Journal: MethodsX

Article Title: Methods for mitochondrial health assessment by High Content Imaging System

doi: 10.1016/j.mex.2022.101685

Figure Lengend Snippet: The CellProfiler program processed fluorescence pictures of untreated fibroblast cells (0 µM H 2 O 2 ) through the pipeline for estimating mitochondrial H 2 O 2 levels. The fibroblast cells were co-stained with Hoechst 33342, MITO-ID® Red, and MitoPY1 fluorescent dyes. Imaging of stained cells was visualized by the Operetta CLS with the 40x NA objective lens. A. The Hoechst 33342 (blue) and MITO-ID® Red (red) channels were dedicated to identifying the Nuclei and cell's boundary outlines for the cytoplasm identification. B. The MitoPY1 channels' green-speckled fluorescence was used to recall hydrogen peroxide (H 2 O 2 ). C. Mitochondrial H 2 O 2 was analyzed by relating the green-speckled fluorescence to the cells identified from Hoechst 33342 and MITO-ID® Red channels. Scale bars represent 10 µm.

Article Snippet: The images of stained cells were visualized using the Operetta CLS TM High-Content Analysis System (PerkinElmer) in the confocal or non-confocal with two peaks of autofocus mode and a water objective lens, 40x high numerical aperture (NA), which had a high refractive index.

Techniques: Fluorescence, Staining, Imaging

Journal: MethodsX

Article Title: Methods for mitochondrial health assessment by High Content Imaging System

doi: 10.1016/j.mex.2022.101685

Figure Lengend Snippet: The CellProfiler program processed fluorescence pictures of fibroblast cells treated with 200 µM of H 2 O 2 for 1 h. The fibroblast cells were co-stained with Hoechst 33342, MITO-ID® Red, and MitoPY1 fluorescent dyes. Imaging of stained cells was visualized by the Operetta CLS with the 40x NA objective lens. A. The Hoechst 33342 (blue) and MITO-ID® Red (red) channels were dedicated to identifying the nuclei and cell's boundary outlines for the cytoplasm identification. B. The MitoPY1 channels' green-speckled fluorescence was used to recall hydrogen peroxide (H 2 O 2 ). C. Mitochondrial H 2 O 2 was analyzed by relating the green-speckled fluorescence to the cells identified from Hoechst 33342 and MITO-ID® Red channels. Scale bars represent 10 µm.

Article Snippet: The images of stained cells were visualized using the Operetta CLS TM High-Content Analysis System (PerkinElmer) in the confocal or non-confocal with two peaks of autofocus mode and a water objective lens, 40x high numerical aperture (NA), which had a high refractive index.

Techniques: Fluorescence, Staining, Imaging

Journal: MethodsX

Article Title: Methods for mitochondrial health assessment by High Content Imaging System

doi: 10.1016/j.mex.2022.101685

Figure Lengend Snippet: The CellProfiler program processed fluorescence pictures of fibroblast cells treated with 400 µM of H 2 O 2 for 1 h. The fibroblast cells were co-stained with Hoechst 33342, MITO-ID® Red, and MitoPY1 fluorescent dyes. Imaging of stained cells was visualized by the Operetta CLS with the 40x NA objective lens. A. The Hoechst 33342 (blue) and MITO-ID® Red (red) channels were dedicated to identifying the Nuclei and cell's boundary outlines for the cytoplasm identification. B. The MitoPY1 channels' green-speckled fluorescence was used to recall hydrogen peroxide (H 2 O 2 ). C. Mitochondrial H 2 O 2 was analyzed by relating the green-speckled fluorescence to the cells identified from Hoechst 33342 and MITO-ID® Red channels. Scale bars represent 10 µm.

Article Snippet: The images of stained cells were visualized using the Operetta CLS TM High-Content Analysis System (PerkinElmer) in the confocal or non-confocal with two peaks of autofocus mode and a water objective lens, 40x high numerical aperture (NA), which had a high refractive index.

