96-well plates Search Results


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  • 93
    Thermo Fisher centrisept 96 well plates
    Centrisept 96 Well Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore 96well plates
    Size-sorted hyperploid subpopulation ( > 6N) showed differential regrowth potential compared to the non-hyperploid (3N-6N) fraction. Serial dilution growth assay where adherent OVCAR3 cells were treated with vehicle, 12.5 µM or 25 µM carboplatin on day 0 for 24 hrs, followed by removal of the drug and further incubation of the cells until day 9. Cells were then detached, flow sorted by forward scatter (FSC) and propidium-iodide negativity into a small or large subpopulation, and then plated into a <t>96-well</t> plate at the indicated density for regrowth until final assessment by imaging on day 30. ( A ) The centroid location of each identified, propidium-iodide excluded, Hoechst stained cell within the wells of a 96-well plate was plotted on day 30, for the post-sorted control or post-sorted 25 µM carboplatin-treated subpopulation. ( B ) Final growth on day 30 as assessed by the total surface area of the well covered by tumor cell nuclei. The three groups were control (post-sorted FSC-small), or 25 µM carboplatin-treated (post-sorted FSC-small, post-sorted FSC-large). Data points were mean total covered surface area (normalized to the well with the largest growth) +/– standard deviations from 3 independent experiments. Nuclei analyzed ranged from 0 to about 27000 nuclei per well. ( C ) Final growth on day 30 from similar experiments as above where the three groups were control (post-sorted FSC-small), or 12.5 µM carboplatin-treated (post-sorted FSC-small, post-sorted FSC-large). Data points were mean total covered surface area (normalized to the well with the largest growth) +/– standard deviations from 3 sorting sessions. Nuclei analyzed ranged from 0 to about 67000 nuclei per well. * p
    96well Plates, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Bio-Rad 96 well plates
    Confirmation of mutation entry clones by barcoded next-gen sequencing Products of the PCR mutagenesis reaction are cloned into the pDONR223 vector via Gateway BP reaction and transformed into competent DH5α bacterial cells. Transformants are spotted on LB + Spectinomycin plates to isolate single colonies. One plate is shown here as a representation. This scheme illustrates the steps involved in mutation sample preparation and next-generation sequencing confirmation. PCR amplification should be done for each mutant at the single colony level prior to sample preparation (illustrated by 96 well plates M1-M4). If a given gene has multiple mutations to clone ( e.g. , four as shown), these mutation PCR products should be separated into different pools (Steps 35-36). A unique barcode adaptor is added to each pool by ligation (Step 37). All pooled mutation PCR products are then sequence-verified by next-generation sequencing technologies (454-FLX, Illumina Solexa, etc) (Steps 38-41).
    96 Well Plates, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 1200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad ddpcr 96 well plates
    Confirmation of mutation entry clones by barcoded next-gen sequencing Products of the PCR mutagenesis reaction are cloned into the pDONR223 vector via Gateway BP reaction and transformed into competent DH5α bacterial cells. Transformants are spotted on LB + Spectinomycin plates to isolate single colonies. One plate is shown here as a representation. This scheme illustrates the steps involved in mutation sample preparation and next-generation sequencing confirmation. PCR amplification should be done for each mutant at the single colony level prior to sample preparation (illustrated by 96 well plates M1-M4). If a given gene has multiple mutations to clone ( e.g. , four as shown), these mutation PCR products should be separated into different pools (Steps 35-36). A unique barcode adaptor is added to each pool by ligation (Step 37). All pooled mutation PCR products are then sequence-verified by next-generation sequencing technologies (454-FLX, Illumina Solexa, etc) (Steps 38-41).
    Ddpcr 96 Well Plates, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ddpcr 96 well plates - by Bioz Stars, 2020-08
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    99
    PerkinElmer 96 well plates
    SM-164 radiosensitizes breast cancer cells. a Sensitivity of breast cancer lines to SM-164: Four lines of breast cancer cells ( left panel ) and MCF10A cells ( right panel ) were seeded in <t>96-well</t> plate in triplicate and treated with various concentrations of SM-164 for 24 h. Cells were then lysed and subjected to ATP-lite assay for cell viability. Shown is mean ± SD ( n = 3). b and c Radiosensitization by SM-164 in SK-BR-3 cells ( b ) and MDA-MB-468 cells ( c ): Cells were seeded in 60-mm dishes and 6 h later treated with SM-164 (200 nM) for 24 h, followed by exposure to radiation at incremental doses up to 8 Gy. SER was calculated as the ratio of the mean inactivation dose under untreated control conditions divided by the mean inactivation dose with SM-164 treatment. Shown is mean ± SEM from three independent experiments, each runs in duplicate
    96 Well Plates, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 598 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Avantor 96 well plates
    SM-164 radiosensitizes breast cancer cells. a Sensitivity of breast cancer lines to SM-164: Four lines of breast cancer cells ( left panel ) and MCF10A cells ( right panel ) were seeded in <t>96-well</t> plate in triplicate and treated with various concentrations of SM-164 for 24 h. Cells were then lysed and subjected to ATP-lite assay for cell viability. Shown is mean ± SD ( n = 3). b and c Radiosensitization by SM-164 in SK-BR-3 cells ( b ) and MDA-MB-468 cells ( c ): Cells were seeded in 60-mm dishes and 6 h later treated with SM-164 (200 nM) for 24 h, followed by exposure to radiation at incremental doses up to 8 Gy. SER was calculated as the ratio of the mean inactivation dose under untreated control conditions divided by the mean inactivation dose with SM-164 treatment. Shown is mean ± SEM from three independent experiments, each runs in duplicate
    96 Well Plates, supplied by Avantor, used in various techniques. Bioz Stars score: 92/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Axygen 96 well plates
    SM-164 radiosensitizes breast cancer cells. a Sensitivity of breast cancer lines to SM-164: Four lines of breast cancer cells ( left panel ) and MCF10A cells ( right panel ) were seeded in <t>96-well</t> plate in triplicate and treated with various concentrations of SM-164 for 24 h. Cells were then lysed and subjected to ATP-lite assay for cell viability. Shown is mean ± SD ( n = 3). b and c Radiosensitization by SM-164 in SK-BR-3 cells ( b ) and MDA-MB-468 cells ( c ): Cells were seeded in 60-mm dishes and 6 h later treated with SM-164 (200 nM) for 24 h, followed by exposure to radiation at incremental doses up to 8 Gy. SER was calculated as the ratio of the mean inactivation dose under untreated control conditions divided by the mean inactivation dose with SM-164 treatment. Shown is mean ± SEM from three independent experiments, each runs in duplicate
    96 Well Plates, supplied by Axygen, used in various techniques. Bioz Stars score: 93/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson 96 well plates
    EF24 shows more potent cytotoxic effect than curcumin on cancer cells. Cells were grown in <t>96-well</t> plates and were treated with EF24 or curcumin as indicated for 48 h. Cell viability was assessed by the sulforhodamine B method and expressed as percentage
    96 Well Plates, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 2034 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    BMG Labtech 96 well plates
    EF24 shows more potent cytotoxic effect than curcumin on cancer cells. Cells were grown in <t>96-well</t> plates and were treated with EF24 or curcumin as indicated for 48 h. Cell viability was assessed by the sulforhodamine B method and expressed as percentage
    96 Well Plates, supplied by BMG Labtech, used in various techniques. Bioz Stars score: 93/100, based on 240 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boster Bio 96 well plates
    EF24 shows more potent cytotoxic effect than curcumin on cancer cells. Cells were grown in <t>96-well</t> plates and were treated with EF24 or curcumin as indicated for 48 h. Cell viability was assessed by the sulforhodamine B method and expressed as percentage
    96 Well Plates, supplied by Boster Bio, used in various techniques. Bioz Stars score: 92/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Corning Life Sciences 96 well plates
