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high throughput screening hts conventional fluorescence microscopy  (Nikon)


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    Nikon high throughput screening hts conventional fluorescence microscopy
    Figure 1. NPC characterization. A) Phase contrast <t>microscopy</t> of a-synuclein gene triplication (SNCA-Tri), control (Ctrl) and a-synuclein knockdown (SNCA-Tri KD) iPSC-derived NPC lines (Scale bar: 50 mm) shows normal cell morphology. B) Mitochondrial and nuclear morphology of NPCs visualized by <t>fluorescence</t> microscopy using Mitotracker Red CMX Ros (red) and Hoechst 33342 (blue) (Scale bar: 10 mm). C) Stem cell marker expression. Immuno-cytochemistry on fixed NPCs detecting cytoplasmic Nestin expression pattern with secondary Alexa 588 conjugated antibody (orange) by fluorescence microscopy (Scale bar: 100 mm). Insert: Immuno-cytochemistry for the nuclear stem cell marker SOX1, detected by
    High Throughput Screening Hts Conventional Fluorescence Microscopy, supplied by Nikon, used in various techniques. Bioz Stars score: 99/100, based on 57101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/high+throughput+screening+hts+conventional+fluorescence+microscopy/pm25390032-103-5-12?v=Nikon
    Average 99 stars, based on 57101 article reviews
    high throughput screening hts conventional fluorescence microscopy - by Bioz Stars, 2026-07
    99/100 stars

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    1) Product Images from "Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication."

    Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

    Journal: PloS one

    doi: 10.1371/journal.pone.0112413

    Figure 1. NPC characterization. A) Phase contrast microscopy of a-synuclein gene triplication (SNCA-Tri), control (Ctrl) and a-synuclein knockdown (SNCA-Tri KD) iPSC-derived NPC lines (Scale bar: 50 mm) shows normal cell morphology. B) Mitochondrial and nuclear morphology of NPCs visualized by fluorescence microscopy using Mitotracker Red CMX Ros (red) and Hoechst 33342 (blue) (Scale bar: 10 mm). C) Stem cell marker expression. Immuno-cytochemistry on fixed NPCs detecting cytoplasmic Nestin expression pattern with secondary Alexa 588 conjugated antibody (orange) by fluorescence microscopy (Scale bar: 100 mm). Insert: Immuno-cytochemistry for the nuclear stem cell marker SOX1, detected by
    Figure Legend Snippet: Figure 1. NPC characterization. A) Phase contrast microscopy of a-synuclein gene triplication (SNCA-Tri), control (Ctrl) and a-synuclein knockdown (SNCA-Tri KD) iPSC-derived NPC lines (Scale bar: 50 mm) shows normal cell morphology. B) Mitochondrial and nuclear morphology of NPCs visualized by fluorescence microscopy using Mitotracker Red CMX Ros (red) and Hoechst 33342 (blue) (Scale bar: 10 mm). C) Stem cell marker expression. Immuno-cytochemistry on fixed NPCs detecting cytoplasmic Nestin expression pattern with secondary Alexa 588 conjugated antibody (orange) by fluorescence microscopy (Scale bar: 100 mm). Insert: Immuno-cytochemistry for the nuclear stem cell marker SOX1, detected by

    Techniques Used: Microscopy, Control, Knockdown, Derivative Assay, Fluorescence, Marker, Expressing, Immunocytochemistry

    Figure 2. NPC viability. A) Cell cycle analysis by propidium-iodine (PI) staining and flow cytometry analysis of Ctrl and SNCA-Tri NPCs with staining grouped by cell cycle phase (G0/1, S and G2/M), showing a reduced percentage of SNCA-Tri NPCs in the S phase (n = 3, mean 6 SD, *p = 0.047). B) Survival under nutritional and toxicant stress. NPCs propagated in medium without glucose (NG) untreated or treated with 20 mM rotenone (R) or 20 mM paraquat (PQ). Survival curves (every 12 hours) for the Ctrl, SNCA-Tri and SNCA-Tri KD cell lines after analysis of adherent cell count (ImageJ). Percentage of surviving cells with time (hrs) (n = 3, mean 6 SEM). C) Cell viability assayed by plate reader based high throughput screen (HTS) of NPCs untreated (HG), treated with 20 mM rotenone (HG+R) or without glucose (NG) for 18 hrs. Live cells were stained with 1 mM of the RedOx indicator C12-Resazurin/Alamar Blue for 15 min before analysis. Graphed are endpoint fluorescence units (RFU) normalized to total cellular protein/well (ug protein) (n = 3, mean 6 SEM, *p#0.05). D) Cell viability assayed by flow cytometry evaluation of apoptosis and cell death in live NPCs treated as under A). Cells stained with C12-Resazurin for cell viability and with Sytox-Green. Graphed are percentages of metabolic active NPCs, determined by Resarufin (Ex./Em. 563/587 nm) fluorescence (viable), apoptotic cells (cell membrane asymmetry detected by an Annexin- V Alexa-660 nm conjugated antibody) (n = 3, mean 6 SD, Ctrl/SNCA-Tri: 5.3%/24.4%, *p = 0.027) or cell death (nuclear fragmentation, detected by Sytox-Green, Ex./Em. 488/530 nm) (n = 3, mean 6 SD, Ctrl/SNCA-Tri: 5.3%/24.4%, **p = 0.004). doi:10.1371/journal.pone.0112413.g002
    Figure Legend Snippet: Figure 2. NPC viability. A) Cell cycle analysis by propidium-iodine (PI) staining and flow cytometry analysis of Ctrl and SNCA-Tri NPCs with staining grouped by cell cycle phase (G0/1, S and G2/M), showing a reduced percentage of SNCA-Tri NPCs in the S phase (n = 3, mean 6 SD, *p = 0.047). B) Survival under nutritional and toxicant stress. NPCs propagated in medium without glucose (NG) untreated or treated with 20 mM rotenone (R) or 20 mM paraquat (PQ). Survival curves (every 12 hours) for the Ctrl, SNCA-Tri and SNCA-Tri KD cell lines after analysis of adherent cell count (ImageJ). Percentage of surviving cells with time (hrs) (n = 3, mean 6 SEM). C) Cell viability assayed by plate reader based high throughput screen (HTS) of NPCs untreated (HG), treated with 20 mM rotenone (HG+R) or without glucose (NG) for 18 hrs. Live cells were stained with 1 mM of the RedOx indicator C12-Resazurin/Alamar Blue for 15 min before analysis. Graphed are endpoint fluorescence units (RFU) normalized to total cellular protein/well (ug protein) (n = 3, mean 6 SEM, *p#0.05). D) Cell viability assayed by flow cytometry evaluation of apoptosis and cell death in live NPCs treated as under A). Cells stained with C12-Resazurin for cell viability and with Sytox-Green. Graphed are percentages of metabolic active NPCs, determined by Resarufin (Ex./Em. 563/587 nm) fluorescence (viable), apoptotic cells (cell membrane asymmetry detected by an Annexin- V Alexa-660 nm conjugated antibody) (n = 3, mean 6 SD, Ctrl/SNCA-Tri: 5.3%/24.4%, *p = 0.027) or cell death (nuclear fragmentation, detected by Sytox-Green, Ex./Em. 488/530 nm) (n = 3, mean 6 SD, Ctrl/SNCA-Tri: 5.3%/24.4%, **p = 0.004). doi:10.1371/journal.pone.0112413.g002

