Combination of small molecule microarray and confocal microscopy techniques for live cell staining fluorescent dye discovery

Discovering new fluorochromes is significantly advanced by high-throughput screening (HTS) methods. In the present study a combination of small molecule microarray (SMM) prescreening and confocal laser scanning microscopy (CLSM) was developed in order to discover novel cell staining fluorescent dyes. Compounds with high native fluorescence were selected from a 14,585-member library and further tested on living cells under the microscope. Eleven compartment- specific, cell-permeable (or plasma membrane-targeted) fluorochromes were identified. Their cytotoxicity was tested and found that between 1-10 micromolar range, they were non-toxic even during long-term incubations. © 2013 by the authors; licensee MDPI, Basel, Switzerland

Prion Protein Does Not Confer Resistance to Hippocampus-Derived Zpl Cells against the Toxic Effects of Cu²⁺, Mn²⁺, Zn²⁺ and Co²⁺ Not Supporting a General Protective Role for PrP in Transition Metal Induced Toxicity

<div><p>The interactions of transition metals with the prion protein (PrP) are well-documented and characterized, however, there is no consensus on their role in either the physiology of PrP or PrP-related neurodegenerative disorders. PrP has been reported to protect cells from the toxic stimuli of metals. By employing a cell viability assay, we examined the effects of various concentrations of Cu²⁺, Zn²⁺, Mn²⁺, and Co²⁺ on Zpl (<i>Prnp</i>^-/-) and ZW (<i>Prnp</i>^+/+) hippocampus-derived mouse neuronal cells. <i>Prnp</i>^-/- Zpl cells were more sensitive to all four metals than PrP-expressing Zw cells. However, when we introduced PrP or only the empty vector into Zpl cells, we could not discern any protective effect associated with the presence of PrP. This observation was further corroborated when assessing the toxic effect of metals by propidium-iodide staining and fluorescence activated cell sorting analysis. Thus, our results on this mouse cell culture model do not seem to support a strong protective role for PrP against transition metal toxicity and also emphasize the necessity of extreme care when comparing cells derived from PrP knock-out and wild type mice.</p></div

Sensitivity to transition metal-induced toxicity of ZW 13–2 and Zpl 2–1 cells.

<p>Cells were tested for survival after treatment with transition metals for 24 h, assessing cell viability by alamarBlue assay. The cell lines ZW 13–2 (open circles) and Zpl 2–1 (black circles) were treated with increasing concentrations of either Cu²⁺-Gly (A) or Zn²⁺ (B), or Mn²⁺ (C) or Co²⁺ (D). Values were compared to those of the untreated controls and are presented as percentage. The data represent the means ± standard deviation (S.D.) of minimum 3 independent experiments performed in 5 replicates. *p<0.05, **p<0.01 and ***p<0.001 indicate significant differences between treated and untreated cells; ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 indicate significant differences between ZW 13–2 and Zpl 2–1 cells.</p

Expression levels of PrP in the generated stable cell lines.

<p>(A) Total cell lysates of stably expressing cells made from <i>Prnp</i>^{<i>−/−</i>} hippocampal neuronal cell line of Zürich I mice (Zpl 2–1) transfected with either the empty vector (Zpl 2-1-vector) or with mouse PrP gene (Zpl 2-1-PrP). Cell lysates were incubated in the absence (lane 1 and lane 3) or presence of PNGase F (lane 2 and lane 4). Western blot analysis was carried out with monoclonal PrP antibody SAF 32. β-actin was used to confirm equal loading of proteins. (B) Immunocytochemistry performed to verify prion protein expression in Zpl 2-1- PrP and Zpl 2-1-vector cells. PrP is immunolabeled by the monoclonal PrP antibody SAF 32 and an Alexa 568 conjugated secondary antibody (red), nucleus is stained with DAPI (cyanine blue) and merged image is shown in the last column. Pictures were recorded using a 60X oil immersion objective with no zooming. (C) Relative expressions of PrP from ZW 13–2 and Zpl 2-1-PrP (left panel). The expression of PrP^C is quantified using densitometry analysis (right panel), the bars represent the average value obtained from three independent western blots and the error bars represent the standard deviations.</p

The effect of the presence of PrP^C on transition metals induced cell death.