    Multiple linear modeling fits relative survivorship and relative growth rate from competitive-aging cultures. Examples of a reference competition (gray) and two deletion strains in co-culture with the WT CFP ( hap3 Δ RFP , orange; swr1 Δ RFP , green). In both panels, dots indicate the experimental ln ( RFP/CFP ) measurements. A , Fitted data is shown in the T timescale (days). Open circles are the data fitted at T i and error bars at those time points are the 95% CI of the fit; their linear regression has slope S (the relative survivorship, our parameter of interest). For each well, parameter A indicates the deviation from ln( RFP/CFP )=0 at T =0, as indicated by the vertical line at T =0. B , Fitted data is shown in the t timescale (hours); nine panels correspond to outgrowths at different age times, T . Crosses shown directly below or above experimental data are the fitted data at the time of each measurement; their linear regression has slope G (the relative growth rate). All samples (wells) in the same <t>96-well</t> plate measurement have the same C T,t (systematic batch error, gray vertical lines).
    96 Well Plates, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 94/100, based on 2734 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Essen Bioscience 96 well plates
    Multiple linear modeling fits relative survivorship and relative growth rate from competitive-aging cultures. Examples of a reference competition (gray) and two deletion strains in co-culture with the WT CFP ( hap3 Δ RFP , orange; swr1 Δ RFP , green). In both panels, dots indicate the experimental ln ( RFP/CFP ) measurements. A , Fitted data is shown in the T timescale (days). Open circles are the data fitted at T i and error bars at those time points are the 95% CI of the fit; their linear regression has slope S (the relative survivorship, our parameter of interest). For each well, parameter A indicates the deviation from ln( RFP/CFP )=0 at T =0, as indicated by the vertical line at T =0. B , Fitted data is shown in the t timescale (hours); nine panels correspond to outgrowths at different age times, T . Crosses shown directly below or above experimental data are the fitted data at the time of each measurement; their linear regression has slope G (the relative growth rate). All samples (wells) in the same <t>96-well</t> plate measurement have the same C T,t (systematic batch error, gray vertical lines).
    96 Well Plates, supplied by Essen Bioscience, used in various techniques. Bioz Stars score: 93/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Greiner Bio 96 well plates
    High-throughput time-resolved microscopy screen for atypical lysis kinetics. (A) Shown is a simplified schematic of the workflow. Cephalexin is added to growing cell cultures in glass-bottom <t>96-well</t> plates and then the plate is kept on the microscope. Images are taken at 4 time-points: 40 min, 85 min, 2 and 4.5 h. Images are then processed to count the number of intact cells at every time-point for each well/strain. Lysed and intact cells are distinguished by thresholding. (B) 96-well plate view of an experiment at T 40 min . Zoomed insets show two wells with different phenotypes. Well marked with ( # ) is representative of most strains, showing intact elongated cells. Well containing the rapidly lysing strain Δ lpoB shows lysed cells at T 40 min . Scale bars correspond to 20 μm. (C) 96-well plate view of an experiment at T 4 . 5h . Zoomed insets show two wells with different phenotypes. Well marked with ( # ) is representative of most strains, showing lysed cells. Well containing the late lysing strain Δ fkpB with intact cells. Scale bars correspond to 100 μm.
    96 Well Plates, supplied by Greiner Bio, used in various techniques. Bioz Stars score: 93/100, based on 1064 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Sarstedt 96 well plates
    High-throughput time-resolved microscopy screen for atypical lysis kinetics. (A) Shown is a simplified schematic of the workflow. Cephalexin is added to growing cell cultures in glass-bottom <t>96-well</t> plates and then the plate is kept on the microscope. Images are taken at 4 time-points: 40 min, 85 min, 2 and 4.5 h. Images are then processed to count the number of intact cells at every time-point for each well/strain. Lysed and intact cells are distinguished by thresholding. (B) 96-well plate view of an experiment at T 40 min . Zoomed insets show two wells with different phenotypes. Well marked with ( # ) is representative of most strains, showing intact elongated cells. Well containing the rapidly lysing strain Δ lpoB shows lysed cells at T 40 min . Scale bars correspond to 20 μm. (C) 96-well plate view of an experiment at T 4 . 5h . Zoomed insets show two wells with different phenotypes. Well marked with ( # ) is representative of most strains, showing lysed cells. Well containing the late lysing strain Δ fkpB with intact cells. Scale bars correspond to 100 μm.
    96 Well Plates, supplied by Sarstedt, used in various techniques. Bioz Stars score: 94/100, based on 263 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    DYNEX tech 96 well plates
    High-throughput time-resolved microscopy screen for atypical lysis kinetics. (A) Shown is a simplified schematic of the workflow. Cephalexin is added to growing cell cultures in glass-bottom <t>96-well</t> plates and then the plate is kept on the microscope. Images are taken at 4 time-points: 40 min, 85 min, 2 and 4.5 h. Images are then processed to count the number of intact cells at every time-point for each well/strain. Lysed and intact cells are distinguished by thresholding. (B) 96-well plate view of an experiment at T 40 min . Zoomed insets show two wells with different phenotypes. Well marked with ( # ) is representative of most strains, showing intact elongated cells. Well containing the rapidly lysing strain Δ lpoB shows lysed cells at T 40 min . Scale bars correspond to 20 μm. (C) 96-well plate view of an experiment at T 4 . 5h . Zoomed insets show two wells with different phenotypes. Well marked with ( # ) is representative of most strains, showing lysed cells. Well containing the late lysing strain Δ fkpB with intact cells. Scale bars correspond to 100 μm.
    96 Well Plates, supplied by DYNEX tech, used in various techniques. Bioz Stars score: 93/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Fisher Scientific 96 well plates
    High-throughput time-resolved microscopy screen for atypical lysis kinetics. (A) Shown is a simplified schematic of the workflow. Cephalexin is added to growing cell cultures in glass-bottom <t>96-well</t> plates and then the plate is kept on the microscope. Images are taken at 4 time-points: 40 min, 85 min, 2 and 4.5 h. Images are then processed to count the number of intact cells at every time-point for each well/strain. Lysed and intact cells are distinguished by thresholding. (B) 96-well plate view of an experiment at T 40 min . Zoomed insets show two wells with different phenotypes. Well marked with ( # ) is representative of most strains, showing intact elongated cells. Well containing the rapidly lysing strain Δ lpoB shows lysed cells at T 40 min . Scale bars correspond to 20 μm. (C) 96-well plate view of an experiment at T 4 . 5h . Zoomed insets show two wells with different phenotypes. Well marked with ( # ) is representative of most strains, showing lysed cells. Well containing the late lysing strain Δ fkpB with intact cells. Scale bars correspond to 100 μm.
    96 Well Plates, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 122 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Innovadyne Technologies 96 well plates
    High-throughput time-resolved microscopy screen for atypical lysis kinetics. (A) Shown is a simplified schematic of the workflow. Cephalexin is added to growing cell cultures in glass-bottom <t>96-well</t> plates and then the plate is kept on the microscope. Images are taken at 4 time-points: 40 min, 85 min, 2 and 4.5 h. Images are then processed to count the number of intact cells at every time-point for each well/strain. Lysed and intact cells are distinguished by thresholding. (B) 96-well plate view of an experiment at T 40 min . Zoomed insets show two wells with different phenotypes. Well marked with ( # ) is representative of most strains, showing intact elongated cells. Well containing the rapidly lysing strain Δ lpoB shows lysed cells at T 40 min . Scale bars correspond to 20 μm. (C) 96-well plate view of an experiment at T 4 . 5h . Zoomed insets show two wells with different phenotypes. Well marked with ( # ) is representative of most strains, showing lysed cells. Well containing the late lysing strain Δ fkpB with intact cells. Scale bars correspond to 100 μm.