    Techniques Used: Cell Cycle Assay, Staining, Flow Cytometry, Cell Counting, High Throughput Screening Assay, Fluorescence, Membrane

    Figure 3. Mitochondrial membrane potential (MMP) and energy balance. A) Fluorescence microscopy of MMP in live NPCs from patient (SNCA-Tri) and control (Ctrl) loaded with 100 nM TMRM in normal growth medium (HG), medium plus 20 mM Rotenone (HG+R) or with 1 mM of the ionophore CCCP (HG+CCCP) as negative control (Scale bar: 10 mm). B) Plate reader based high throughput screen (HTS) of MMP in live NPCs loaded with 20 mM JC-10 for 45 min. Cells were also treated with medium w/o glucose (NG). Shown are log ratios of reduced (Ex./Em. 540 nm/590 nm) to oxidized JC-10 (Ex./Em. 488 nm/520 nm) normalized to Hoechst 33342 (Log Norm. JC-10 Ratio) after 60 min. (n = 8, mean 6 SEM, Ctrl/SNCA-
    Figure Legend Snippet: Figure 3. Mitochondrial membrane potential (MMP) and energy balance. A) Fluorescence microscopy of MMP in live NPCs from patient (SNCA-Tri) and control (Ctrl) loaded with 100 nM TMRM in normal growth medium (HG), medium plus 20 mM Rotenone (HG+R) or with 1 mM of the ionophore CCCP (HG+CCCP) as negative control (Scale bar: 10 mm). B) Plate reader based high throughput screen (HTS) of MMP in live NPCs loaded with 20 mM JC-10 for 45 min. Cells were also treated with medium w/o glucose (NG). Shown are log ratios of reduced (Ex./Em. 540 nm/590 nm) to oxidized JC-10 (Ex./Em. 488 nm/520 nm) normalized to Hoechst 33342 (Log Norm. JC-10 Ratio) after 60 min. (n = 8, mean 6 SEM, Ctrl/SNCA-

    Techniques Used: Membrane, Fluorescence, Microscopy, Control, Negative Control, High Throughput Screening Assay

    Figure 4. Protein biosynthesis and proteasome function. A) Mitochondrial protein biosynthesis and protein import. Fluorescent protein expression patterns in confluent adherent NPC cultures (PC: Phase Contrast) transduced with two baculoviral vectors expressing fluorescent proteins targeted to either the peroxisomal (Perox.; Green) or the mitochondrial (Mito.; Red) compartment. Shown are fluorescent protein expression patterns in live confluent Ctrl and SNCA-Tri cell lines grown under normal growth conditions (HG) and evaluated 20 hrs post transduction (Scale bar: 200 mm, 5 mm). B) Time resolved peroxisomal and mitochondrial protein biosynthesis. Fluorescent protein expression patterns as under A), but imaged at 8 and 18 hrs post viral transduction. C) Proteasome activity measured by fluorescence microscopy of adherent NPCs cultured with 20 mM rotenone alone or with 10 mM of the proteasome inhibitor MG132. Depicted are fixed cells stained with 5 mM of the aggresome/ proteasome specific dye Bodipy TMR-AHX3L3VS (red). Hoechst 33342 was used as nuclear counter stain (blue) (Scale bar: 20 mm). D) Proteasome activity measured by flow cytometry evaluation of cells treated and stained as under B). Charted are the aggresome propensity factors (APF) of NPCs calculated from the mean RFU (MRFU) of Bodipy-TMR fluorescence (APF = 1006[MRFU MG132 treated2MRFU untreated]/MRFU MG132 treated (n = 3, mean 6 SD, APF Ctrl/SNCA-Tri: 51/120, *p = 0.041). doi:10.1371/journal.pone.0112413.g004
    Figure Legend Snippet: Figure 4. Protein biosynthesis and proteasome function. A) Mitochondrial protein biosynthesis and protein import. Fluorescent protein expression patterns in confluent adherent NPC cultures (PC: Phase Contrast) transduced with two baculoviral vectors expressing fluorescent proteins targeted to either the peroxisomal (Perox.; Green) or the mitochondrial (Mito.; Red) compartment. Shown are fluorescent protein expression patterns in live confluent Ctrl and SNCA-Tri cell lines grown under normal growth conditions (HG) and evaluated 20 hrs post transduction (Scale bar: 200 mm, 5 mm). B) Time resolved peroxisomal and mitochondrial protein biosynthesis. Fluorescent protein expression patterns as under A), but imaged at 8 and 18 hrs post viral transduction. C) Proteasome activity measured by fluorescence microscopy of adherent NPCs cultured with 20 mM rotenone alone or with 10 mM of the proteasome inhibitor MG132. Depicted are fixed cells stained with 5 mM of the aggresome/ proteasome specific dye Bodipy TMR-AHX3L3VS (red). Hoechst 33342 was used as nuclear counter stain (blue) (Scale bar: 20 mm). D) Proteasome activity measured by flow cytometry evaluation of cells treated and stained as under B). Charted are the aggresome propensity factors (APF) of NPCs calculated from the mean RFU (MRFU) of Bodipy-TMR fluorescence (APF = 1006[MRFU MG132 treated2MRFU untreated]/MRFU MG132 treated (n = 3, mean 6 SD, APF Ctrl/SNCA-Tri: 51/120, *p = 0.041). doi:10.1371/journal.pone.0112413.g004