Techniques: Fluorescence, Staining, Imaging

Journal: MethodsX

Article Title: Methods for mitochondrial health assessment by High Content Imaging System

doi: 10.1016/j.mex.2022.101685

Figure Lengend Snippet: CellProfiler program processed fluorescence pictures of untreated fibroblast cells (ethanol, 0 µM of FCCP) via the pipeline for measuring MMP levels. The fibroblast cells were co-stained with Hoechst 33342 and JC-10 fluorescent dyes. Imaging of stained cells was visualized by the Operetta CLS with the 40x NA objective lens. A. J-monomers (green-fluorescence) from JC-10 Alexa Fluor 488 channels associated with nuclei were specified and segmented. B. J-aggregates (orange-fluorescence) indicating polarized MMP from the JC-10 Rhodamine channels were inferred. C. The areas of J-aggregates masked by J-monomers (middle image) representing orange-fluorescence and the J-monomers masked by J-aggregates (right image) representing green-fluorescence were identified to calculate the intensity by the Mask Object module. The MMP alterations can be evaluated quantitatively using the intensity ratio of orange-fluorescence aggregates to green-fluorescence monomers. Scale bars represent 10 µm.

Article Snippet: The images of stained cells were visualized using the Operetta CLS TM High-Content Analysis System (PerkinElmer) in the confocal or non-confocal with two peaks of autofocus mode and a water objective lens, 40x high numerical aperture (NA), which had a high refractive index.

Techniques: Fluorescence, Staining, Imaging

Journal: MethodsX

Article Title: Methods for mitochondrial health assessment by High Content Imaging System

doi: 10.1016/j.mex.2022.101685

Figure Lengend Snippet: CellProfiler program processed fluorescence pictures of fibroblast cells treated with 0.01 µM of FCCP for 1 h via the pipeline for measuring MMP levels. The fibroblast cells were co-stained with Hoechst 33342 and JC-10 fluorescent dyes. Imaging of stained cells was visualized by the Operetta CLS with the 40x NA objective lens. A. J-monomers (green-fluorescence) from JC-10 Alexa Fluor 488 channels associated with nuclei were specified and segmented. B. J-aggregates (orange-fluorescence) indicating polarized MMP from the JC-10 Rhodamine channels were inferred. C. The areas of J-aggregates masked by J-monomers (middle image) representing orange-fluorescence and the J-monomers masked by J-aggregates (right image) representing green-fluorescence were identified to calculate the intensity by the Mask Object module. The MMP alterations can be evaluated quantitatively using the intensity ratio of orange-fluorescence aggregates to green-fluorescence monomers. Scale bars represent 10 µm.

Article Snippet: The images of stained cells were visualized using the Operetta CLS TM High-Content Analysis System (PerkinElmer) in the confocal or non-confocal with two peaks of autofocus mode and a water objective lens, 40x high numerical aperture (NA), which had a high refractive index.

Techniques: Fluorescence, Staining, Imaging

Journal: MethodsX

Article Title: Methods for mitochondrial health assessment by High Content Imaging System

doi: 10.1016/j.mex.2022.101685

Figure Lengend Snippet: CellProfiler program processed fluorescence pictures of fibroblast cells treated with 0.1 µM of FCCP for 1 h via the pipeline for measuring MMP levels. The fibroblast cells were co-stained with Hoechst 33342 and JC-10 fluorescent dyes. Imaging of stained cells was visualized by the Operetta CLS with the 40x NA objective lens. A. J-monomers (green-fluorescence) from JC-10 Alexa Fluor 488 channels associated with nuclei were specified and segmented. B. J-aggregates (orange-fluorescence) indicating polarized MMP from the JC-10 Rhodamine channels were inferred. C. The areas of J-aggregates masked by J-monomers (middle image) representing orange-fluorescence and the J-monomers masked by J-aggregates (right image) representing green-fluorescence were identified to calculate the intensity by the Mask Object module. The MMP alterations can be evaluated quantitatively using the intensity ratio of orange-fluorescence aggregates to green-fluorescence monomers. Scale bars represent 10 µm.

Article Snippet: The images of stained cells were visualized using the Operetta CLS TM High-Content Analysis System (PerkinElmer) in the confocal or non-confocal with two peaks of autofocus mode and a water objective lens, 40x high numerical aperture (NA), which had a high refractive index.