    96 Well Plates, supplied by Innovadyne Technologies, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GE Healthcare 96 well plates
    High-throughput time-resolved microscopy screen for atypical lysis kinetics. (A) Shown is a simplified schematic of the workflow. Cephalexin is added to growing cell cultures in glass-bottom <t>96-well</t> plates and then the plate is kept on the microscope. Images are taken at 4 time-points: 40 min, 85 min, 2 and 4.5 h. Images are then processed to count the number of intact cells at every time-point for each well/strain. Lysed and intact cells are distinguished by thresholding. (B) 96-well plate view of an experiment at T 40 min . Zoomed insets show two wells with different phenotypes. Well marked with ( # ) is representative of most strains, showing intact elongated cells. Well containing the rapidly lysing strain Δ lpoB shows lysed cells at T 40 min . Scale bars correspond to 20 μm. (C) 96-well plate view of an experiment at T 4 . 5h . Zoomed insets show two wells with different phenotypes. Well marked with ( # ) is representative of most strains, showing lysed cells. Well containing the late lysing strain Δ fkpB with intact cells. Scale bars correspond to 100 μm.
    96 Well Plates, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    ibidi 96 well plates
    Quantitative CRISPR/Cas9 editing strategy A) Tagging strategy to allow quantitation of PC-I in NIH3T3 fibroblasts with introduction of a multifunctional tag at the N-terminus of proα2(I) to allow detection of edited cells, and the small luciferase, NLuc. B) After introduction of the multifunctional tag in exon 1 of Col1a2 using CRISPR/Cas9, edited cells were sorted based on GFP fluorescence. C) PCR validation of edited DNA in cells isolated from B) using primers in Supplementary table 1 . D) Real-time PCR of total Col1a2 transcripts (Black) and edited transcripts in NIH3T3 and nluc::Col1a2 cells. Primers at the 5’ and 3’ ends of the introduced nluc confirmed insertion into the Col1a2 transcript. Bars show mean ± SD, n=3 independent experiments. E) Boxes show exon positions of the edited Col1a2 RNA transcript, sequencing of PCR products in D) demonstrate insertion of nluc in frame with Col1a2 . F) Boxes show exon positions of the edited Col1a2 RNA transcript and the amino acid sequence of the expected tagged protein. Conditioned medium from nluc::Col1a2 cells was affinity purified ( Supplementary Fig. 1 ) and subjected to mass spectrometry analysis. A peptide spanning NLuc and protein encoded by Col1a2 exons 1-3 was identified. G) In-gel detection of NLuc tagged proα2(I) chain under reducing conditions identified NLuc activity at approximately 140 kDa. H) Imaging and quantitation of the light produced by nluc::Col1a2 and unedited NIH3T3 cells incubated with the NLuc substrate, Furimazine (Nano-Glo). On a <t>96-well</t> plate, single wells were imaged using a single-lens reflex camera and quantitation of photon counts using a multi-well plate luminometer. Bar shows means ± SD from n=30 replicate measurements. **** represents p=0.0001, paired Student’s t-Test.
    96 Well Plates, supplied by ibidi, used in various techniques. Bioz Stars score: 93/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Iwaki America 96 well plates
    Quantitative CRISPR/Cas9 editing strategy A) Tagging strategy to allow quantitation of PC-I in NIH3T3 fibroblasts with introduction of a multifunctional tag at the N-terminus of proα2(I) to allow detection of edited cells, and the small luciferase, NLuc. B) After introduction of the multifunctional tag in exon 1 of Col1a2 using CRISPR/Cas9, edited cells were sorted based on GFP fluorescence. C) PCR validation of edited DNA in cells isolated from B) using primers in Supplementary table 1 . D) Real-time PCR of total Col1a2 transcripts (Black) and edited transcripts in NIH3T3 and nluc::Col1a2 cells. Primers at the 5’ and 3’ ends of the introduced nluc confirmed insertion into the Col1a2 transcript. Bars show mean ± SD, n=3 independent experiments. E) Boxes show exon positions of the edited Col1a2 RNA transcript, sequencing of PCR products in D) demonstrate insertion of nluc in frame with Col1a2 . F) Boxes show exon positions of the edited Col1a2 RNA transcript and the amino acid sequence of the expected tagged protein. Conditioned medium from nluc::Col1a2 cells was affinity purified ( Supplementary Fig. 1 ) and subjected to mass spectrometry analysis. A peptide spanning NLuc and protein encoded by Col1a2 exons 1-3 was identified. G) In-gel detection of NLuc tagged proα2(I) chain under reducing conditions identified NLuc activity at approximately 140 kDa. H) Imaging and quantitation of the light produced by nluc::Col1a2 and unedited NIH3T3 cells incubated with the NLuc substrate, Furimazine (Nano-Glo). On a <t>96-well</t> plate, single wells were imaged using a single-lens reflex camera and quantitation of photon counts using a multi-well plate luminometer. Bar shows means ± SD from n=30 replicate measurements. **** represents p=0.0001, paired Student’s t-Test.
    96 Well Plates, supplied by Iwaki America, used in various techniques. Bioz Stars score: 93/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Nest Biotechnology 96 well plates
    Quantitative CRISPR/Cas9 editing strategy A) Tagging strategy to allow quantitation of PC-I in NIH3T3 fibroblasts with introduction of a multifunctional tag at the N-terminus of proα2(I) to allow detection of edited cells, and the small luciferase, NLuc. B) After introduction of the multifunctional tag in exon 1 of Col1a2 using CRISPR/Cas9, edited cells were sorted based on GFP fluorescence. C) PCR validation of edited DNA in cells isolated from B) using primers in Supplementary table 1 . D) Real-time PCR of total Col1a2 transcripts (Black) and edited transcripts in NIH3T3 and nluc::Col1a2 cells. Primers at the 5’ and 3’ ends of the introduced nluc confirmed insertion into the Col1a2 transcript. Bars show mean ± SD, n=3 independent experiments. E) Boxes show exon positions of the edited Col1a2 RNA transcript, sequencing of PCR products in D) demonstrate insertion of nluc in frame with Col1a2 . F) Boxes show exon positions of the edited Col1a2 RNA transcript and the amino acid sequence of the expected tagged protein. Conditioned medium from nluc::Col1a2 cells was affinity purified ( Supplementary Fig. 1 ) and subjected to mass spectrometry analysis. A peptide spanning NLuc and protein encoded by Col1a2 exons 1-3 was identified. G) In-gel detection of NLuc tagged proα2(I) chain under reducing conditions identified NLuc activity at approximately 140 kDa. H) Imaging and quantitation of the light produced by nluc::Col1a2 and unedited NIH3T3 cells incubated with the NLuc substrate, Furimazine (Nano-Glo). On a <t>96-well</t> plate, single wells were imaged using a single-lens reflex camera and quantitation of photon counts using a multi-well plate luminometer. Bar shows means ± SD from n=30 replicate measurements. **** represents p=0.0001, paired Student’s t-Test.
    96 Well Plates, supplied by Nest Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Size-sorted hyperploid subpopulation ( > 6N) showed differential regrowth potential compared to the non-hyperploid (3N-6N) fraction. Serial dilution growth assay where adherent OVCAR3 cells were treated with vehicle, 12.5 µM or 25 µM carboplatin on day 0 for 24 hrs, followed by removal of the drug and further incubation of the cells until day 9. Cells were then detached, flow sorted by forward scatter (FSC) and propidium-iodide negativity into a small or large subpopulation, and then plated into a 96-well plate at the indicated density for regrowth until final assessment by imaging on day 30. ( A ) The centroid location of each identified, propidium-iodide excluded, Hoechst stained cell within the wells of a 96-well plate was plotted on day 30, for the post-sorted control or post-sorted 25 µM carboplatin-treated subpopulation. ( B ) Final growth on day 30 as assessed by the total surface area of the well covered by tumor cell nuclei. The three groups were control (post-sorted FSC-small), or 25 µM carboplatin-treated (post-sorted FSC-small, post-sorted FSC-large). Data points were mean total covered surface area (normalized to the well with the largest growth) +/– standard deviations from 3 independent experiments. Nuclei analyzed ranged from 0 to about 27000 nuclei per well. ( C ) Final growth on day 30 from similar experiments as above where the three groups were control (post-sorted FSC-small), or 12.5 µM carboplatin-treated (post-sorted FSC-small, post-sorted FSC-large). Data points were mean total covered surface area (normalized to the well with the largest growth) +/– standard deviations from 3 sorting sessions. Nuclei analyzed ranged from 0 to about 67000 nuclei per well. * p