    Techniques Used: Expressing, Transduction, Activity Assay, Fluorescence, Microscopy, Cell Culture, Staining, Flow Cytometry

    Figure 5. Reactive oxygen species (ROS) production. A) Fluorescence microscopy of live adherent NPCs untreated (HG) or treated with 100 mM TBHP (HG+TBHP), loaded with CM-H2DCFDA and imaged under controlled exposure conditions (10 sec fluorescent light exposure before image acquisition). Hoechst 33342 was used as counter stain (Scale bar: 20 mm). B) Plate reader based HTS of ROS levels in adherent NPC in 96- well plates and treated as under A). Relative CM-H2DCFDA fluorescence intensities (RFU) were normalized to Hoechst 33342 (H33342) (n = 12, mean 6 SEM, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 0.5/1/0.75, HG+R: 0.7/1.3/0.6, NG: 0.4/1.1/0.7, *p#0.046, **p#0.009, ***#0.001). C) ROS production rates by HTS plate reader analysis of CM-H2DCFDA fluorescence development over time (D RFU CM-H2DCFDA/sec + H33342) in cells exposed to TBHP as under A), measured with normal medium (HG) with or without rotenone (R) and in medium without glucose (NG) (n = 12, mean 6 SEM, Ctrl/SNCA-Tri/ SNCA-Tri KD: HG: 22/75/68, HG+R: 177/367/178, NG: 80/353/184, *p#0.010, **p#0.007, ***p#0.001). D) Mitochondrial superoxide production rates assayed by HTS plate reader analysis of the mitochondrial targeted fluorescent superoxide indicator MitoSOX. Depicted are changes in relative fluorescence units normalized to H33342) (D RFU MitoSOX/min + H33342) (n = 4, mean 6 SD, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 0.28/1.2/0.3, HG+R: 2.1/ 5.5/3.7, NG: 2.3/5.2/0.8,*p#0.038, **p#0.007). doi:10.1371/journal.pone.0112413.g005
    Figure Legend Snippet: Figure 5. Reactive oxygen species (ROS) production. A) Fluorescence microscopy of live adherent NPCs untreated (HG) or treated with 100 mM TBHP (HG+TBHP), loaded with CM-H2DCFDA and imaged under controlled exposure conditions (10 sec fluorescent light exposure before image acquisition). Hoechst 33342 was used as counter stain (Scale bar: 20 mm). B) Plate reader based HTS of ROS levels in adherent NPC in 96- well plates and treated as under A). Relative CM-H2DCFDA fluorescence intensities (RFU) were normalized to Hoechst 33342 (H33342) (n = 12, mean 6 SEM, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 0.5/1/0.75, HG+R: 0.7/1.3/0.6, NG: 0.4/1.1/0.7, *p#0.046, **p#0.009, ***#0.001). C) ROS production rates by HTS plate reader analysis of CM-H2DCFDA fluorescence development over time (D RFU CM-H2DCFDA/sec + H33342) in cells exposed to TBHP as under A), measured with normal medium (HG) with or without rotenone (R) and in medium without glucose (NG) (n = 12, mean 6 SEM, Ctrl/SNCA-Tri/ SNCA-Tri KD: HG: 22/75/68, HG+R: 177/367/178, NG: 80/353/184, *p#0.010, **p#0.007, ***p#0.001). D) Mitochondrial superoxide production rates assayed by HTS plate reader analysis of the mitochondrial targeted fluorescent superoxide indicator MitoSOX. Depicted are changes in relative fluorescence units normalized to H33342) (D RFU MitoSOX/min + H33342) (n = 4, mean 6 SD, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 0.28/1.2/0.3, HG+R: 2.1/ 5.5/3.7, NG: 2.3/5.2/0.8,*p#0.038, **p#0.007). doi:10.1371/journal.pone.0112413.g005