Techniques: Fluorescence, Staining, Imaging

Journal: MethodsX

Article Title: Methods for mitochondrial health assessment by High Content Imaging System

doi: 10.1016/j.mex.2022.101685

Figure Lengend Snippet: CellProfiler program processed fluorescence pictures of untreated fibroblast cells (ethanol, 0 µM of phenanthroline) through the pipeline for analyzing the levels of mitochondrial fragmentation and length. The fibroblast cells were co-stained with Hoechst 33342 and MITO-ID® Red fluorescent dyes. Imaging of stained cells was visualized by the Operetta CLS with the 40x NA objective lens. A. The image from the MITO-ID® Red channel was altered to Grayscale and enhanced the red fluorescent intensity. B. Mitochondria were identified by tagging the enhanced red fluorescence using the Thresholding strategy. C. The Morph module was utilized to shrink mitochondria into the skeleton and mark the endpoints of the skeleton. D. The mean mitochondrial length and fragmentation per cell were estimated through the Measure Object Skeleton module by measuring the distance between the shrunken endpoints within the mitochondrial skeleton. Scale bars represent 10 µm.

Article Snippet: The images of stained cells were visualized using the Operetta CLS TM High-Content Analysis System (PerkinElmer) in the confocal or non-confocal with two peaks of autofocus mode and a water objective lens, 40x high numerical aperture (NA), which had a high refractive index.

Techniques: Fluorescence, Staining, Imaging

Journal: MethodsX

Article Title: Methods for mitochondrial health assessment by High Content Imaging System

doi: 10.1016/j.mex.2022.101685

Figure Lengend Snippet: CellProfiler program processed fluorescence pictures of fibroblast cells treated with 50 µM of phenanthroline for 4 through the pipeline for analyzing the levels of mitochondrial fragmentation and length. The fibroblast cells were co-stained with Hoechst 33342 and MITO-ID® Red fluorescent dyes. Imaging of stained cells was visualized by the Operetta CLS with the 40x NA objective lens. A. The image from the MITO-ID® Red channel was altered to Grayscale and enhanced the red fluorescent intensity. B. Mitochondria were identified by tagging the enhanced red fluorescence using the Thresholding strategy. C. The Morph module was utilized to shrink mitochondria into the skeleton and mark the endpoints of the skeleton. D. The mean mitochondrial length and fragmentation per cell were estimated through the Measure Object Skeleton module by measuring the distance between the shrunken endpoints within the mitochondrial skeleton. Scale bars represent 10 µm.

Article Snippet: The images of stained cells were visualized using the Operetta CLS TM High-Content Analysis System (PerkinElmer) in the confocal or non-confocal with two peaks of autofocus mode and a water objective lens, 40x high numerical aperture (NA), which had a high refractive index.

Techniques: Fluorescence, Staining, Imaging

Journal: Nucleic Acids Research

Article Title: Methyl-CpG binding domain protein 1 regulates localization and activity of Tet1 in a CXXC3 domain-dependent manner

doi: 10.1093/nar/gkx281

Figure Lengend Snippet: Mbd1 enhances Tet1-mediated 5hmC formation. ( A ) Experimental rationale. Mouse tail fibroblasts (MTF) were transfected with mCherry-Tet1CD (catalytic domain of Tet1) or mCherry-Tet1FL (full length Tet1) together with Mbd1-GFP (methyl-CpG binding domain protein 1), Mbd2-GFP or GFP. Twenty-four hours after transfection the cells were immunostained with a 5-hydroxymethylcytosine (5hmC) specific antibody. The signals of Tet1, Mbd/GFP and 5hmC were detected by high content screening microscopy and low Tet1 expressing cells were used for plotting. ( B ) Effect of Mbd1, Mbd2 and GFP on Tet1CD-mediated 5hmC formation. The cells were first grouped according to Tet1 protein levels, then, in each group, cells were further binned by Mbd/GFP protein levels and single cell intensities were plotted. The 5hmC intensities in the cells with high levels of Mbds/GFP and high levels of Tet1CD were used to perform student's t -test and P -values are indicated. Experiments were repeated at least three times. Cell numbers for Tet1CD + Mbd1, Tet1CD + GFP and Tet1CD + Mbd2 are 10817, 7271 and 4375, respectively. ( C ) Effect of Mbd1 and GFP on Tet1FL-mediated 5hmC formation in mouse embryonic fibroblast cells (MEF). Intensities of single cells are plotted and the P -value of student's t -test is indicated. Experiments were repeated at least two times. Cell numbers for Tet1FL + Mbd1 and Tet1FL + GFP are 7515 and 3429, respectively. ( D ) Effect of Mbd1 and GFP on Tet1-mediated 5-carboxylcytosine (5caC) formation in MTF cells. MTF cells were transfected with mCherry-Tet1CD together with Mbd1-GFP or GFP. Twenty-four hours after transfection, the cells were immunostained with an 5caC antibody. The average intensity of single cells was plotted according to Tet1CD protein levels. Experiments were repeated twice and mean values were plotted. Cell numbers for Tet1CD + Mbd1 and Tet1CD + GFP are 1770 and 582, respectively. The 5caC intensities in the cells with high levels of Tet1CD (dashed line box) were used to perform student's t -test and the P -value is indicated. 95% confidence intervals were also indicated in the plot.