    Journal: Oncotarget

    Article Title: Avoidance of apoptotic death via a hyperploid salvage survival pathway after platinum treatment in high grade serous carcinoma cell line models

    doi: 10.18632/oncotarget.27330

    Figure Lengend Snippet: Size-sorted hyperploid subpopulation ( > 6N) showed differential regrowth potential compared to the non-hyperploid (3N-6N) fraction. Serial dilution growth assay where adherent OVCAR3 cells were treated with vehicle, 12.5 µM or 25 µM carboplatin on day 0 for 24 hrs, followed by removal of the drug and further incubation of the cells until day 9. Cells were then detached, flow sorted by forward scatter (FSC) and propidium-iodide negativity into a small or large subpopulation, and then plated into a 96-well plate at the indicated density for regrowth until final assessment by imaging on day 30. ( A ) The centroid location of each identified, propidium-iodide excluded, Hoechst stained cell within the wells of a 96-well plate was plotted on day 30, for the post-sorted control or post-sorted 25 µM carboplatin-treated subpopulation. ( B ) Final growth on day 30 as assessed by the total surface area of the well covered by tumor cell nuclei. The three groups were control (post-sorted FSC-small), or 25 µM carboplatin-treated (post-sorted FSC-small, post-sorted FSC-large). Data points were mean total covered surface area (normalized to the well with the largest growth) +/– standard deviations from 3 independent experiments. Nuclei analyzed ranged from 0 to about 27000 nuclei per well. ( C ) Final growth on day 30 from similar experiments as above where the three groups were control (post-sorted FSC-small), or 12.5 µM carboplatin-treated (post-sorted FSC-small, post-sorted FSC-large). Data points were mean total covered surface area (normalized to the well with the largest growth) +/– standard deviations from 3 sorting sessions. Nuclei analyzed ranged from 0 to about 67000 nuclei per well. * p

    Article Snippet: Immunofluorescence protocol Adherent OVCAR3 tumor cells within 96-well plates were fixed with 4% paraformaldehyde (Electron Microscopy Science 15710) for 30 min and then permeabilized with 0.15% Triton X-100 (Sigma-Aldrich X100) overnight at 4C.

    Techniques: Serial Dilution, Growth Assay, Incubation, Flow Cytometry, Imaging, Staining

    Platinum-induced nuclear enlargement as a distinct cellular response among surviving tumor cells at intermediate to late time points. Time course experiment where adherent OVCAR3 cells maintained within a set of four similarly prepared 96-well plates were all treated with 0 µM to 160 µM carboplatin for 24 hrs on day 0, followed by drug removal. One plate from the set of four was then fixed on day 1, 4, 7 or 11, respectively. ( A – D ) Hoechst 33342-stained nuclei, and where presented, the corresponding nuclear masks as identified by automated image analysis software for control vs. carboplatin treated cells on day 1 (A), day 4 (B), day 7 (C) or day 11 (D). Scale bar = 20 µm. ( E ) Time course of median nuclear area for control vs. carboplatin treated cells. Data points were median nuclear area from each individual experiment and the connected line passed through the average of the three biological replicates. Median nuclear area assessed from well with at least 5 to about 20000 nuclei. ** p

    Journal: Oncotarget

    Article Title: Avoidance of apoptotic death via a hyperploid salvage survival pathway after platinum treatment in high grade serous carcinoma cell line models

    doi: 10.18632/oncotarget.27330

    Figure Lengend Snippet: Platinum-induced nuclear enlargement as a distinct cellular response among surviving tumor cells at intermediate to late time points. Time course experiment where adherent OVCAR3 cells maintained within a set of four similarly prepared 96-well plates were all treated with 0 µM to 160 µM carboplatin for 24 hrs on day 0, followed by drug removal. One plate from the set of four was then fixed on day 1, 4, 7 or 11, respectively. ( A – D ) Hoechst 33342-stained nuclei, and where presented, the corresponding nuclear masks as identified by automated image analysis software for control vs. carboplatin treated cells on day 1 (A), day 4 (B), day 7 (C) or day 11 (D). Scale bar = 20 µm. ( E ) Time course of median nuclear area for control vs. carboplatin treated cells. Data points were median nuclear area from each individual experiment and the connected line passed through the average of the three biological replicates. Median nuclear area assessed from well with at least 5 to about 20000 nuclei. ** p

    Article Snippet: Immunofluorescence protocol Adherent OVCAR3 tumor cells within 96-well plates were fixed with 4% paraformaldehyde (Electron Microscopy Science 15710) for 30 min and then permeabilized with 0.15% Triton X-100 (Sigma-Aldrich X100) overnight at 4C.

    Techniques: Staining, Software

    Nuclear area and DNA ploidy analysis revealed the de novo generation of a subpopulation of large hyperploid tumor cells due to G2-M checkpoint deregulation. Time course or dose-response experiment where adherent OVCAR3 cells maintained within 96-well plates were treated with carboplatin from 0 µM and up to 1000 µM for 24 hrs on day 0, followed by removal of the drug. For the time course experiment, one plate from a set of four was then fixed on day 1, 4, 7 or 11 for immunofluorescence or Hoechst staining. Only propidium-iodide excluded nuclei were used for quantification. ( A ) Time course of cell cycle phase/DNA ploidy distribution by total Hoechst DNA content. Data bars were the mean percentage of each subpopulation +/– standard deviations from 3 independent experiments. Nuclei analyzed ranged from 0 to about 20000 nuclei per well. ( B ) Median nuclear area of different subpopulations among the surviving cells on day 11 after carboplatin treatment. Data bars were the average of the median nuclear area of the indicated subpopulation (normalized to the Go/G1 of control) +/– standard deviations from 3 independent experiments. Median nuclear area assessed from well with at least 5 to about 20000 nuclei. Wells from the 160 µM carboplatin treatment were not included for analysis due to the very low number of surviving cells. ** p

