mitochondria-specific (tetramethylrhodamine, red) fluorescent dyes. <b>B,</b> Quantification of the mean intensity of TMRM in naive and TGF-β1-treated cells using Image<i>J</i>. <b>C,</b> Quantification of α-SMA (marker of myofibroblasts), adenine nucleotide transporter (ANT) and voltage-dependent anion channels (VDAC) expression in naive (TGF-β1- untreated) and differentiated (TGF-β1-treated) NIH/3T3 cells. Densities of western blot bands were normalized to glyceraldehyde-6-phosphate dehydrogenase (GAPDH), a house-keeping protein. <b>D</b>, Quantification of mitochondrial DNA in naive and TGF-β1-treated cells. Shown are averages of at least three independent measurements ± standard error of mean, and the asterisk shows significance, p<0.05. E, Quantification of ATP/ADP ratio in naive and TGF-β1 treated cells (n = 3, p<0.05).</p

Propidium Iodide-based quantification of the number of viable cells in a 96-well Seahorse microplate.

<p><b>A</b>, Propidium Iodide fluorescence (Relative Fluorescence Unit) measured from individual wells of a XF 96-well microplate (excitation 535 nm; emission 617 nm) versus the number of cells plated into the well (cells/cm²). <b>B</b>, Protein content measured in each well versus cell density in these wells (number of cells plated into well, cells/cm²). <b>C</b>, Linear relationship between cell density (number of cells plated into well, cells/cm²) and OCR (oxygen consumption rate in corresponding wells). Shown are averages of at least three independent measurements ± standard error of mean.</p

Functional and structural differences in fibroblasts from atria of patients with and without atrial fibrillation

Conclusion: This study, for the first time, identifies differences in fibroblasts from human atria between AR (atrial fibrillation) and non-AF patients with regard to size, shape, motility, and time to wound closure. Further investigation into the functional significance of these differences on cardiac repair after injury and progression of AF and its complications is warranted