    Techniques Used: Fluorescence, Microscopy, Staining

    Figure 6. Mitochondrial integrity, MPT opening, and apoptosis. A) Mitochondrial calcein loading by fluorescent plate reader HTS of in NPCs grown in 96 well micro plates. Relative fluorescent signal intensities (RFU) for calcein acquired after 30 min loading with Calcein AM and CoCl2 were normalized to mitochondrial content (Mitotracker) and to cell number by Hoechst 33342 (H33342). 1 mM ionomycin was added directly before HTS analysis as negative control (Iono) (n = 8, mean 6 SD, Ctrl/SNCA-Tri: 3.4/4.9, *p = 0.039). B) MPT-induced mitochondrial calcein loss in Ctrl and SNCA-Tri NPCs after mitochondrial calcein–AM loading. Representative fluorescence microscopy images of Ctrl and SNCA-Tri NPCs loaded with calcein (green), Mitotracker (red) and CoCl2 were assayed 1 hr. after treatment with 4 mM staurosporine under NG conditions. MPT opening results in entry of CoCl2 into mitochondria and loss of calcein signal (nuclear counter stain: Hoechst 33342; scale bar: 100 mm). Inserts: Higher magnification images obtained by conventional fluorescence microscopy (Scale bar: 10 mm). C) HCI automated fluorescence microscopy analysis of MPT in NPCs treated with 4 mM staurosporine as under B). Images (see B) were analyzed using MetaXpress image processing software. Depicted are data of cellular calcein signal intensities normalized to mitochondrial content (Norm. RFU Calcein/RFU Mitotracker) from two replicate wells with four image sites/well per treatment condition (n = 16, mean 6 SD, Ctrl/SNCA-Tri, HG: 834/457, HG+R: 1425/1011, NG: 864/574, HG+Iono: 187/190, *p#0.01). D) Kinetic evaluation of MPT opening and loss of mitochondrial calcein signal after induction of MTP using fluorescence plate reader based HTS
    Figure Legend Snippet: Figure 6. Mitochondrial integrity, MPT opening, and apoptosis. A) Mitochondrial calcein loading by fluorescent plate reader HTS of in NPCs grown in 96 well micro plates. Relative fluorescent signal intensities (RFU) for calcein acquired after 30 min loading with Calcein AM and CoCl2 were normalized to mitochondrial content (Mitotracker) and to cell number by Hoechst 33342 (H33342). 1 mM ionomycin was added directly before HTS analysis as negative control (Iono) (n = 8, mean 6 SD, Ctrl/SNCA-Tri: 3.4/4.9, *p = 0.039). B) MPT-induced mitochondrial calcein loss in Ctrl and SNCA-Tri NPCs after mitochondrial calcein–AM loading. Representative fluorescence microscopy images of Ctrl and SNCA-Tri NPCs loaded with calcein (green), Mitotracker (red) and CoCl2 were assayed 1 hr. after treatment with 4 mM staurosporine under NG conditions. MPT opening results in entry of CoCl2 into mitochondria and loss of calcein signal (nuclear counter stain: Hoechst 33342; scale bar: 100 mm). Inserts: Higher magnification images obtained by conventional fluorescence microscopy (Scale bar: 10 mm). C) HCI automated fluorescence microscopy analysis of MPT in NPCs treated with 4 mM staurosporine as under B). Images (see B) were analyzed using MetaXpress image processing software. Depicted are data of cellular calcein signal intensities normalized to mitochondrial content (Norm. RFU Calcein/RFU Mitotracker) from two replicate wells with four image sites/well per treatment condition (n = 16, mean 6 SD, Ctrl/SNCA-Tri, HG: 834/457, HG+R: 1425/1011, NG: 864/574, HG+Iono: 187/190, *p#0.01). D) Kinetic evaluation of MPT opening and loss of mitochondrial calcein signal after induction of MTP using fluorescence plate reader based HTS

    Techniques Used: Negative Control, Fluorescence, Microscopy, Staining, Software

    Figure 7. Apoptosis sensitivity and caspase activation. A) Caspase 3 activity in cell lysates from adherent NPCs either left untreated or treated with 20 mM rotenone (R) for 18 hrs and then exposed to 1 uM staurosporine for 120 min before analysis. HTS analysis for caspase 3 activity from cell lysates was by activation of the fluorescent caspase substrate 7-amino-4-methylcoumarin (AMC) (Ex./Em. 340/440 nm) (n = 9, mean 6 SEM, Ctrl/SNCA-Tri/SNCA-Tri KD, HG: 33/69/42, HG+R: 42/129/87, NG: 55/138/85, *p#0.050, **p#0.0035; from three independent experiments). B) Kinetics of caspase 3/7 activity in permeabilized NPCs pretreated as described under B) and assayed 15 min after staurosporine treatment. Changes in caspase 3 activity are depicted as DmM AMC fluorescence/min + mg cellular protein (detected by Bradford protein assay) (n = 9, mean 6 SEM). doi:10.1371/journal.pone.0112413.g007
    Figure Legend Snippet: Figure 7. Apoptosis sensitivity and caspase activation. A) Caspase 3 activity in cell lysates from adherent NPCs either left untreated or treated with 20 mM rotenone (R) for 18 hrs and then exposed to 1 uM staurosporine for 120 min before analysis. HTS analysis for caspase 3 activity from cell lysates was by activation of the fluorescent caspase substrate 7-amino-4-methylcoumarin (AMC) (Ex./Em. 340/440 nm) (n = 9, mean 6 SEM, Ctrl/SNCA-Tri/SNCA-Tri KD, HG: 33/69/42, HG+R: 42/129/87, NG: 55/138/85, *p#0.050, **p#0.0035; from three independent experiments). B) Kinetics of caspase 3/7 activity in permeabilized NPCs pretreated as described under B) and assayed 15 min after staurosporine treatment. Changes in caspase 3 activity are depicted as DmM AMC fluorescence/min + mg cellular protein (detected by Bradford protein assay) (n = 9, mean 6 SEM). doi:10.1371/journal.pone.0112413.g007

    Techniques Used: Activation Assay, Activity Assay, Fluorescence, Bradford Protein Assay