    Journal: Oncotarget

    Article Title: Avoidance of apoptotic death via a hyperploid salvage survival pathway after platinum treatment in high grade serous carcinoma cell line models

    doi: 10.18632/oncotarget.27330

    Figure Lengend Snippet: Nuclear area and DNA ploidy analysis revealed the de novo generation of a subpopulation of large hyperploid tumor cells due to G2-M checkpoint deregulation. Time course or dose-response experiment where adherent OVCAR3 cells maintained within 96-well plates were treated with carboplatin from 0 µM and up to 1000 µM for 24 hrs on day 0, followed by removal of the drug. For the time course experiment, one plate from a set of four was then fixed on day 1, 4, 7 or 11 for immunofluorescence or Hoechst staining. Only propidium-iodide excluded nuclei were used for quantification. ( A ) Time course of cell cycle phase/DNA ploidy distribution by total Hoechst DNA content. Data bars were the mean percentage of each subpopulation +/– standard deviations from 3 independent experiments. Nuclei analyzed ranged from 0 to about 20000 nuclei per well. ( B ) Median nuclear area of different subpopulations among the surviving cells on day 11 after carboplatin treatment. Data bars were the average of the median nuclear area of the indicated subpopulation (normalized to the Go/G1 of control) +/– standard deviations from 3 independent experiments. Median nuclear area assessed from well with at least 5 to about 20000 nuclei. Wells from the 160 µM carboplatin treatment were not included for analysis due to the very low number of surviving cells. ** p

    Article Snippet: Immunofluorescence protocol Adherent OVCAR3 tumor cells within 96-well plates were fixed with 4% paraformaldehyde (Electron Microscopy Science 15710) for 30 min and then permeabilized with 0.15% Triton X-100 (Sigma-Aldrich X100) overnight at 4C.

    Techniques: Immunofluorescence, Staining

    Hyperploid tumor cells remained proliferative and retained capacity to generate viable daughter cells. Immunofluorescence staining or time-lapse microscopy experiments where adherent OVCAR3 cells maintained within 96-well plates were treated with 0 µM to 160 µM carboplatin for 24 hrs on day 0, followed by removal of the drug. Residual cells within the wells were then fixed on day 11 for immunofluorescence and Hoechst staining ( A – C , F – G ), or imaged live by time-lapse microscopy between day 1–2, 4–5, 8–9, 11–12 ( D – E ). (A) Carboplatin concentration-dependent effect on percentage of non-hyperploid (3N-6N) or hyperploid cells with cleaved-PARP positivity on day 11. Cleaved-PARP positivity was defined using mean nuclear intensity and nuclear-to-cytoplasmic ratio of the antibody staining to identify cells (~2%) separated from the main viable cluster in the control bulk population. Data points were mean percentage of cleaved-PARP positivity +/– standard deviations from 3 independent experiments. Nuclei analyzed ranged from 20 to about 17000 nuclei per well. * p

    Journal: Oncotarget

    Article Title: Avoidance of apoptotic death via a hyperploid salvage survival pathway after platinum treatment in high grade serous carcinoma cell line models

    doi: 10.18632/oncotarget.27330

    Figure Lengend Snippet: Hyperploid tumor cells remained proliferative and retained capacity to generate viable daughter cells. Immunofluorescence staining or time-lapse microscopy experiments where adherent OVCAR3 cells maintained within 96-well plates were treated with 0 µM to 160 µM carboplatin for 24 hrs on day 0, followed by removal of the drug. Residual cells within the wells were then fixed on day 11 for immunofluorescence and Hoechst staining ( A – C , F – G ), or imaged live by time-lapse microscopy between day 1–2, 4–5, 8–9, 11–12 ( D – E ). (A) Carboplatin concentration-dependent effect on percentage of non-hyperploid (3N-6N) or hyperploid cells with cleaved-PARP positivity on day 11. Cleaved-PARP positivity was defined using mean nuclear intensity and nuclear-to-cytoplasmic ratio of the antibody staining to identify cells (~2%) separated from the main viable cluster in the control bulk population. Data points were mean percentage of cleaved-PARP positivity +/– standard deviations from 3 independent experiments. Nuclei analyzed ranged from 20 to about 17000 nuclei per well. * p

    Article Snippet: Immunofluorescence protocol Adherent OVCAR3 tumor cells within 96-well plates were fixed with 4% paraformaldehyde (Electron Microscopy Science 15710) for 30 min and then permeabilized with 0.15% Triton X-100 (Sigma-Aldrich X100) overnight at 4C.

    Techniques: Immunofluorescence, Staining, Time-lapse Microscopy, Concentration Assay

    Platinum treatment resulted in apoptotic and necrotic cell death in the bulk tumor cell population. Time course, dose response or live imaging experiment where adherent OVCAR3 cells maintained within 96-well plates were treated with carboplatin from 0 µM and up to 1000 µM for 24 hrs on day 0, followed by drug removal. For a typical time course experiment, a set of four similarly prepared 96-well plates were all treated on day 0 and then one plate within the set would be fixed on day 1, 4, 7 or 11. ( A ) Time course of cell survival showing propidium-iodide excluded, Hoechst-stained nuclei count at 1, 4, 7 or 11 days after carboplatin treatment with the indicated concentration. Data points were mean nuclei count per well indicating cell survival (normalized to control of day 1) +/– standard deviations from 3 independent experiments. Nuclei analyzed ranged from 0 to about 20000 nuclei per well. ** p

    Journal: Oncotarget

    Article Title: Avoidance of apoptotic death via a hyperploid salvage survival pathway after platinum treatment in high grade serous carcinoma cell line models

    doi: 10.18632/oncotarget.27330

    Figure Lengend Snippet: Platinum treatment resulted in apoptotic and necrotic cell death in the bulk tumor cell population. Time course, dose response or live imaging experiment where adherent OVCAR3 cells maintained within 96-well plates were treated with carboplatin from 0 µM and up to 1000 µM for 24 hrs on day 0, followed by drug removal. For a typical time course experiment, a set of four similarly prepared 96-well plates were all treated on day 0 and then one plate within the set would be fixed on day 1, 4, 7 or 11. ( A ) Time course of cell survival showing propidium-iodide excluded, Hoechst-stained nuclei count at 1, 4, 7 or 11 days after carboplatin treatment with the indicated concentration. Data points were mean nuclei count per well indicating cell survival (normalized to control of day 1) +/– standard deviations from 3 independent experiments. Nuclei analyzed ranged from 0 to about 20000 nuclei per well. ** p

    Article Snippet: Immunofluorescence protocol Adherent OVCAR3 tumor cells within 96-well plates were fixed with 4% paraformaldehyde (Electron Microscopy Science 15710) for 30 min and then permeabilized with 0.15% Triton X-100 (Sigma-Aldrich X100) overnight at 4C.

    Techniques: Imaging, Staining, Concentration Assay

    Confirmation of mutation entry clones by barcoded next-gen sequencing Products of the PCR mutagenesis reaction are cloned into the pDONR223 vector via Gateway BP reaction and transformed into competent DH5α bacterial cells. Transformants are spotted on LB + Spectinomycin plates to isolate single colonies. One plate is shown here as a representation. This scheme illustrates the steps involved in mutation sample preparation and next-generation sequencing confirmation. PCR amplification should be done for each mutant at the single colony level prior to sample preparation (illustrated by 96 well plates M1-M4). If a given gene has multiple mutations to clone ( e.g. , four as shown), these mutation PCR products should be separated into different pools (Steps 35-36). A unique barcode adaptor is added to each pool by ligation (Step 37). All pooled mutation PCR products are then sequence-verified by next-generation sequencing technologies (454-FLX, Illumina Solexa, etc) (Steps 38-41).