<p>Cells were tested for cell death after treatment with transition metals for 24 h, cell death was measured by propidium iodide (PI) exclusion assay. (A) Zpl 2–1, ZW 13–2, Zpl 2-1-vector and Zpl 2-1-PrP cells were treated either with 750 μM of Cu²⁺-Gly (1:4 mol/mol), or with 100 μM of Zn²⁺, or 500 μM of Mn²⁺ or 750 μM of Co²⁺. Dead cells were stained with PI, and histograms were obtained by flow cytometric analysis. M1 represents the population of PI positive cells. The bar graphs on the panels b through e indicate the average of PI positive cells in case of 750 μM of Cu²⁺ treatment (B), 100 μM of Zn²⁺ treatment (C), 500 μM of Mn²⁺ treatment (D) and 750 μM of Co²⁺ treatment (E), respectively. Experiments were performed three times in duplicates, and data represent the mean ± standard deviation (S.D.). *p<0.05, **p<0.01 and ***p<0.001 indicate significant differences between treated (+) and untreated (-) cells; ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 indicate significant differences between the ratios obtained for treated ZW 13–2 cells to treated Zpl 2–1 cells, and treated Zpl 2-1-vector cells to treated Zpl 2-1-PrP cells, respectively.</p

The effect of the presence of PrP^C on the susceptibility of cells to transition metal-induced toxicity.

<p>Cells were tested for survival after treatment with transition metals for 24 h and cell viability was determined using alamarBlue assay. Values are compared to those of the untreated controls and are expressed as percentage. The cells Zpl 2-1-PrP (open triangles) and Zpl 2-1-vector (black triangles) are compared (panels A through D) during treatments with increasing concentrations of metals, as follows: Cu²⁺-Gly (A), of Zn²⁺ (B), Mn²⁺ (C) and Co²⁺ (D). The cell lines ZW 13–2 (open circles), Zpl 2–1 (black circles) were treated similarly with increasing concentrations of metals and all four types of cells are compared on panels E through H, as follows: increasing concentration of Cu²⁺-Gly (E), of Zn²⁺ (F), Mn²⁺ (G) and of Co²⁺(H). The data are presented as means ± standard deviation (S.D.) of minimum 3 independent experiments performed in 5 replicates. *p<0.05, **p<0.01 and ***p<0.001 indicate significant differences between treated and untreated cells on panels A through D; ^μp<0.05, ^μμp<0.01 and ^μμμp<0.001 indicate significant differences between treated Zpl 2-1-PrP cells and treated Zpl 2-1-vector cells; ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 indicate significant differences between the treated ZW 13–2 cells and treated Zpl 2–1 cells; ^δp<0.05, ^δδp<0.01 and ^δδδp<0.001indicate significant differences between the treated ZW 13–2 cells and treated Zpl 2-1-vector cells, and ^φp<0.05, ^φφp<0.01 and ^φφφp<0.001 indicate significant differences between the treated ZW 13–2 cells and treated Zpl 2-1-PrP cells.</p

Expression levels of PrP in the ZW 13–2 and Zpl 2–1 cell lines.

<p>(A) Western blot of total cell lysates from wild type hippocampal neuronal cell line of ICR mice (ZW 13–2) and <i>Prnp</i>^{<i>−/−</i>} hippocampal neuronal cell line of Zürich I mice (Zpl 2–1). Cell lysates were incubated either in the absence (lane 1 and lane 3) or in the presence of PNGase F (lane 2 and lane 4). Western blot analysis of PrP was carried out with monoclonal PrP antibody SAF 32. β-actin was used to confirm equal loading of proteins. (B) Immunocytochemistry verification of prion protein expression in ZW 13–2 and Zpl 2–1 cells. PrP is immunolabeled by using the monoclonal PrP antibody SAF 32 with an Alexa 568 conjugated secondary antibody (red), nucleus is stained with DAPI (cyanine blue) and merged images are shown in the last column. Pictures were recorded by using a 60X oil immersion objective and a zooming factor of 4.</p