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    high throughput screening hts conventional fluorescence microscopy  (Nikon)
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    Nikon high throughput screening hts conventional fluorescence microscopy
    Figure 1. NPC characterization. A) Phase contrast <t>microscopy</t> of a-synuclein gene triplication (SNCA-Tri), control (Ctrl) and a-synuclein knockdown (SNCA-Tri KD) iPSC-derived NPC lines (Scale bar: 50 mm) shows normal cell morphology. B) Mitochondrial and nuclear morphology of NPCs visualized by <t>fluorescence</t> microscopy using Mitotracker Red CMX Ros (red) and Hoechst 33342 (blue) (Scale bar: 10 mm). C) Stem cell marker expression. Immuno-cytochemistry on fixed NPCs detecting cytoplasmic Nestin expression pattern with secondary Alexa 588 conjugated antibody (orange) by fluorescence microscopy (Scale bar: 100 mm). Insert: Immuno-cytochemistry for the nuclear stem cell marker SOX1, detected by
    High Throughput Screening Hts Conventional Fluorescence Microscopy, supplied by Nikon, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/high+throughput+screening+hts+conventional+fluorescence+microscopy/pm25390032-103-5-12?v=Nikon
    Average 99 stars, based on 1 article reviews
    high throughput screening hts conventional fluorescence microscopy - by Bioz Stars, 2026-07
    99/100 stars
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    Figure 1. NPC characterization. A) Phase contrast microscopy of a-synuclein gene triplication (SNCA-Tri), control (Ctrl) and a-synuclein knockdown (SNCA-Tri KD) iPSC-derived NPC lines (Scale bar: 50 mm) shows normal cell morphology. B) Mitochondrial and nuclear morphology of NPCs visualized by fluorescence microscopy using Mitotracker Red CMX Ros (red) and Hoechst 33342 (blue) (Scale bar: 10 mm). C) Stem cell marker expression. Immuno-cytochemistry on fixed NPCs detecting cytoplasmic Nestin expression pattern with secondary Alexa 588 conjugated antibody (orange) by fluorescence microscopy (Scale bar: 100 mm). Insert: Immuno-cytochemistry for the nuclear stem cell marker SOX1, detected by

    Journal: PloS one

    Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

    doi: 10.1371/journal.pone.0112413

    Figure Lengend Snippet: Figure 1. NPC characterization. A) Phase contrast microscopy of a-synuclein gene triplication (SNCA-Tri), control (Ctrl) and a-synuclein knockdown (SNCA-Tri KD) iPSC-derived NPC lines (Scale bar: 50 mm) shows normal cell morphology. B) Mitochondrial and nuclear morphology of NPCs visualized by fluorescence microscopy using Mitotracker Red CMX Ros (red) and Hoechst 33342 (blue) (Scale bar: 10 mm). C) Stem cell marker expression. Immuno-cytochemistry on fixed NPCs detecting cytoplasmic Nestin expression pattern with secondary Alexa 588 conjugated antibody (orange) by fluorescence microscopy (Scale bar: 100 mm). Insert: Immuno-cytochemistry for the nuclear stem cell marker SOX1, detected by

    Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

    Techniques: Microscopy, Control, Knockdown, Derivative Assay, Fluorescence, Marker, Expressing, Immunocytochemistry

    Figure 2. NPC viability. A) Cell cycle analysis by propidium-iodine (PI) staining and flow cytometry analysis of Ctrl and SNCA-Tri NPCs with staining grouped by cell cycle phase (G0/1, S and G2/M), showing a reduced percentage of SNCA-Tri NPCs in the S phase (n = 3, mean 6 SD, *p = 0.047). B) Survival under nutritional and toxicant stress. NPCs propagated in medium without glucose (NG) untreated or treated with 20 mM rotenone (R) or 20 mM paraquat (PQ). Survival curves (every 12 hours) for the Ctrl, SNCA-Tri and SNCA-Tri KD cell lines after analysis of adherent cell count (ImageJ). Percentage of surviving cells with time (hrs) (n = 3, mean 6 SEM). C) Cell viability assayed by plate reader based high throughput screen (HTS) of NPCs untreated (HG), treated with 20 mM rotenone (HG+R) or without glucose (NG) for 18 hrs. Live cells were stained with 1 mM of the RedOx indicator C12-Resazurin/Alamar Blue for 15 min before analysis. Graphed are endpoint fluorescence units (RFU) normalized to total cellular protein/well (ug protein) (n = 3, mean 6 SEM, *p#0.05). D) Cell viability assayed by flow cytometry evaluation of apoptosis and cell death in live NPCs treated as under A). Cells stained with C12-Resazurin for cell viability and with Sytox-Green. Graphed are percentages of metabolic active NPCs, determined by Resarufin (Ex./Em. 563/587 nm) fluorescence (viable), apoptotic cells (cell membrane asymmetry detected by an Annexin- V Alexa-660 nm conjugated antibody) (n = 3, mean 6 SD, Ctrl/SNCA-Tri: 5.3%/24.4%, *p = 0.027) or cell death (nuclear fragmentation, detected by Sytox-Green, Ex./Em. 488/530 nm) (n = 3, mean 6 SD, Ctrl/SNCA-Tri: 5.3%/24.4%, **p = 0.004). doi:10.1371/journal.pone.0112413.g002