    Journal: Nature protocols

    Article Title: Base-resolution stratification of cancer mutations using functional variomics

    doi: 10.1038/nprot.2017.086

    Figure Lengend Snippet: Confirmation of mutation entry clones by barcoded next-gen sequencing Products of the PCR mutagenesis reaction are cloned into the pDONR223 vector via Gateway BP reaction and transformed into competent DH5α bacterial cells. Transformants are spotted on LB + Spectinomycin plates to isolate single colonies. One plate is shown here as a representation. This scheme illustrates the steps involved in mutation sample preparation and next-generation sequencing confirmation. PCR amplification should be done for each mutant at the single colony level prior to sample preparation (illustrated by 96 well plates M1-M4). If a given gene has multiple mutations to clone ( e.g. , four as shown), these mutation PCR products should be separated into different pools (Steps 35-36). A unique barcode adaptor is added to each pool by ligation (Step 37). All pooled mutation PCR products are then sequence-verified by next-generation sequencing technologies (454-FLX, Illumina Solexa, etc) (Steps 38-41).

    Article Snippet: Round-bottom 96-well plates (Corning, cat. no. 3788) 50 ml sterile reagent troughs (Corning, cat. no. 4871) Thin-walled 96-well PCR microtiter plates (Bio-Rad, cat. no. HSP-9601) 15 cm sterile petri dishes (Fisher Scientific, cat. no. 08-757-14) Aluminum foil microplate seal (Bio-Rad, cat. no. MSF-1001) 50 ml polypropylene tubes (BD Falcon, cat. no. 352070) Tissue culture 96 wells flat bottom plate (BD Falcon, cat. no. 353075) PIPETMAN P10, 1 to 10 µl (Gibson, cat. no. F144802) PIPETMAN P200, 50 to 200 µl (Gibson, cat. no. F123601) Corning™ 4084 P10 multichannel pipettes 1 - 10 µl (Thermofisher scientific, cat. no. 07-764-710) Corning™ 4085 P50 multichannel pipettes 5 - 50 µl (Thermofisher scientific, cat. no. 07-764-711) Corning™ 4085 P200 multichannel pipettes 20 - 200 µl (Thermofisher scientific, cat. no. 07-764-712) Corning® Stripettor™ Ultra Pipet Controller (Thermofisher scientific, cat. no. 4099) Thermocycler capable of handling 96-well plates (Bio-Rad, cat. no. T100™) Temperature controlled incubator (30°C) (Thermofisher scientific, cat. no. 15-103-0515) Temperature controlled water bath (42°C) (Thermofisher scientific, cat. no. FSGPD10) 1% E-Gel® 96 Agarose Gels (Thermofisher scientific, cat. no. G700801) E-gel Mother E-Base capable of running 96 samples (Thermofisher scientific, cat. no. EBM03) Temperature controlled shaker for 250 ml flasks and 50 ml tubes (MaxQ™ 6000 Shaker, Thermofisher scientific, cat. no. SHKE6000) Benchtop Centrifuge suitable for microplates and 50 ml tubes (Sorvall™ Legend™ XT/XF centrifuge, Thermofisher scientific, cat. no. 75-004-505) Plate shaker (Mixmate, Eppendorf, cat. no. 022674200) GENESYS™ 10S UV-Vis Spectrophotometer (Thermofisher scientific, cat. no. 840-208100) Vortexer (Thermofisher scientific, cat. no. 88880017TS) Centro XS3 Luminescence Microplate Reader (Berthold Technologies, cat. no. 46970) 1300 Series A2 Class II, Type A2 Bio Safety Cabinets (Thermofisher scientific, cat. no. 13-261-221) Optional: liquid handling robot with 96-well multichannel head and individual well cherry-picking capability (Tecan, cat. no. Fluent 780) Optional: BioRobot Universal System Robot with 96-well DNA miniprep capability (Qiagen, cat. no. 9001094)

    Techniques: Mutagenesis, Clone Assay, Sequencing, Polymerase Chain Reaction, Plasmid Preparation, Transformation Assay, Sample Prep, Next-Generation Sequencing, Amplification, Ligation

    SM-164 radiosensitizes breast cancer cells. a Sensitivity of breast cancer lines to SM-164: Four lines of breast cancer cells ( left panel ) and MCF10A cells ( right panel ) were seeded in 96-well plate in triplicate and treated with various concentrations of SM-164 for 24 h. Cells were then lysed and subjected to ATP-lite assay for cell viability. Shown is mean ± SD ( n = 3). b and c Radiosensitization by SM-164 in SK-BR-3 cells ( b ) and MDA-MB-468 cells ( c ): Cells were seeded in 60-mm dishes and 6 h later treated with SM-164 (200 nM) for 24 h, followed by exposure to radiation at incremental doses up to 8 Gy. SER was calculated as the ratio of the mean inactivation dose under untreated control conditions divided by the mean inactivation dose with SM-164 treatment. Shown is mean ± SEM from three independent experiments, each runs in duplicate

    Journal: Breast cancer research and treatment

    Article Title: Smac-mimetic compound SM-164 induces radiosensitization in breast cancer cells through activation of caspases and induction of apoptosis

    doi: 10.1007/s10549-011-1752-3

    Figure Lengend Snippet: SM-164 radiosensitizes breast cancer cells. a Sensitivity of breast cancer lines to SM-164: Four lines of breast cancer cells ( left panel ) and MCF10A cells ( right panel ) were seeded in 96-well plate in triplicate and treated with various concentrations of SM-164 for 24 h. Cells were then lysed and subjected to ATP-lite assay for cell viability. Shown is mean ± SD ( n = 3). b and c Radiosensitization by SM-164 in SK-BR-3 cells ( b ) and MDA-MB-468 cells ( c ): Cells were seeded in 60-mm dishes and 6 h later treated with SM-164 (200 nM) for 24 h, followed by exposure to radiation at incremental doses up to 8 Gy. SER was calculated as the ratio of the mean inactivation dose under untreated control conditions divided by the mean inactivation dose with SM-164 treatment. Shown is mean ± SEM from three independent experiments, each runs in duplicate

    Article Snippet: Cell were seeded in 96-well plates in triplicate and treated the next day with SM-164 in various doses ranging from 0.03 to 10 μM for 24 h. The viability of the cells was then measured using one-step ATPlite cell proliferation assay (Perkin Elmer) [ ].

    Techniques: Multiple Displacement Amplification

    EF24 shows more potent cytotoxic effect than curcumin on cancer cells. Cells were grown in 96-well plates and were treated with EF24 or curcumin as indicated for 48 h. Cell viability was assessed by the sulforhodamine B method and expressed as percentage

    Journal:

    Article Title: Inhibition of IκB Kinase-Nuclear Factor-κB Signaling Pathway by 3,5-Bis(2-flurobenzylidene)piperidin-4-one (EF24), a Novel Monoketone Analog of Curcumin *

    doi: 10.1124/mol.108.046201

    Figure Lengend Snippet: EF24 shows more potent cytotoxic effect than curcumin on cancer cells. Cells were grown in 96-well plates and were treated with EF24 or curcumin as indicated for 48 h. Cell viability was assessed by the sulforhodamine B method and expressed as percentage

    Article Snippet: A549 cells were plated in 96-well plates (BD Biosciences Discovery Labware, Bedford, MA) at 10,000 cells/90 μ l/well and grown for 20 h. Test compounds were added to each well and incubated at 37°C.