Article Snippet: Then, DNA was counterstained with 4΄,6-Diamidine-2-phenylindole dihydrochloride (DAPI, 1 μg/ml). mCherry, GFP or Alexa Fluor 488 and AMCA signals were imaged using high content screening microscopy with 20× 0.45 numerical aperture long working distance objective (PerkinElmer, UK), a Xenon fiber optic as light source, 360–400, 460–490 and 560–580 nm excitation filters and 410–480 (AMCA, DAPI), 500–550 (GFP, Alexa Fluor 488) and 590–640 (mCherry) emission filters, respectively.

Techniques: Transfection, Binding Assay, High Content Screening, Microscopy, Expressing

Journal: Nucleic Acids Research

Article Title: Methyl-CpG binding domain protein 1 regulates localization and activity of Tet1 in a CXXC3 domain-dependent manner

doi: 10.1093/nar/gkx281

Figure Lengend Snippet: The CXXC3 domain of Mbd1 is necessary for Mbd1 enhanced 5hmC formation. ( A ) Structure of full length Mbd1 and Mbd1ΔCXXC3. ( B ) Examples of Tet1CD localization in the presence of Mbd1 and Mbd1ΔCXX3. MEF cells were transfected with mCherry-Tet1CD and Mbd1-GFP or GFP-Mbd1ΔCXXC3. Eight hours after transfection, the cells were imaged using confocal microscopy. Scale bars: 5 μm. ( C ) The effect of the CXXC3 on Tet1 accumulation to heterochromatic DNA. The ratio of mean intensity inside and outside heterochromatic DNA was plotted. Cell number and P -value of student's t -test are indicated on the plot. ( D ) Effect of the CXXC3 on Tet1-mediated 5hmC formation. MEF cells were transfected with mCherry-Tet1CD and Mbd1-GFP or Flag-Mbd1ΔCXXC3. Twenty-four hours after transfection, cells were stained for 5hmC. Then, cells with low Tet1CD level were used for plotting. The cells were first subgrouped by Tet1 level and then, in each group, cells were further binned by Mbd1/Mbd1ΔCXXC3 protein level. Single cell 5hmC intensity was plotted. The 5hmC intensity in the cells with high levels of Mbds and high levels of Tet1CD were used to perform student's t -test and P -values are indicated. Experiments were repeated at least three times. Cell numbers for Tet1CD + Mbd1 and Tet1CD + Mbd1ΔCXXC3 are 16757 and 2589, respectively.

Article Snippet: Then, DNA was counterstained with 4΄,6-Diamidine-2-phenylindole dihydrochloride (DAPI, 1 μg/ml). mCherry, GFP or Alexa Fluor 488 and AMCA signals were imaged using high content screening microscopy with 20× 0.45 numerical aperture long working distance objective (PerkinElmer, UK), a Xenon fiber optic as light source, 360–400, 460–490 and 560–580 nm excitation filters and 410–480 (AMCA, DAPI), 500–550 (GFP, Alexa Fluor 488) and 590–640 (mCherry) emission filters, respectively.

Techniques: Transfection, Confocal Microscopy, Staining

Journal: Nucleic Acids Research

Article Title: Methyl-CpG binding domain protein 1 regulates localization and activity of Tet1 in a CXXC3 domain-dependent manner