    Journal: PloS one

    Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

    doi: 10.1371/journal.pone.0112413

    Figure Lengend Snippet: Figure 2. NPC viability. A) Cell cycle analysis by propidium-iodine (PI) staining and flow cytometry analysis of Ctrl and SNCA-Tri NPCs with staining grouped by cell cycle phase (G0/1, S and G2/M), showing a reduced percentage of SNCA-Tri NPCs in the S phase (n = 3, mean 6 SD, *p = 0.047). B) Survival under nutritional and toxicant stress. NPCs propagated in medium without glucose (NG) untreated or treated with 20 mM rotenone (R) or 20 mM paraquat (PQ). Survival curves (every 12 hours) for the Ctrl, SNCA-Tri and SNCA-Tri KD cell lines after analysis of adherent cell count (ImageJ). Percentage of surviving cells with time (hrs) (n = 3, mean 6 SEM). C) Cell viability assayed by plate reader based high throughput screen (HTS) of NPCs untreated (HG), treated with 20 mM rotenone (HG+R) or without glucose (NG) for 18 hrs. Live cells were stained with 1 mM of the RedOx indicator C12-Resazurin/Alamar Blue for 15 min before analysis. Graphed are endpoint fluorescence units (RFU) normalized to total cellular protein/well (ug protein) (n = 3, mean 6 SEM, *p#0.05). D) Cell viability assayed by flow cytometry evaluation of apoptosis and cell death in live NPCs treated as under A). Cells stained with C12-Resazurin for cell viability and with Sytox-Green. Graphed are percentages of metabolic active NPCs, determined by Resarufin (Ex./Em. 563/587 nm) fluorescence (viable), apoptotic cells (cell membrane asymmetry detected by an Annexin- V Alexa-660 nm conjugated antibody) (n = 3, mean 6 SD, Ctrl/SNCA-Tri: 5.3%/24.4%, *p = 0.027) or cell death (nuclear fragmentation, detected by Sytox-Green, Ex./Em. 488/530 nm) (n = 3, mean 6 SD, Ctrl/SNCA-Tri: 5.3%/24.4%, **p = 0.004). doi:10.1371/journal.pone.0112413.g002

    Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

    Techniques: Cell Cycle Assay, Staining, Flow Cytometry, Cell Counting, High Throughput Screening Assay, Fluorescence, Membrane

    Figure 3. Mitochondrial membrane potential (MMP) and energy balance. A) Fluorescence microscopy of MMP in live NPCs from patient (SNCA-Tri) and control (Ctrl) loaded with 100 nM TMRM in normal growth medium (HG), medium plus 20 mM Rotenone (HG+R) or with 1 mM of the ionophore CCCP (HG+CCCP) as negative control (Scale bar: 10 mm). B) Plate reader based high throughput screen (HTS) of MMP in live NPCs loaded with 20 mM JC-10 for 45 min. Cells were also treated with medium w/o glucose (NG). Shown are log ratios of reduced (Ex./Em. 540 nm/590 nm) to oxidized JC-10 (Ex./Em. 488 nm/520 nm) normalized to Hoechst 33342 (Log Norm. JC-10 Ratio) after 60 min. (n = 8, mean 6 SEM, Ctrl/SNCA-

    Journal: PloS one

    Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

    doi: 10.1371/journal.pone.0112413

    Figure Lengend Snippet: Figure 3. Mitochondrial membrane potential (MMP) and energy balance. A) Fluorescence microscopy of MMP in live NPCs from patient (SNCA-Tri) and control (Ctrl) loaded with 100 nM TMRM in normal growth medium (HG), medium plus 20 mM Rotenone (HG+R) or with 1 mM of the ionophore CCCP (HG+CCCP) as negative control (Scale bar: 10 mm). B) Plate reader based high throughput screen (HTS) of MMP in live NPCs loaded with 20 mM JC-10 for 45 min. Cells were also treated with medium w/o glucose (NG). Shown are log ratios of reduced (Ex./Em. 540 nm/590 nm) to oxidized JC-10 (Ex./Em. 488 nm/520 nm) normalized to Hoechst 33342 (Log Norm. JC-10 Ratio) after 60 min. (n = 8, mean 6 SEM, Ctrl/SNCA-

    Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

    Techniques: Membrane, Fluorescence, Microscopy, Control, Negative Control, High Throughput Screening Assay

    Figure 4. Protein biosynthesis and proteasome function. A) Mitochondrial protein biosynthesis and protein import. Fluorescent protein expression patterns in confluent adherent NPC cultures (PC: Phase Contrast) transduced with two baculoviral vectors expressing fluorescent proteins targeted to either the peroxisomal (Perox.; Green) or the mitochondrial (Mito.; Red) compartment. Shown are fluorescent protein expression patterns in live confluent Ctrl and SNCA-Tri cell lines grown under normal growth conditions (HG) and evaluated 20 hrs post transduction (Scale bar: 200 mm, 5 mm). B) Time resolved peroxisomal and mitochondrial protein biosynthesis. Fluorescent protein expression patterns as under A), but imaged at 8 and 18 hrs post viral transduction. C) Proteasome activity measured by fluorescence microscopy of adherent NPCs cultured with 20 mM rotenone alone or with 10 mM of the proteasome inhibitor MG132. Depicted are fixed cells stained with 5 mM of the aggresome/ proteasome specific dye Bodipy TMR-AHX3L3VS (red). Hoechst 33342 was used as nuclear counter stain (blue) (Scale bar: 20 mm). D) Proteasome activity measured by flow cytometry evaluation of cells treated and stained as under B). Charted are the aggresome propensity factors (APF) of NPCs calculated from the mean RFU (MRFU) of Bodipy-TMR fluorescence (APF = 1006[MRFU MG132 treated2MRFU untreated]/MRFU MG132 treated (n = 3, mean 6 SD, APF Ctrl/SNCA-Tri: 51/120, *p = 0.041). doi:10.1371/journal.pone.0112413.g004