    Techniques:

    EF24 impairs TNF- α induced NF- κ B nuclear translocation. A549 cells were grown in 96-well plates and treated with TNF- α or control (DMSO) for 30 min before sample processing for the detection of NF- κ B as described under

    Journal:

    Article Title: Inhibition of IκB Kinase-Nuclear Factor-κB Signaling Pathway by 3,5-Bis(2-flurobenzylidene)piperidin-4-one (EF24), a Novel Monoketone Analog of Curcumin *

    doi: 10.1124/mol.108.046201

    Figure Lengend Snippet: EF24 impairs TNF- α induced NF- κ B nuclear translocation. A549 cells were grown in 96-well plates and treated with TNF- α or control (DMSO) for 30 min before sample processing for the detection of NF- κ B as described under

    Article Snippet: A549 cells were plated in 96-well plates (BD Biosciences Discovery Labware, Bedford, MA) at 10,000 cells/90 μ l/well and grown for 20 h. Test compounds were added to each well and incubated at 37°C.

    Techniques: Translocation Assay

    Multiple linear modeling fits relative survivorship and relative growth rate from competitive-aging cultures. Examples of a reference competition (gray) and two deletion strains in co-culture with the WT CFP ( hap3 Δ RFP , orange; swr1 Δ RFP , green). In both panels, dots indicate the experimental ln ( RFP/CFP ) measurements. A , Fitted data is shown in the T timescale (days). Open circles are the data fitted at T i and error bars at those time points are the 95% CI of the fit; their linear regression has slope S (the relative survivorship, our parameter of interest). For each well, parameter A indicates the deviation from ln( RFP/CFP )=0 at T =0, as indicated by the vertical line at T =0. B , Fitted data is shown in the t timescale (hours); nine panels correspond to outgrowths at different age times, T . Crosses shown directly below or above experimental data are the fitted data at the time of each measurement; their linear regression has slope G (the relative growth rate). All samples (wells) in the same 96-well plate measurement have the same C T,t (systematic batch error, gray vertical lines).

    Journal: bioRxiv

    Article Title: An optimized competitive-aging method reveals gene-drug interactions underlying the chronological lifespan of Saccharomyces cerevisiae

    doi: 10.1101/696682

    Figure Lengend Snippet: Multiple linear modeling fits relative survivorship and relative growth rate from competitive-aging cultures. Examples of a reference competition (gray) and two deletion strains in co-culture with the WT CFP ( hap3 Δ RFP , orange; swr1 Δ RFP , green). In both panels, dots indicate the experimental ln ( RFP/CFP ) measurements. A , Fitted data is shown in the T timescale (days). Open circles are the data fitted at T i and error bars at those time points are the 95% CI of the fit; their linear regression has slope S (the relative survivorship, our parameter of interest). For each well, parameter A indicates the deviation from ln( RFP/CFP )=0 at T =0, as indicated by the vertical line at T =0. B , Fitted data is shown in the t timescale (hours); nine panels correspond to outgrowths at different age times, T . Crosses shown directly below or above experimental data are the fitted data at the time of each measurement; their linear regression has slope G (the relative growth rate). All samples (wells) in the same 96-well plate measurement have the same C T,t (systematic batch error, gray vertical lines).

    Article Snippet: Live/dead cell viability assays Stationary-phase cultures (5 µL) were transferred to 50 µL of BD FACSFlow (BD #342003) with 0.1% propidium iodide (PI) and 0.1% SYTO9 green (ThermoFisher L34952) in 96-well plates (Corning 3585), shaken for 1 min at 1,000 rpm in a plate shaker (Heidolph Titramax 1000), and incubated at room temperature in the dark for 20 minutes.

    Techniques: Co-Culture Assay

    High-throughput time-resolved microscopy screen for atypical lysis kinetics. (A) Shown is a simplified schematic of the workflow. Cephalexin is added to growing cell cultures in glass-bottom 96-well plates and then the plate is kept on the microscope. Images are taken at 4 time-points: 40 min, 85 min, 2 and 4.5 h. Images are then processed to count the number of intact cells at every time-point for each well/strain. Lysed and intact cells are distinguished by thresholding. (B) 96-well plate view of an experiment at T 40 min . Zoomed insets show two wells with different phenotypes. Well marked with ( # ) is representative of most strains, showing intact elongated cells. Well containing the rapidly lysing strain Δ lpoB shows lysed cells at T 40 min . Scale bars correspond to 20 μm. (C) 96-well plate view of an experiment at T 4 . 5h . Zoomed insets show two wells with different phenotypes. Well marked with ( # ) is representative of most strains, showing lysed cells. Well containing the late lysing strain Δ fkpB with intact cells. Scale bars correspond to 100 μm.

    Journal: Frontiers in Microbiology

    Article Title: Image-Based Dynamic Phenotyping Reveals Genetic Determinants of Filamentation-Mediated β-Lactam Tolerance

    doi: 10.3389/fmicb.2020.00374

    Figure Lengend Snippet: High-throughput time-resolved microscopy screen for atypical lysis kinetics. (A) Shown is a simplified schematic of the workflow. Cephalexin is added to growing cell cultures in glass-bottom 96-well plates and then the plate is kept on the microscope. Images are taken at 4 time-points: 40 min, 85 min, 2 and 4.5 h. Images are then processed to count the number of intact cells at every time-point for each well/strain. Lysed and intact cells are distinguished by thresholding. (B) 96-well plate view of an experiment at T 40 min . Zoomed insets show two wells with different phenotypes. Well marked with ( # ) is representative of most strains, showing intact elongated cells. Well containing the rapidly lysing strain Δ lpoB shows lysed cells at T 40 min . Scale bars correspond to 20 μm. (C) 96-well plate view of an experiment at T 4 . 5h . Zoomed insets show two wells with different phenotypes. Well marked with ( # ) is representative of most strains, showing lysed cells. Well containing the late lysing strain Δ fkpB with intact cells. Scale bars correspond to 100 μm.

    Article Snippet: Briefly, strains were grown overnight in 96-well plates (Greiner, 96 Well Polystyrene Microplate, clear) containing LB with 30 μg/ml Kanamycin (MP Biomedicals), and then diluted 5000 times in a 96-well plate with glass bottom (Brooks Automation Ltd., Ref. MGB096-1-2-LG-L) containing fresh media.

    Techniques: High Throughput Screening Assay, Microscopy, Lysis

    Measuring β-lactam induced lysis kinetics by phase contrast microscopy. (A) Time-lapse analysis of wild-type cells on agar pads containing cephalexin 100 μg/ml. Cells immediately stop dividing after seeded on the cephalexin containing agar but continue to elongate. After growing to variable lengths, the cells develop a bulge (shown by red arrow heads) and lyse. Scale bar corresponds to 25 μm. (B) Graph shows wild-type E. coli cell counts at different time intervals after treatment with cephalexin 100 μg/ml at t = 0 in glass-bottom imaging microplates. Cell counts were determined simultaneously by image analysis (left axis in red) and by CFU counting on agar plates (right axis in blue). (C) Wild-type E. coli was treated with cephalexin 200 μg/ml in glass-bottom 96-well plates at different cell densities and cell counts were determined based on image analysis. All data points correspond to 3 replicates in (B,C) .