doi: 10.1093/nar/gkx281

Figure Lengend Snippet: Mbd1 facilitates the localization of Tet1 to heterochromatic DNA. ( A–C ) Localization of Tet1CD and Mbd1 or Mbd2 in MTF cells. (A) MTF cells were transfected with Tet1CD together with GFP-tagged Mbd1 or Mbd2 constructs. Eight hours after transfection, Tet1 and Mbd1/Mbd2 signals were detected by confocal microscopy and images were used for H -coefficient colocalization (B) and protein accumulation (C) analysis. To calculate protein accumulation at heterochromatin, mean fluorescence intensities inside and outside heterochromatin were measured with ImageJ and the ratio of the mean gray value inside and outside heterochromatin was plotted. Scale bars: 5 μm. The cell number and P -values of the student's t -test are indicated. Two independent experiments were performed. ( D ) Fluorescence recovery after photobleaching (FRAP) analysis of Tet1 protein mobility. MEF cells were transfected with constructs coding for mCherry-Tet1CD together with Mbd1-GFP or GFP. The mCherry-Tet1CD signal at heterochromatin regions was photobleached with a 488 nm laser. The mean recovery curve was plotted. Cell numbers and P -value from Wilcoxon test are indicated on the plot. Two independent experiments were performed. ( E ) Accumulation of Tet1CD and Tet1CDmut (catalytic mutant of Tet1) to heterochromatic DNA. Shown are quantification results of the accumulation of Tet1 proteins at heterochromatic DNA using images from high content screening time-lapse microscopy. To better visualize the accumulation changes, the median accumulation value of Tet1CD at an early time point (8 h after transfection) is indicated and P -values of the student's t -test are indicated. Two independent experiments were performed. For each time point, more than 100 cells were analyzed.

Article Snippet: Then, DNA was counterstained with 4΄,6-Diamidine-2-phenylindole dihydrochloride (DAPI, 1 μg/ml). mCherry, GFP or Alexa Fluor 488 and AMCA signals were imaged using high content screening microscopy with 20× 0.45 numerical aperture long working distance objective (PerkinElmer, UK), a Xenon fiber optic as light source, 360–400, 460–490 and 560–580 nm excitation filters and 410–480 (AMCA, DAPI), 500–550 (GFP, Alexa Fluor 488) and 590–640 (mCherry) emission filters, respectively.

Techniques: Transfection, Construct, Confocal Microscopy, Fluorescence, Mutagenesis, High Content Screening, Time-lapse Microscopy

Journal: Nucleic Acids Research

Article Title: Methyl-CpG binding domain protein 1 regulates localization and activity of Tet1 in a CXXC3 domain-dependent manner

doi: 10.1093/nar/gkx281

Figure Lengend Snippet: Mbd1 enhanced Tet1 localization to heterochromatic DNA is cell cycle independent. ( A ) Distribution of Tet1, Mbd1 and PCNA (proliferating cell nuclear antigen) in MEF cells. MEF cells were transfected with mCherry-Tet1CD, Mbd1-GFP and CFP-PCNA constructs. Eight hours after transfection, the live cells were imaged using confocal microscopy. The cell populations were discriminated by PCNA distribution to either (late) S-phase or G-phase. ( B ) Distribution of Tet1 and Mbd1 in G1-phase. Time-lapse microscopy was performed to follow cell division using the cells transfected with mCherry-Tet1CD and Mbd1-GFP (for full time lapse see ). Shown is an example of a cell in G1-phase. Scale bars: 5 μm.

Article Snippet: Then, DNA was counterstained with 4΄,6-Diamidine-2-phenylindole dihydrochloride (DAPI, 1 μg/ml). mCherry, GFP or Alexa Fluor 488 and AMCA signals were imaged using high content screening microscopy with 20× 0.45 numerical aperture long working distance objective (PerkinElmer, UK), a Xenon fiber optic as light source, 360–400, 460–490 and 560–580 nm excitation filters and 410–480 (AMCA, DAPI), 500–550 (GFP, Alexa Fluor 488) and 590–640 (mCherry) emission filters, respectively.

Techniques: Transfection, Construct, Confocal Microscopy, Time-lapse Microscopy

Journal: Nucleic Acids Research

Article Title: Methyl-CpG binding domain protein 1 regulates localization and activity of Tet1 in a CXXC3 domain-dependent manner

doi: 10.1093/nar/gkx281

Figure Lengend Snippet: The interaction of Mbd1 and Tet1 enhances Tet1 localization to heterochromatic DNA. ( A ) Dnmt1 −/− MEF ( p53 −/− and Dnmt1 −/− ) cells were transfected with mCherry-Tet1CD and Mbd1-GFP or Mbd2-GFP. Eight hours after transfection, the cells were imaged using confocal microscopy. The left panel shows the schematic of the experiment and the right panel shows exemplary cells with Tet1 and Mbd1/Mbd2 expression. ( B ) Accumulation of Mbd1/Mbd2 and Tet1 proteins to heterochromatic DNA measured as in Figure . ( C ) Dnmt1 −/− MEF cells were triple-transfected with mCherry-Tet1CD, GBP-MaSat (GFP binding protein, polydactyl zinc finger major satellite binder) and Mbd1-GFP or Mbd2-GFP plasmids. Eight hours after transfection, cells were imaged using confocal microscopy. The left panel shows the schematic of the experiment and how GBP-MaSat targets GFP-tagged proteins to heterochromatic DNA. The right panel shows exemplary cells with Tet1 and Mbd1/Mbd2 expression in the presence of GBP-MaSat. ( D ) Accumulation of Tet1 and Mbd1/2 proteins to heterochromatic DNA. Cell number and P -value of t -test are indicated. ( E ) Interaction between Tet1CD and subdomains of Mbd1. HEK (human embryonic kidney) cells co-expressing mCherry-Tet1CD and GFP tagged Mbds were used for co-immunoprecipitation assays using immobilized RFP binding protein. The input and bound GFP signals on the membrane were detected using a GFP-specific antibody. Left panels are input for mCherry and GFP fusion proteins and rightmost panel shows interaction bands. The middle panel depicts the structures of Mbd1 fusions. The right panel summarizes the results of two independent biological replicates.

Article Snippet: Then, DNA was counterstained with 4΄,6-Diamidine-2-phenylindole dihydrochloride (DAPI, 1 μg/ml). mCherry, GFP or Alexa Fluor 488 and AMCA signals were imaged using high content screening microscopy with 20× 0.45 numerical aperture long working distance objective (PerkinElmer, UK), a Xenon fiber optic as light source, 360–400, 460–490 and 560–580 nm excitation filters and 410–480 (AMCA, DAPI), 500–550 (GFP, Alexa Fluor 488) and 590–640 (mCherry) emission filters, respectively.

Techniques: Transfection, Confocal Microscopy, Expressing, Binding Assay, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Methyl-CpG binding domain protein 1 regulates localization and activity of Tet1 in a CXXC3 domain-dependent manner

doi: 10.1093/nar/gkx281

Figure Lengend Snippet: Tet1 displaces Mbd1 binding to heterochromatic DNA. ( A ) Mobility of Mbd1 and Mbd1ΔCXXC3. MEF cells were transfected with Mbd1-GFP or GFP-Mbd1ΔCXXC3. Eight hours after transfection, the mobility of Mbd proteins was measured by FRAP. The mean values are plotted as a recovery curve and the cell number is indicated on the plot. T-half values were extracted from mean exponential fitting. Solid and dashed lines indicate recovery curve of Mbd1ΔCXXC3 and Mbd1, respectively. ( B ) Mobility of Mbd1 (right) and Mbd1ΔCXXC3 (left) in the presence of Tet1CD. MEF cells co-expressing mCherry-Tet1CD and GFP-Mbd1 or GFP-Mbd1ΔCXX3 were used for FRAP analysis. One heterochromatic region was photobleached with a 488 nm laser, and after photobleaching the intensities of GFP and mCherry were measured. The mean values are plotted as a recovery curve and cell numbers are indicated on the plot. T-half values were extracted from the mean exponential fitting. T-half values for single cells are shown as box plots and P -values (Wilcoxon test) are indicated. At least two independent experiments were performed. ( C ) Scheme summarizing the Mbd1 and Tet1 binding modes to heterochromatic DNA. Black and white circles indicate methylated and unmethylated CpG, respectively.

Article Snippet: Then, DNA was counterstained with 4΄,6-Diamidine-2-phenylindole dihydrochloride (DAPI, 1 μg/ml). mCherry, GFP or Alexa Fluor 488 and AMCA signals were imaged using high content screening microscopy with 20× 0.45 numerical aperture long working distance objective (PerkinElmer, UK), a Xenon fiber optic as light source, 360–400, 460–490 and 560–580 nm excitation filters and 410–480 (AMCA, DAPI), 500–550 (GFP, Alexa Fluor 488) and 590–640 (mCherry) emission filters, respectively.

Techniques: Binding Assay, Transfection, Expressing, Methylation