    Journal: PloS one

    Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

    doi: 10.1371/journal.pone.0112413

    Figure Lengend Snippet: Figure 4. Protein biosynthesis and proteasome function. A) Mitochondrial protein biosynthesis and protein import. Fluorescent protein expression patterns in confluent adherent NPC cultures (PC: Phase Contrast) transduced with two baculoviral vectors expressing fluorescent proteins targeted to either the peroxisomal (Perox.; Green) or the mitochondrial (Mito.; Red) compartment. Shown are fluorescent protein expression patterns in live confluent Ctrl and SNCA-Tri cell lines grown under normal growth conditions (HG) and evaluated 20 hrs post transduction (Scale bar: 200 mm, 5 mm). B) Time resolved peroxisomal and mitochondrial protein biosynthesis. Fluorescent protein expression patterns as under A), but imaged at 8 and 18 hrs post viral transduction. C) Proteasome activity measured by fluorescence microscopy of adherent NPCs cultured with 20 mM rotenone alone or with 10 mM of the proteasome inhibitor MG132. Depicted are fixed cells stained with 5 mM of the aggresome/ proteasome specific dye Bodipy TMR-AHX3L3VS (red). Hoechst 33342 was used as nuclear counter stain (blue) (Scale bar: 20 mm). D) Proteasome activity measured by flow cytometry evaluation of cells treated and stained as under B). Charted are the aggresome propensity factors (APF) of NPCs calculated from the mean RFU (MRFU) of Bodipy-TMR fluorescence (APF = 1006[MRFU MG132 treated2MRFU untreated]/MRFU MG132 treated (n = 3, mean 6 SD, APF Ctrl/SNCA-Tri: 51/120, *p = 0.041). doi:10.1371/journal.pone.0112413.g004

    Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

    Techniques: Expressing, Transduction, Activity Assay, Fluorescence, Microscopy, Cell Culture, Staining, Flow Cytometry

    Figure 5. Reactive oxygen species (ROS) production. A) Fluorescence microscopy of live adherent NPCs untreated (HG) or treated with 100 mM TBHP (HG+TBHP), loaded with CM-H2DCFDA and imaged under controlled exposure conditions (10 sec fluorescent light exposure before image acquisition). Hoechst 33342 was used as counter stain (Scale bar: 20 mm). B) Plate reader based HTS of ROS levels in adherent NPC in 96- well plates and treated as under A). Relative CM-H2DCFDA fluorescence intensities (RFU) were normalized to Hoechst 33342 (H33342) (n = 12, mean 6 SEM, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 0.5/1/0.75, HG+R: 0.7/1.3/0.6, NG: 0.4/1.1/0.7, *p#0.046, **p#0.009, ***#0.001). C) ROS production rates by HTS plate reader analysis of CM-H2DCFDA fluorescence development over time (D RFU CM-H2DCFDA/sec + H33342) in cells exposed to TBHP as under A), measured with normal medium (HG) with or without rotenone (R) and in medium without glucose (NG) (n = 12, mean 6 SEM, Ctrl/SNCA-Tri/ SNCA-Tri KD: HG: 22/75/68, HG+R: 177/367/178, NG: 80/353/184, *p#0.010, **p#0.007, ***p#0.001). D) Mitochondrial superoxide production rates assayed by HTS plate reader analysis of the mitochondrial targeted fluorescent superoxide indicator MitoSOX. Depicted are changes in relative fluorescence units normalized to H33342) (D RFU MitoSOX/min + H33342) (n = 4, mean 6 SD, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 0.28/1.2/0.3, HG+R: 2.1/ 5.5/3.7, NG: 2.3/5.2/0.8,*p#0.038, **p#0.007). doi:10.1371/journal.pone.0112413.g005

    Journal: PloS one

    Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

    doi: 10.1371/journal.pone.0112413

    Figure Lengend Snippet: Figure 5. Reactive oxygen species (ROS) production. A) Fluorescence microscopy of live adherent NPCs untreated (HG) or treated with 100 mM TBHP (HG+TBHP), loaded with CM-H2DCFDA and imaged under controlled exposure conditions (10 sec fluorescent light exposure before image acquisition). Hoechst 33342 was used as counter stain (Scale bar: 20 mm). B) Plate reader based HTS of ROS levels in adherent NPC in 96- well plates and treated as under A). Relative CM-H2DCFDA fluorescence intensities (RFU) were normalized to Hoechst 33342 (H33342) (n = 12, mean 6 SEM, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 0.5/1/0.75, HG+R: 0.7/1.3/0.6, NG: 0.4/1.1/0.7, *p#0.046, **p#0.009, ***#0.001). C) ROS production rates by HTS plate reader analysis of CM-H2DCFDA fluorescence development over time (D RFU CM-H2DCFDA/sec + H33342) in cells exposed to TBHP as under A), measured with normal medium (HG) with or without rotenone (R) and in medium without glucose (NG) (n = 12, mean 6 SEM, Ctrl/SNCA-Tri/ SNCA-Tri KD: HG: 22/75/68, HG+R: 177/367/178, NG: 80/353/184, *p#0.010, **p#0.007, ***p#0.001). D) Mitochondrial superoxide production rates assayed by HTS plate reader analysis of the mitochondrial targeted fluorescent superoxide indicator MitoSOX. Depicted are changes in relative fluorescence units normalized to H33342) (D RFU MitoSOX/min + H33342) (n = 4, mean 6 SD, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 0.28/1.2/0.3, HG+R: 2.1/ 5.5/3.7, NG: 2.3/5.2/0.8,*p#0.038, **p#0.007). doi:10.1371/journal.pone.0112413.g005

    Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

    Techniques: Fluorescence, Microscopy, Staining

    Figure 6. Mitochondrial integrity, MPT opening, and apoptosis. A) Mitochondrial calcein loading by fluorescent plate reader HTS of in NPCs grown in 96 well micro plates. Relative fluorescent signal intensities (RFU) for calcein acquired after 30 min loading with Calcein AM and CoCl2 were normalized to mitochondrial content (Mitotracker) and to cell number by Hoechst 33342 (H33342). 1 mM ionomycin was added directly before HTS analysis as negative control (Iono) (n = 8, mean 6 SD, Ctrl/SNCA-Tri: 3.4/4.9, *p = 0.039). B) MPT-induced mitochondrial calcein loss in Ctrl and SNCA-Tri NPCs after mitochondrial calcein–AM loading. Representative fluorescence microscopy images of Ctrl and SNCA-Tri NPCs loaded with calcein (green), Mitotracker (red) and CoCl2 were assayed 1 hr. after treatment with 4 mM staurosporine under NG conditions. MPT opening results in entry of CoCl2 into mitochondria and loss of calcein signal (nuclear counter stain: Hoechst 33342; scale bar: 100 mm). Inserts: Higher magnification images obtained by conventional fluorescence microscopy (Scale bar: 10 mm). C) HCI automated fluorescence microscopy analysis of MPT in NPCs treated with 4 mM staurosporine as under B). Images (see B) were analyzed using MetaXpress image processing software. Depicted are data of cellular calcein signal intensities normalized to mitochondrial content (Norm. RFU Calcein/RFU Mitotracker) from two replicate wells with four image sites/well per treatment condition (n = 16, mean 6 SD, Ctrl/SNCA-Tri, HG: 834/457, HG+R: 1425/1011, NG: 864/574, HG+Iono: 187/190, *p#0.01). D) Kinetic evaluation of MPT opening and loss of mitochondrial calcein signal after induction of MTP using fluorescence plate reader based HTS

    Journal: PloS one

    Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

    doi: 10.1371/journal.pone.0112413

    Figure Lengend Snippet: Figure 6. Mitochondrial integrity, MPT opening, and apoptosis. A) Mitochondrial calcein loading by fluorescent plate reader HTS of in NPCs grown in 96 well micro plates. Relative fluorescent signal intensities (RFU) for calcein acquired after 30 min loading with Calcein AM and CoCl2 were normalized to mitochondrial content (Mitotracker) and to cell number by Hoechst 33342 (H33342). 1 mM ionomycin was added directly before HTS analysis as negative control (Iono) (n = 8, mean 6 SD, Ctrl/SNCA-Tri: 3.4/4.9, *p = 0.039). B) MPT-induced mitochondrial calcein loss in Ctrl and SNCA-Tri NPCs after mitochondrial calcein–AM loading. Representative fluorescence microscopy images of Ctrl and SNCA-Tri NPCs loaded with calcein (green), Mitotracker (red) and CoCl2 were assayed 1 hr. after treatment with 4 mM staurosporine under NG conditions. MPT opening results in entry of CoCl2 into mitochondria and loss of calcein signal (nuclear counter stain: Hoechst 33342; scale bar: 100 mm). Inserts: Higher magnification images obtained by conventional fluorescence microscopy (Scale bar: 10 mm). C) HCI automated fluorescence microscopy analysis of MPT in NPCs treated with 4 mM staurosporine as under B). Images (see B) were analyzed using MetaXpress image processing software. Depicted are data of cellular calcein signal intensities normalized to mitochondrial content (Norm. RFU Calcein/RFU Mitotracker) from two replicate wells with four image sites/well per treatment condition (n = 16, mean 6 SD, Ctrl/SNCA-Tri, HG: 834/457, HG+R: 1425/1011, NG: 864/574, HG+Iono: 187/190, *p#0.01). D) Kinetic evaluation of MPT opening and loss of mitochondrial calcein signal after induction of MTP using fluorescence plate reader based HTS

    Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

    Techniques: Negative Control, Fluorescence, Microscopy, Staining, Software

    Figure 7. Apoptosis sensitivity and caspase activation. A) Caspase 3 activity in cell lysates from adherent NPCs either left untreated or treated with 20 mM rotenone (R) for 18 hrs and then exposed to 1 uM staurosporine for 120 min before analysis. HTS analysis for caspase 3 activity from cell lysates was by activation of the fluorescent caspase substrate 7-amino-4-methylcoumarin (AMC) (Ex./Em. 340/440 nm) (n = 9, mean 6 SEM, Ctrl/SNCA-Tri/SNCA-Tri KD, HG: 33/69/42, HG+R: 42/129/87, NG: 55/138/85, *p#0.050, **p#0.0035; from three independent experiments). B) Kinetics of caspase 3/7 activity in permeabilized NPCs pretreated as described under B) and assayed 15 min after staurosporine treatment. Changes in caspase 3 activity are depicted as DmM AMC fluorescence/min + mg cellular protein (detected by Bradford protein assay) (n = 9, mean 6 SEM). doi:10.1371/journal.pone.0112413.g007

    Journal: PloS one

    Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

    doi: 10.1371/journal.pone.0112413

    Figure Lengend Snippet: Figure 7. Apoptosis sensitivity and caspase activation. A) Caspase 3 activity in cell lysates from adherent NPCs either left untreated or treated with 20 mM rotenone (R) for 18 hrs and then exposed to 1 uM staurosporine for 120 min before analysis. HTS analysis for caspase 3 activity from cell lysates was by activation of the fluorescent caspase substrate 7-amino-4-methylcoumarin (AMC) (Ex./Em. 340/440 nm) (n = 9, mean 6 SEM, Ctrl/SNCA-Tri/SNCA-Tri KD, HG: 33/69/42, HG+R: 42/129/87, NG: 55/138/85, *p#0.050, **p#0.0035; from three independent experiments). B) Kinetics of caspase 3/7 activity in permeabilized NPCs pretreated as described under B) and assayed 15 min after staurosporine treatment. Changes in caspase 3 activity are depicted as DmM AMC fluorescence/min + mg cellular protein (detected by Bradford protein assay) (n = 9, mean 6 SEM). doi:10.1371/journal.pone.0112413.g007

    Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

    Techniques: Activation Assay, Activity Assay, Fluorescence, Bradford Protein Assay