    Journal: Frontiers in Microbiology

    Article Title: Image-Based Dynamic Phenotyping Reveals Genetic Determinants of Filamentation-Mediated β-Lactam Tolerance

    doi: 10.3389/fmicb.2020.00374

    Figure Lengend Snippet: Measuring β-lactam induced lysis kinetics by phase contrast microscopy. (A) Time-lapse analysis of wild-type cells on agar pads containing cephalexin 100 μg/ml. Cells immediately stop dividing after seeded on the cephalexin containing agar but continue to elongate. After growing to variable lengths, the cells develop a bulge (shown by red arrow heads) and lyse. Scale bar corresponds to 25 μm. (B) Graph shows wild-type E. coli cell counts at different time intervals after treatment with cephalexin 100 μg/ml at t = 0 in glass-bottom imaging microplates. Cell counts were determined simultaneously by image analysis (left axis in red) and by CFU counting on agar plates (right axis in blue). (C) Wild-type E. coli was treated with cephalexin 200 μg/ml in glass-bottom 96-well plates at different cell densities and cell counts were determined based on image analysis. All data points correspond to 3 replicates in (B,C) .

    Article Snippet: Briefly, strains were grown overnight in 96-well plates (Greiner, 96 Well Polystyrene Microplate, clear) containing LB with 30 μg/ml Kanamycin (MP Biomedicals), and then diluted 5000 times in a 96-well plate with glass bottom (Brooks Automation Ltd., Ref. MGB096-1-2-LG-L) containing fresh media.

    Techniques: Lysis, Microscopy, Imaging

    Quantitative CRISPR/Cas9 editing strategy A) Tagging strategy to allow quantitation of PC-I in NIH3T3 fibroblasts with introduction of a multifunctional tag at the N-terminus of proα2(I) to allow detection of edited cells, and the small luciferase, NLuc. B) After introduction of the multifunctional tag in exon 1 of Col1a2 using CRISPR/Cas9, edited cells were sorted based on GFP fluorescence. C) PCR validation of edited DNA in cells isolated from B) using primers in Supplementary table 1 . D) Real-time PCR of total Col1a2 transcripts (Black) and edited transcripts in NIH3T3 and nluc::Col1a2 cells. Primers at the 5’ and 3’ ends of the introduced nluc confirmed insertion into the Col1a2 transcript. Bars show mean ± SD, n=3 independent experiments. E) Boxes show exon positions of the edited Col1a2 RNA transcript, sequencing of PCR products in D) demonstrate insertion of nluc in frame with Col1a2 . F) Boxes show exon positions of the edited Col1a2 RNA transcript and the amino acid sequence of the expected tagged protein. Conditioned medium from nluc::Col1a2 cells was affinity purified ( Supplementary Fig. 1 ) and subjected to mass spectrometry analysis. A peptide spanning NLuc and protein encoded by Col1a2 exons 1-3 was identified. G) In-gel detection of NLuc tagged proα2(I) chain under reducing conditions identified NLuc activity at approximately 140 kDa. H) Imaging and quantitation of the light produced by nluc::Col1a2 and unedited NIH3T3 cells incubated with the NLuc substrate, Furimazine (Nano-Glo). On a 96-well plate, single wells were imaged using a single-lens reflex camera and quantitation of photon counts using a multi-well plate luminometer. Bar shows means ± SD from n=30 replicate measurements. **** represents p=0.0001, paired Student’s t-Test.

    Journal: bioRxiv

    Article Title: Dynamic protein quantitation (DyProQ) of procollagen-I by CRISPR-Cas9 NanoLuciferase tagging

    doi: 10.1101/2020.05.17.099119

    Figure Lengend Snippet: Quantitative CRISPR/Cas9 editing strategy A) Tagging strategy to allow quantitation of PC-I in NIH3T3 fibroblasts with introduction of a multifunctional tag at the N-terminus of proα2(I) to allow detection of edited cells, and the small luciferase, NLuc. B) After introduction of the multifunctional tag in exon 1 of Col1a2 using CRISPR/Cas9, edited cells were sorted based on GFP fluorescence. C) PCR validation of edited DNA in cells isolated from B) using primers in Supplementary table 1 . D) Real-time PCR of total Col1a2 transcripts (Black) and edited transcripts in NIH3T3 and nluc::Col1a2 cells. Primers at the 5’ and 3’ ends of the introduced nluc confirmed insertion into the Col1a2 transcript. Bars show mean ± SD, n=3 independent experiments. E) Boxes show exon positions of the edited Col1a2 RNA transcript, sequencing of PCR products in D) demonstrate insertion of nluc in frame with Col1a2 . F) Boxes show exon positions of the edited Col1a2 RNA transcript and the amino acid sequence of the expected tagged protein. Conditioned medium from nluc::Col1a2 cells was affinity purified ( Supplementary Fig. 1 ) and subjected to mass spectrometry analysis. A peptide spanning NLuc and protein encoded by Col1a2 exons 1-3 was identified. G) In-gel detection of NLuc tagged proα2(I) chain under reducing conditions identified NLuc activity at approximately 140 kDa. H) Imaging and quantitation of the light produced by nluc::Col1a2 and unedited NIH3T3 cells incubated with the NLuc substrate, Furimazine (Nano-Glo). On a 96-well plate, single wells were imaged using a single-lens reflex camera and quantitation of photon counts using a multi-well plate luminometer. Bar shows means ± SD from n=30 replicate measurements. **** represents p=0.0001, paired Student’s t-Test.

    Article Snippet: Bioluminescence Imaging For bioluminescence imaging of recombinant NLuc and nluc::Col1a2 cells was performed in black walled μ-Plate 96-well plates (iBidi).

    Techniques: CRISPR, Quantitation Assay, Luciferase, Fluorescence, Polymerase Chain Reaction, Isolation, Real-time Polymerase Chain Reaction, Sequencing, Affinity Purification, Mass Spectrometry, Activity Assay, Imaging, Produced, Incubation

    Heatmaps of 96-well plates seeded with different numbers of nluc::Col1a2 cells. The colour scale (right) indicates the number of counts in each well. Three plates were compared, clear (A) , black (B) and white (C) . Cells were seed in triplicate as indicated. A) In clear plates the spill over of luminescence could be observed with large numbers of cells, indicated by significant luminescence being recorded in empty wells neighbouring wells containing cells. B) Black plates limited spill over of signal between wells but also suppressed luminescence in the wells. C) White plates limited spill over but also enhanced sensitivity of detection.

    Journal: bioRxiv

    Article Title: Dynamic protein quantitation (DyProQ) of procollagen-I by CRISPR-Cas9 NanoLuciferase tagging

    doi: 10.1101/2020.05.17.099119

    Figure Lengend Snippet: Heatmaps of 96-well plates seeded with different numbers of nluc::Col1a2 cells. The colour scale (right) indicates the number of counts in each well. Three plates were compared, clear (A) , black (B) and white (C) . Cells were seed in triplicate as indicated. A) In clear plates the spill over of luminescence could be observed with large numbers of cells, indicated by significant luminescence being recorded in empty wells neighbouring wells containing cells. B) Black plates limited spill over of signal between wells but also suppressed luminescence in the wells. C) White plates limited spill over but also enhanced sensitivity of detection.

    Article Snippet: Bioluminescence Imaging For bioluminescence imaging of recombinant NLuc and nluc::Col1a2 cells was performed in black walled μ-Plate 96-well plates (iBidi).

    Techniques: