96 well flat bottom plates  (Thermo Fisher)


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    96 Well Plate Individual
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    Thermo Scientific Abgene polypropylene microplates and deepwell plates offer storage security for assays compound libraries or storing samples for either intermediate or long term use Abgene microtiter plates are manufactured to exacting specifications in our Class 100 000 clean room ISO 9001 conditions using high quality medical grade virgin polypropylene resins which ensure confidence in the quality and performance of the storage plates Designed to ANSI standards these microplates and deepwell plates are compatible with a variety of automated liquid handling for high throughput workflows The polypropylene storage plates are offered with spindle pyramidal U and V bottom wells along with a wide range of volumes to accommodate most applications
    Catalog Number:
    ab0765
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    Structured Review

    Thermo Fisher 96 well flat bottom plates
    Effect of TPT on the proliferative response of bidirectional MLR and anti-CD3/CD28–stimulated CD4 + T cells. Allogeneic human PBMCs from 2 different donors (3 × 10 5 cells/well each) were cocultured in <t>96-well</t> flat-bottom plates in the presence
    Thermo Scientific Abgene polypropylene microplates and deepwell plates offer storage security for assays compound libraries or storing samples for either intermediate or long term use Abgene microtiter plates are manufactured to exacting specifications in our Class 100 000 clean room ISO 9001 conditions using high quality medical grade virgin polypropylene resins which ensure confidence in the quality and performance of the storage plates Designed to ANSI standards these microplates and deepwell plates are compatible with a variety of automated liquid handling for high throughput workflows The polypropylene storage plates are offered with spindle pyramidal U and V bottom wells along with a wide range of volumes to accommodate most applications
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    Images

    1) Product Images from "Triptolide, a constituent of immunosuppressive Chinese herbal medicine, is a potent suppressor of dendritic-cell maturation and trafficking"

    Article Title: Triptolide, a constituent of immunosuppressive Chinese herbal medicine, is a potent suppressor of dendritic-cell maturation and trafficking

    Journal:

    doi: 10.1182/blood-2005-03-0854

    Effect of TPT on the proliferative response of bidirectional MLR and anti-CD3/CD28–stimulated CD4 + T cells. Allogeneic human PBMCs from 2 different donors (3 × 10 5 cells/well each) were cocultured in 96-well flat-bottom plates in the presence
    Figure Legend Snippet: Effect of TPT on the proliferative response of bidirectional MLR and anti-CD3/CD28–stimulated CD4 + T cells. Allogeneic human PBMCs from 2 different donors (3 × 10 5 cells/well each) were cocultured in 96-well flat-bottom plates in the presence

    Techniques Used:

    2) Product Images from "Identification and characterization of noncalcemic, tissue-selective, nonsecosteroidal vitamin D receptor modulators"

    Article Title: Identification and characterization of noncalcemic, tissue-selective, nonsecosteroidal vitamin D receptor modulators

    Journal: Journal of Clinical Investigation

    doi: 10.1172/JCI25901

    Nonsecosteroidal VDR ligands are potent agonists in keratinocytes and PBMCs. ( A ) LY2108491 and LY2109866 are potent inhibitors of keratinocyte proliferation. KerTr cells plated in 96-well plates were dosed with various concentrations of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 72 hours at 37°C before BrdU incorporation into DNA was analyzed as a measure of cell proliferation. Results (mean α SEM) of experiments performed in triplicate are shown. ( B ) Nonsecosteroidal VDR ligands are potent inducers of CYP24 gene expression in keratinocytes. TaqMan quantitative RT-PCR (Q-PCR) was performed on total RNA prepared from KerTr cells treated with various concentrations of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 24 hours. Levels of GAPDH mRNA were measured in all the samples, and the results were normalized and presented as fold induction (α SEM) compared with normalized CYP24 levels in vehicle-treated cells. ( C ) Nonsecosteroidal VDR ligands are efficacious in TPA- and PHA-activated PBMCs. Primary cells isolated from donors were stimulated with TPA (100 ng/ml) and PHA (25 μl/ml) and treated with vehicle or 100 nM each of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 24 hours. TaqMan Q-PCR was performed on RNA obtained from vehicle-treated or VDR ligand–treated samples, using primer pairs and probes for IL-2, IL-4, IL-10, GATA3, and GAPDH. The amount of IL-2, IL-4, IL-10, and GATA3 transcripts relative to GAPDH transcripts is shown as mean α SEM of quadruplicate experimes.
    Figure Legend Snippet: Nonsecosteroidal VDR ligands are potent agonists in keratinocytes and PBMCs. ( A ) LY2108491 and LY2109866 are potent inhibitors of keratinocyte proliferation. KerTr cells plated in 96-well plates were dosed with various concentrations of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 72 hours at 37°C before BrdU incorporation into DNA was analyzed as a measure of cell proliferation. Results (mean α SEM) of experiments performed in triplicate are shown. ( B ) Nonsecosteroidal VDR ligands are potent inducers of CYP24 gene expression in keratinocytes. TaqMan quantitative RT-PCR (Q-PCR) was performed on total RNA prepared from KerTr cells treated with various concentrations of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 24 hours. Levels of GAPDH mRNA were measured in all the samples, and the results were normalized and presented as fold induction (α SEM) compared with normalized CYP24 levels in vehicle-treated cells. ( C ) Nonsecosteroidal VDR ligands are efficacious in TPA- and PHA-activated PBMCs. Primary cells isolated from donors were stimulated with TPA (100 ng/ml) and PHA (25 μl/ml) and treated with vehicle or 100 nM each of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 24 hours. TaqMan Q-PCR was performed on RNA obtained from vehicle-treated or VDR ligand–treated samples, using primer pairs and probes for IL-2, IL-4, IL-10, GATA3, and GAPDH. The amount of IL-2, IL-4, IL-10, and GATA3 transcripts relative to GAPDH transcripts is shown as mean α SEM of quadruplicate experimes.

    Techniques Used: BrdU Incorporation Assay, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, Isolation

    3) Product Images from "Effect of azithromycin on a red complex polymicrobial biofilm"

    Article Title: Effect of azithromycin on a red complex polymicrobial biofilm

    Journal: Journal of Oral Microbiology

    doi: 10.1080/20002297.2017.1339579

    Effect of azithromycin up to 5.0 mg/L on the red complex mono- and polymicrobial biofilms in a 96-well plate model. Azithromycin at concentrations 0–100 mg/L was incubated with bacterial cultures for 48 h under anaerobic conditions. Data points represent the mean AU 620 value of a minimum of three biological replicates. Note the categorical scale.
    Figure Legend Snippet: Effect of azithromycin up to 5.0 mg/L on the red complex mono- and polymicrobial biofilms in a 96-well plate model. Azithromycin at concentrations 0–100 mg/L was incubated with bacterial cultures for 48 h under anaerobic conditions. Data points represent the mean AU 620 value of a minimum of three biological replicates. Note the categorical scale.

    Techniques Used: Incubation

    Formation of mono- and polymicrobial biofilms in a 96-well plate model after 48 h of incubation at 37°C under anaerobic condition. Native bacterial growth with addition of uncultured growth medium and no antibiotic served as controls. Adherent biofilms were stained with 0.1% crystal violet and the optical density at AU 620 was measured. Data represent the mean AU 620 value of a minimum of three biological replicates.
    Figure Legend Snippet: Formation of mono- and polymicrobial biofilms in a 96-well plate model after 48 h of incubation at 37°C under anaerobic condition. Native bacterial growth with addition of uncultured growth medium and no antibiotic served as controls. Adherent biofilms were stained with 0.1% crystal violet and the optical density at AU 620 was measured. Data represent the mean AU 620 value of a minimum of three biological replicates.

    Techniques Used: Incubation, Staining

    Effects of azithromycin and amoxicillin + metronidazole (1:1 ratio) up to 5.0 mg/L on formation polymicrobial biofilms after 48 h of anaerobic incubation at 37°C in a 96-well plate model. Azithromycin and amoxicillin + metronidazole (1:1 ratio) at concentrations 0–100 mg/L were incubated with bacterial cultures. Data points represent the mean AU 620 value of a minimum of three biological replicates and the standard deviation. * p
    Figure Legend Snippet: Effects of azithromycin and amoxicillin + metronidazole (1:1 ratio) up to 5.0 mg/L on formation polymicrobial biofilms after 48 h of anaerobic incubation at 37°C in a 96-well plate model. Azithromycin and amoxicillin + metronidazole (1:1 ratio) at concentrations 0–100 mg/L were incubated with bacterial cultures. Data points represent the mean AU 620 value of a minimum of three biological replicates and the standard deviation. * p

    Techniques Used: Incubation, Standard Deviation

    Effect of amoxicillin + metronidazole up to 5.0 mg/L on the red complex mono- and polymicrobial biofilms in a 96-well plate model. Amoxicillin + metronidazole in a 1:1 ratio at concentrations 0–100 mg/L was incubated with bacterial cultures for 48 h at 37°C anaerobically. Data points represent the mean AU 620 value of a minimum of three biological replicates. Note the categorical scale.
    Figure Legend Snippet: Effect of amoxicillin + metronidazole up to 5.0 mg/L on the red complex mono- and polymicrobial biofilms in a 96-well plate model. Amoxicillin + metronidazole in a 1:1 ratio at concentrations 0–100 mg/L was incubated with bacterial cultures for 48 h at 37°C anaerobically. Data points represent the mean AU 620 value of a minimum of three biological replicates. Note the categorical scale.

    Techniques Used: Incubation

    4) Product Images from "Effect of azithromycin on a red complex polymicrobial biofilm"

    Article Title: Effect of azithromycin on a red complex polymicrobial biofilm

    Journal: Journal of Oral Microbiology

    doi: 10.1080/20002297.2017.1339579

    Effect of azithromycin up to 5.0 mg/L on the red complex mono- and polymicrobial biofilms in a 96-well plate model. Azithromycin at concentrations 0–100 mg/L was incubated with bacterial cultures for 48 h under anaerobic conditions. Data points represent the mean AU 620 value of a minimum of three biological replicates. Note the categorical scale.
    Figure Legend Snippet: Effect of azithromycin up to 5.0 mg/L on the red complex mono- and polymicrobial biofilms in a 96-well plate model. Azithromycin at concentrations 0–100 mg/L was incubated with bacterial cultures for 48 h under anaerobic conditions. Data points represent the mean AU 620 value of a minimum of three biological replicates. Note the categorical scale.

    Techniques Used: Incubation

    Formation of mono- and polymicrobial biofilms in a 96-well plate model after 48 h of incubation at 37°C under anaerobic condition. Native bacterial growth with addition of uncultured growth medium and no antibiotic served as controls. Adherent biofilms were stained with 0.1% crystal violet and the optical density at AU 620 was measured. Data represent the mean AU 620 value of a minimum of three biological replicates.
    Figure Legend Snippet: Formation of mono- and polymicrobial biofilms in a 96-well plate model after 48 h of incubation at 37°C under anaerobic condition. Native bacterial growth with addition of uncultured growth medium and no antibiotic served as controls. Adherent biofilms were stained with 0.1% crystal violet and the optical density at AU 620 was measured. Data represent the mean AU 620 value of a minimum of three biological replicates.

    Techniques Used: Incubation, Staining

    Effects of azithromycin and amoxicillin + metronidazole (1:1 ratio) up to 5.0 mg/L on formation polymicrobial biofilms after 48 h of anaerobic incubation at 37°C in a 96-well plate model. Azithromycin and amoxicillin + metronidazole (1:1 ratio) at concentrations 0–100 mg/L were incubated with bacterial cultures. Data points represent the mean AU 620 value of a minimum of three biological replicates and the standard deviation. * p
    Figure Legend Snippet: Effects of azithromycin and amoxicillin + metronidazole (1:1 ratio) up to 5.0 mg/L on formation polymicrobial biofilms after 48 h of anaerobic incubation at 37°C in a 96-well plate model. Azithromycin and amoxicillin + metronidazole (1:1 ratio) at concentrations 0–100 mg/L were incubated with bacterial cultures. Data points represent the mean AU 620 value of a minimum of three biological replicates and the standard deviation. * p

    Techniques Used: Incubation, Standard Deviation

    Effect of amoxicillin + metronidazole up to 5.0 mg/L on the red complex mono- and polymicrobial biofilms in a 96-well plate model. Amoxicillin + metronidazole in a 1:1 ratio at concentrations 0–100 mg/L was incubated with bacterial cultures for 48 h at 37°C anaerobically. Data points represent the mean AU 620 value of a minimum of three biological replicates. Note the categorical scale.
    Figure Legend Snippet: Effect of amoxicillin + metronidazole up to 5.0 mg/L on the red complex mono- and polymicrobial biofilms in a 96-well plate model. Amoxicillin + metronidazole in a 1:1 ratio at concentrations 0–100 mg/L was incubated with bacterial cultures for 48 h at 37°C anaerobically. Data points represent the mean AU 620 value of a minimum of three biological replicates. Note the categorical scale.

    Techniques Used: Incubation

    5) Product Images from "Intranasal Delivery of Cationic PLGA Nano/Microparticles- Loaded FMDV DNA Vaccine Encoding IL-6 Elicited Protective Immunity against FMDV Challenge"

    Article Title: Intranasal Delivery of Cationic PLGA Nano/Microparticles- Loaded FMDV DNA Vaccine Encoding IL-6 Elicited Protective Immunity against FMDV Challenge

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0027605

    Lymphocyte proliferation. Single lymphocyte suspensions were isolated from rats (n = 6) 7 days after the second immunization, plated in triplicate in a 96-well plate and stimulated in vitro for 48 h with inactivated FMDV, Con A (positive control) or with BSA. Means were compared by non-parametric ANOVA. Proliferation was analyzed using the MTT colorimetric assay and proliferation expressed as stimulated index. Significant results between pA-immunized animals and control groups are indicated by #.
    Figure Legend Snippet: Lymphocyte proliferation. Single lymphocyte suspensions were isolated from rats (n = 6) 7 days after the second immunization, plated in triplicate in a 96-well plate and stimulated in vitro for 48 h with inactivated FMDV, Con A (positive control) or with BSA. Means were compared by non-parametric ANOVA. Proliferation was analyzed using the MTT colorimetric assay and proliferation expressed as stimulated index. Significant results between pA-immunized animals and control groups are indicated by #.

    Techniques Used: Isolation, In Vitro, Positive Control, MTT Assay, Colorimetric Assay

    6) Product Images from "Activated neutrophils exert myeloid-derived suppressor cell activity damaging T cells beyond repair"

    Article Title: Activated neutrophils exert myeloid-derived suppressor cell activity damaging T cells beyond repair

    Journal: Blood Advances

    doi: 10.1182/bloodadvances.2019031609

    Activated neutrophils suppress T-cell proliferation. Purified T cells (either CD4 + or CD8 + ) were cultured in the presence or absence of anti-CD3 antibody or anti-CD28 antibody with unstimulated or fMLF-activated neutrophils (unless otherwise indicated). Cells were harvested after 5 to 6 days and analyzed by flow cytometry for CFSE dilution. (A) Representative fluorescence-activated cell sorting (FACS) plots of CFSE dilution of CD4 + T cells. (B) Quantification of CD4 + (left) and CD8 + (right) T-cell proliferation (n = 17). (C) Titration of the cell ratio with 4000 (5:1 ratio), 20 000 (1:1), 40 000 (1:2), 60 000 (1:3), 100 000 (1:5), or 160 000 (1:8) neutrophils per well of a 96-well plate (n = 3-17). (D) Purified T cells were cultured in the presence or absence of IL-15 with unstimulated or fMLF-activated neutrophils (n = 5). (E) Purified T cells were cultured with anti-CD3 and anti-CD28 antibodies (red bars), and in the presence of neutrophils (blue bars) and/or indicated stimuli. Three to 19 donors were tested in duplicate per stimulus. Error bars indicate standard error of the mean (SEM); **** P
    Figure Legend Snippet: Activated neutrophils suppress T-cell proliferation. Purified T cells (either CD4 + or CD8 + ) were cultured in the presence or absence of anti-CD3 antibody or anti-CD28 antibody with unstimulated or fMLF-activated neutrophils (unless otherwise indicated). Cells were harvested after 5 to 6 days and analyzed by flow cytometry for CFSE dilution. (A) Representative fluorescence-activated cell sorting (FACS) plots of CFSE dilution of CD4 + T cells. (B) Quantification of CD4 + (left) and CD8 + (right) T-cell proliferation (n = 17). (C) Titration of the cell ratio with 4000 (5:1 ratio), 20 000 (1:1), 40 000 (1:2), 60 000 (1:3), 100 000 (1:5), or 160 000 (1:8) neutrophils per well of a 96-well plate (n = 3-17). (D) Purified T cells were cultured in the presence or absence of IL-15 with unstimulated or fMLF-activated neutrophils (n = 5). (E) Purified T cells were cultured with anti-CD3 and anti-CD28 antibodies (red bars), and in the presence of neutrophils (blue bars) and/or indicated stimuli. Three to 19 donors were tested in duplicate per stimulus. Error bars indicate standard error of the mean (SEM); **** P

    Techniques Used: Purification, Cell Culture, Flow Cytometry, Cytometry, Fluorescence, FACS, Titration

    7) Product Images from "Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 in Vitro"

    Article Title: Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 in Vitro

    Journal: bioRxiv

    doi: 10.1101/2020.12.02.408112

    Potency of selected HCV PI against SARS-CoV-2 was confirmed in Huh7.5 cells. Huh7.5 cells were seeded in 96-well plates and the following day infected with SARS-CoV-2 followed by treatment with specified concentrations of the PI boceprevir, telaprevir and simeprevir, as described in Materials and Methods. After 70-74 hours incubation SARS-CoV-2 infected cells were visualized by immunostaining for the SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Datapoints (red dots) are means of 7 replicates ± SEM and represent % residual infectivity, determined as % SARS-CoV-2 positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration response curves (red lines) were fitted and EC50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay as described in Materials and Methods. Data points (blue triangles) are means of 3 replicate cultures ± SEM and represent % cell viability relative to mean absorbance of 12 nontreated controls. Sigmoidal concentration response curves were fitted and CC50 values were determined, as shown in Supplementary Figure 4. The blue stippled line represents the drug concentrations at which DMSO is expected to induce cytotoxicity with reduction of cell viability to
    Figure Legend Snippet: Potency of selected HCV PI against SARS-CoV-2 was confirmed in Huh7.5 cells. Huh7.5 cells were seeded in 96-well plates and the following day infected with SARS-CoV-2 followed by treatment with specified concentrations of the PI boceprevir, telaprevir and simeprevir, as described in Materials and Methods. After 70-74 hours incubation SARS-CoV-2 infected cells were visualized by immunostaining for the SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Datapoints (red dots) are means of 7 replicates ± SEM and represent % residual infectivity, determined as % SARS-CoV-2 positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration response curves (red lines) were fitted and EC50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay as described in Materials and Methods. Data points (blue triangles) are means of 3 replicate cultures ± SEM and represent % cell viability relative to mean absorbance of 12 nontreated controls. Sigmoidal concentration response curves were fitted and CC50 values were determined, as shown in Supplementary Figure 4. The blue stippled line represents the drug concentrations at which DMSO is expected to induce cytotoxicity with reduction of cell viability to

    Techniques Used: Infection, Incubation, Immunostaining, Concentration Assay, Colorimetric Assay

    Analysis of interactions of selected HCV PI with remdesivir. VeroE6 cells seeded in 96-well plates were infected the following day with SARS-CoV-2 followed by treatment with specified concentrations of the linear PI boceprevir (BOC) and narlaprevir (NAR), or the macrocyclic PI simeprevir (SIM), paritaprevir (PAR) and grazoprevir (GRA), or polymerase inhibitor remdesivir (REM), or a combination of these PI and remdesivir, as described in Materials and Methods. After 46-50 hours incubation SARS-CoV-2 infected cells were visualized by immunostaining for SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Fractional effect (Fa) values were calculated by relating counts from infected and treated cultures to the mean count from at least 21 infected nontreated cultures and were entered into CompuSyn software. Datapoints are means of 6 to 7 replicates, and for each treatment experiment 6 to 10 datapoints were entered. For each inhibitor combination depicted per row, the following curves were fitted using Compusyn: (A) concentration-Fa curves plotting Fa values ranging from 0.01 to 0.99 against specified inhibitor concentrations. (B) Fa-CI curves plotting CI values ranging from 0 to 2 against Fa values ranging from 0.01 to 0.99. (C) Fa-Log 10 CI curves plotting logarithmic CI values ranging from 0.01 to 100 against Fa values ranging from 0.01 to 0.99. (B and C) Overall, CI values ≥1.1 suggest antagonism “A”, while CI values
    Figure Legend Snippet: Analysis of interactions of selected HCV PI with remdesivir. VeroE6 cells seeded in 96-well plates were infected the following day with SARS-CoV-2 followed by treatment with specified concentrations of the linear PI boceprevir (BOC) and narlaprevir (NAR), or the macrocyclic PI simeprevir (SIM), paritaprevir (PAR) and grazoprevir (GRA), or polymerase inhibitor remdesivir (REM), or a combination of these PI and remdesivir, as described in Materials and Methods. After 46-50 hours incubation SARS-CoV-2 infected cells were visualized by immunostaining for SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Fractional effect (Fa) values were calculated by relating counts from infected and treated cultures to the mean count from at least 21 infected nontreated cultures and were entered into CompuSyn software. Datapoints are means of 6 to 7 replicates, and for each treatment experiment 6 to 10 datapoints were entered. For each inhibitor combination depicted per row, the following curves were fitted using Compusyn: (A) concentration-Fa curves plotting Fa values ranging from 0.01 to 0.99 against specified inhibitor concentrations. (B) Fa-CI curves plotting CI values ranging from 0 to 2 against Fa values ranging from 0.01 to 0.99. (C) Fa-Log 10 CI curves plotting logarithmic CI values ranging from 0.01 to 100 against Fa values ranging from 0.01 to 0.99. (B and C) Overall, CI values ≥1.1 suggest antagonism “A”, while CI values

    Techniques Used: Infection, Incubation, Immunostaining, Software, Concentration Assay

    Potency of a panel of HCV PI and an HCV NS4A inhibitor against SARS-CoV-2 in VeroE6 cells. VeroE6 cells were seeded in 96-well plates and the following day infected with SARS-CoV-2 followed by treatment with specified concentrations of the PI boceprevir, telaprevir, narlaprevir, simeprevir, paritaprevir, grazoprevir, glecaprevir, voxilaprevir, vaniprevir, danoprevir, deldeprevir, asunaprevir and faldaprevir, as well as HCV NS4A inhibitor ACH-806, as described in Materials and Methods. After 46-50 hours of incubation, SARS-CoV-2 infected cells were visualized by immunostaining for the SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Datapoints (red dots) are means of counts from 7 replicate cultures ± standard errors of the means (SEM) and represent % residual infectivity, determined as % SARS-CoV-2 positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration response curves (red lines) were fitted and EC50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay, as described in Materials and Methods. Datapoints (blue triangles) are means of 3 replicate cultures ± SEM and represent % cell viability relative to mean absorbance from 12 replicate nontreated control cultures. Sigmoidal concentration response curves were fitted and CC50 values were determined as shown in Supplementary Figure 3. The red / blue stippled line represents the drug concentrations at which DMSO is expected to induce antiviral effects with reduction of residual infectivity to
    Figure Legend Snippet: Potency of a panel of HCV PI and an HCV NS4A inhibitor against SARS-CoV-2 in VeroE6 cells. VeroE6 cells were seeded in 96-well plates and the following day infected with SARS-CoV-2 followed by treatment with specified concentrations of the PI boceprevir, telaprevir, narlaprevir, simeprevir, paritaprevir, grazoprevir, glecaprevir, voxilaprevir, vaniprevir, danoprevir, deldeprevir, asunaprevir and faldaprevir, as well as HCV NS4A inhibitor ACH-806, as described in Materials and Methods. After 46-50 hours of incubation, SARS-CoV-2 infected cells were visualized by immunostaining for the SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Datapoints (red dots) are means of counts from 7 replicate cultures ± standard errors of the means (SEM) and represent % residual infectivity, determined as % SARS-CoV-2 positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration response curves (red lines) were fitted and EC50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay, as described in Materials and Methods. Datapoints (blue triangles) are means of 3 replicate cultures ± SEM and represent % cell viability relative to mean absorbance from 12 replicate nontreated control cultures. Sigmoidal concentration response curves were fitted and CC50 values were determined as shown in Supplementary Figure 3. The red / blue stippled line represents the drug concentrations at which DMSO is expected to induce antiviral effects with reduction of residual infectivity to

    Techniques Used: Infection, Incubation, Immunostaining, Concentration Assay, Colorimetric Assay

    8) Product Images from "Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity"

    Article Title: Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity

    Journal: Journal of Immunology Research

    doi: 10.1155/2016/9540975

    The luciferase IVT RNA-based assay efficiently assesses mAb-induced ADCC and CDC of tumour cell lines. ADCC assay using (a) KATO-III and (b) NUGC-4 cells. KATO-III and NUGC-4 cells endogenously expressing hCLDN18.2 were transfected with 7 μ g luc2 IVT RNA and seeded into 96-well plates independently. 4 h later, IMAB 362 at different concentrations and human PBMCs (E : T ratio = 40 : 1) from 6 different donors were added to the target cells and incubated for 24 h. ADCC was determined 40 and 45 min after addition of D-luciferin substrate to the KATO-III and NUGC-4 cells, respectively. (c) CDC assay. CHO-K1 cells stably expressing hCLDN18.2 were transfected with 7 μ g luc2 IVT RNA and seeded into 96-well plates. 24 h later, cells were incubated for 80 min with IMAB 362 diluted in human serum (final concentration of 20%) from 6 different healthy donors. CDC was determined 45 min after addition of D-luciferin substrate. Results are the mean ± SD ( n = 3).
    Figure Legend Snippet: The luciferase IVT RNA-based assay efficiently assesses mAb-induced ADCC and CDC of tumour cell lines. ADCC assay using (a) KATO-III and (b) NUGC-4 cells. KATO-III and NUGC-4 cells endogenously expressing hCLDN18.2 were transfected with 7 μ g luc2 IVT RNA and seeded into 96-well plates independently. 4 h later, IMAB 362 at different concentrations and human PBMCs (E : T ratio = 40 : 1) from 6 different donors were added to the target cells and incubated for 24 h. ADCC was determined 40 and 45 min after addition of D-luciferin substrate to the KATO-III and NUGC-4 cells, respectively. (c) CDC assay. CHO-K1 cells stably expressing hCLDN18.2 were transfected with 7 μ g luc2 IVT RNA and seeded into 96-well plates. 24 h later, cells were incubated for 80 min with IMAB 362 diluted in human serum (final concentration of 20%) from 6 different healthy donors. CDC was determined 45 min after addition of D-luciferin substrate. Results are the mean ± SD ( n = 3).

    Techniques Used: Luciferase, ADCC Assay, Expressing, Transfection, Incubation, CDC Assay, Stable Transfection, Concentration Assay

    Electroporation of firefly luciferase IVT RNA into DCs and K562-A2 cells is nontoxic and leads to strong and long-lasting gene expression without affecting target cell phenotype. (a) Optimised luc2 reporter vector: composed of a gene-optimized synthetic firefly luciferase reporter gene cloned in front of two human β -globin 3′ untranslated regions (UTRs) fused head to tail and an unmasked free poly(A) tail of 120 bp. (b) Kinetics of luc2 expression in K562-A2 cells ( n = 1), human iDCs ( n = 3), and mDCs ( n = 3). Cells transfected with 8 pmol of luc2 -encoding IVT RNA were harvested at different time points to measure luminescence from 1 × 10 4 cells (Bright-Glo Luciferase Assay Kit for 96-well plates (Promega)). Results are the mean ± SD luminescence. Percent luminescence is relative to the highest luminescence signal obtained in each experiment. (c) Viability, reporter gene expression of iDCs and mDCs after eGFP and luc2 electroporation and phenotype after electroporation are depicted in descending order, respectively. iDCs (left panel) and mDCs (right panel) of 2 different donors were transfected with 10 μ g eGFP- or luc2 -encoding IVT RNA. Negative controls: cells electroporated without RNA (mock) and unelectroporated (no e'p) cells. Cells were harvested at different time points. Viability and HLA-DR, CD83, and eGFP expression levels were determined by flow cytometry. Luciferase activity of 1 × 10 4 viable cells was measured by luminescence in triplicate.
    Figure Legend Snippet: Electroporation of firefly luciferase IVT RNA into DCs and K562-A2 cells is nontoxic and leads to strong and long-lasting gene expression without affecting target cell phenotype. (a) Optimised luc2 reporter vector: composed of a gene-optimized synthetic firefly luciferase reporter gene cloned in front of two human β -globin 3′ untranslated regions (UTRs) fused head to tail and an unmasked free poly(A) tail of 120 bp. (b) Kinetics of luc2 expression in K562-A2 cells ( n = 1), human iDCs ( n = 3), and mDCs ( n = 3). Cells transfected with 8 pmol of luc2 -encoding IVT RNA were harvested at different time points to measure luminescence from 1 × 10 4 cells (Bright-Glo Luciferase Assay Kit for 96-well plates (Promega)). Results are the mean ± SD luminescence. Percent luminescence is relative to the highest luminescence signal obtained in each experiment. (c) Viability, reporter gene expression of iDCs and mDCs after eGFP and luc2 electroporation and phenotype after electroporation are depicted in descending order, respectively. iDCs (left panel) and mDCs (right panel) of 2 different donors were transfected with 10 μ g eGFP- or luc2 -encoding IVT RNA. Negative controls: cells electroporated without RNA (mock) and unelectroporated (no e'p) cells. Cells were harvested at different time points. Viability and HLA-DR, CD83, and eGFP expression levels were determined by flow cytometry. Luciferase activity of 1 × 10 4 viable cells was measured by luminescence in triplicate.

    Techniques Used: Electroporation, Luciferase, Expressing, Plasmid Preparation, Clone Assay, Transfection, Flow Cytometry, Cytometry, Activity Assay

    9) Product Images from "Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 in Vitro"

    Article Title: Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 in Vitro

    Journal: bioRxiv

    doi: 10.1101/2020.12.02.408112

    Potency of selected HCV PI against SARS-CoV-2 was confirmed in Huh7.5 cells. Huh7.5 cells were seeded in 96-well plates and the following day infected with SARS-CoV-2 followed by treatment with specified concentrations of the PI boceprevir, telaprevir and simeprevir, as described in Materials and Methods. After 70-74 hours incubation SARS-CoV-2 infected cells were visualized by immunostaining for the SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Datapoints (red dots) are means of 7 replicates ± SEM and represent % residual infectivity, determined as % SARS-CoV-2 positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration response curves (red lines) were fitted and EC50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay as described in Materials and Methods. Data points (blue triangles) are means of 3 replicate cultures ± SEM and represent % cell viability relative to mean absorbance of 12 nontreated controls. Sigmoidal concentration response curves were fitted and CC50 values were determined, as shown in Supplementary Figure 4. The blue stippled line represents the drug concentrations at which DMSO is expected to induce cytotoxicity with reduction of cell viability to
    Figure Legend Snippet: Potency of selected HCV PI against SARS-CoV-2 was confirmed in Huh7.5 cells. Huh7.5 cells were seeded in 96-well plates and the following day infected with SARS-CoV-2 followed by treatment with specified concentrations of the PI boceprevir, telaprevir and simeprevir, as described in Materials and Methods. After 70-74 hours incubation SARS-CoV-2 infected cells were visualized by immunostaining for the SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Datapoints (red dots) are means of 7 replicates ± SEM and represent % residual infectivity, determined as % SARS-CoV-2 positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration response curves (red lines) were fitted and EC50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay as described in Materials and Methods. Data points (blue triangles) are means of 3 replicate cultures ± SEM and represent % cell viability relative to mean absorbance of 12 nontreated controls. Sigmoidal concentration response curves were fitted and CC50 values were determined, as shown in Supplementary Figure 4. The blue stippled line represents the drug concentrations at which DMSO is expected to induce cytotoxicity with reduction of cell viability to

    Techniques Used: Infection, Incubation, Immunostaining, Concentration Assay, Colorimetric Assay

    Analysis of interactions of selected HCV PI with remdesivir. VeroE6 cells seeded in 96-well plates were infected the following day with SARS-CoV-2 followed by treatment with specified concentrations of the linear PI boceprevir (BOC) and narlaprevir (NAR), or the macrocyclic PI simeprevir (SIM), paritaprevir (PAR) and grazoprevir (GRA), or polymerase inhibitor remdesivir (REM), or a combination of these PI and remdesivir, as described in Materials and Methods. After 46-50 hours incubation SARS-CoV-2 infected cells were visualized by immunostaining for SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Fractional effect (Fa) values were calculated by relating counts from infected and treated cultures to the mean count from at least 21 infected nontreated cultures and were entered into CompuSyn software. Datapoints are means of 6 to 7 replicates, and for each treatment experiment 6 to 10 datapoints were entered. For each inhibitor combination depicted per row, the following curves were fitted using Compusyn: (A) concentration-Fa curves plotting Fa values ranging from 0.01 to 0.99 against specified inhibitor concentrations. (B) Fa-CI curves plotting CI values ranging from 0 to 2 against Fa values ranging from 0.01 to 0.99. (C) Fa-Log 10 CI curves plotting logarithmic CI values ranging from 0.01 to 100 against Fa values ranging from 0.01 to 0.99. (B and C) Overall, CI values ≥1.1 suggest antagonism “A”, while CI values
    Figure Legend Snippet: Analysis of interactions of selected HCV PI with remdesivir. VeroE6 cells seeded in 96-well plates were infected the following day with SARS-CoV-2 followed by treatment with specified concentrations of the linear PI boceprevir (BOC) and narlaprevir (NAR), or the macrocyclic PI simeprevir (SIM), paritaprevir (PAR) and grazoprevir (GRA), or polymerase inhibitor remdesivir (REM), or a combination of these PI and remdesivir, as described in Materials and Methods. After 46-50 hours incubation SARS-CoV-2 infected cells were visualized by immunostaining for SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Fractional effect (Fa) values were calculated by relating counts from infected and treated cultures to the mean count from at least 21 infected nontreated cultures and were entered into CompuSyn software. Datapoints are means of 6 to 7 replicates, and for each treatment experiment 6 to 10 datapoints were entered. For each inhibitor combination depicted per row, the following curves were fitted using Compusyn: (A) concentration-Fa curves plotting Fa values ranging from 0.01 to 0.99 against specified inhibitor concentrations. (B) Fa-CI curves plotting CI values ranging from 0 to 2 against Fa values ranging from 0.01 to 0.99. (C) Fa-Log 10 CI curves plotting logarithmic CI values ranging from 0.01 to 100 against Fa values ranging from 0.01 to 0.99. (B and C) Overall, CI values ≥1.1 suggest antagonism “A”, while CI values

    Techniques Used: Infection, Incubation, Immunostaining, Software, Concentration Assay

    Potency of a panel of HCV PI and an HCV NS4A inhibitor against SARS-CoV-2 in VeroE6 cells. VeroE6 cells were seeded in 96-well plates and the following day infected with SARS-CoV-2 followed by treatment with specified concentrations of the PI boceprevir, telaprevir, narlaprevir, simeprevir, paritaprevir, grazoprevir, glecaprevir, voxilaprevir, vaniprevir, danoprevir, deldeprevir, asunaprevir and faldaprevir, as well as HCV NS4A inhibitor ACH-806, as described in Materials and Methods. After 46-50 hours of incubation, SARS-CoV-2 infected cells were visualized by immunostaining for the SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Datapoints (red dots) are means of counts from 7 replicate cultures ± standard errors of the means (SEM) and represent % residual infectivity, determined as % SARS-CoV-2 positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration response curves (red lines) were fitted and EC50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay, as described in Materials and Methods. Datapoints (blue triangles) are means of 3 replicate cultures ± SEM and represent % cell viability relative to mean absorbance from 12 replicate nontreated control cultures. Sigmoidal concentration response curves were fitted and CC50 values were determined as shown in Supplementary Figure 3. The red / blue stippled line represents the drug concentrations at which DMSO is expected to induce antiviral effects with reduction of residual infectivity to
    Figure Legend Snippet: Potency of a panel of HCV PI and an HCV NS4A inhibitor against SARS-CoV-2 in VeroE6 cells. VeroE6 cells were seeded in 96-well plates and the following day infected with SARS-CoV-2 followed by treatment with specified concentrations of the PI boceprevir, telaprevir, narlaprevir, simeprevir, paritaprevir, grazoprevir, glecaprevir, voxilaprevir, vaniprevir, danoprevir, deldeprevir, asunaprevir and faldaprevir, as well as HCV NS4A inhibitor ACH-806, as described in Materials and Methods. After 46-50 hours of incubation, SARS-CoV-2 infected cells were visualized by immunostaining for the SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Datapoints (red dots) are means of counts from 7 replicate cultures ± standard errors of the means (SEM) and represent % residual infectivity, determined as % SARS-CoV-2 positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration response curves (red lines) were fitted and EC50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay, as described in Materials and Methods. Datapoints (blue triangles) are means of 3 replicate cultures ± SEM and represent % cell viability relative to mean absorbance from 12 replicate nontreated control cultures. Sigmoidal concentration response curves were fitted and CC50 values were determined as shown in Supplementary Figure 3. The red / blue stippled line represents the drug concentrations at which DMSO is expected to induce antiviral effects with reduction of residual infectivity to

    Techniques Used: Infection, Incubation, Immunostaining, Concentration Assay, Colorimetric Assay

    10) Product Images from "Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 in Vitro"

    Article Title: Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 in Vitro

    Journal: bioRxiv

    doi: 10.1101/2020.12.02.408112

    Potency of selected HCV PI against SARS-CoV-2 was confirmed in Huh7.5 cells. Huh7.5 cells were seeded in 96-well plates and the following day infected with SARS-CoV-2 followed by treatment with specified concentrations of the PI boceprevir, telaprevir and simeprevir, as described in Materials and Methods. After 70-74 hours incubation SARS-CoV-2 infected cells were visualized by immunostaining for the SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Datapoints (red dots) are means of 7 replicates ± SEM and represent % residual infectivity, determined as % SARS-CoV-2 positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration response curves (red lines) were fitted and EC50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay as described in Materials and Methods. Data points (blue triangles) are means of 3 replicate cultures ± SEM and represent % cell viability relative to mean absorbance of 12 nontreated controls. Sigmoidal concentration response curves were fitted and CC50 values were determined, as shown in Supplementary Figure 4. The blue stippled line represents the drug concentrations at which DMSO is expected to induce cytotoxicity with reduction of cell viability to
    Figure Legend Snippet: Potency of selected HCV PI against SARS-CoV-2 was confirmed in Huh7.5 cells. Huh7.5 cells were seeded in 96-well plates and the following day infected with SARS-CoV-2 followed by treatment with specified concentrations of the PI boceprevir, telaprevir and simeprevir, as described in Materials and Methods. After 70-74 hours incubation SARS-CoV-2 infected cells were visualized by immunostaining for the SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Datapoints (red dots) are means of 7 replicates ± SEM and represent % residual infectivity, determined as % SARS-CoV-2 positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration response curves (red lines) were fitted and EC50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay as described in Materials and Methods. Data points (blue triangles) are means of 3 replicate cultures ± SEM and represent % cell viability relative to mean absorbance of 12 nontreated controls. Sigmoidal concentration response curves were fitted and CC50 values were determined, as shown in Supplementary Figure 4. The blue stippled line represents the drug concentrations at which DMSO is expected to induce cytotoxicity with reduction of cell viability to

    Techniques Used: Infection, Incubation, Immunostaining, Concentration Assay, Colorimetric Assay

    Analysis of interactions of selected HCV PI with remdesivir. VeroE6 cells seeded in 96-well plates were infected the following day with SARS-CoV-2 followed by treatment with specified concentrations of the linear PI boceprevir (BOC) and narlaprevir (NAR), or the macrocyclic PI simeprevir (SIM), paritaprevir (PAR) and grazoprevir (GRA), or polymerase inhibitor remdesivir (REM), or a combination of these PI and remdesivir, as described in Materials and Methods. After 46-50 hours incubation SARS-CoV-2 infected cells were visualized by immunostaining for SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Fractional effect (Fa) values were calculated by relating counts from infected and treated cultures to the mean count from at least 21 infected nontreated cultures and were entered into CompuSyn software. Datapoints are means of 6 to 7 replicates, and for each treatment experiment 6 to 10 datapoints were entered. For each inhibitor combination depicted per row, the following curves were fitted using Compusyn: (A) concentration-Fa curves plotting Fa values ranging from 0.01 to 0.99 against specified inhibitor concentrations. (B) Fa-CI curves plotting CI values ranging from 0 to 2 against Fa values ranging from 0.01 to 0.99. (C) Fa-Log 10 CI curves plotting logarithmic CI values ranging from 0.01 to 100 against Fa values ranging from 0.01 to 0.99. (B and C) Overall, CI values ≥1.1 suggest antagonism “A”, while CI values
    Figure Legend Snippet: Analysis of interactions of selected HCV PI with remdesivir. VeroE6 cells seeded in 96-well plates were infected the following day with SARS-CoV-2 followed by treatment with specified concentrations of the linear PI boceprevir (BOC) and narlaprevir (NAR), or the macrocyclic PI simeprevir (SIM), paritaprevir (PAR) and grazoprevir (GRA), or polymerase inhibitor remdesivir (REM), or a combination of these PI and remdesivir, as described in Materials and Methods. After 46-50 hours incubation SARS-CoV-2 infected cells were visualized by immunostaining for SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Fractional effect (Fa) values were calculated by relating counts from infected and treated cultures to the mean count from at least 21 infected nontreated cultures and were entered into CompuSyn software. Datapoints are means of 6 to 7 replicates, and for each treatment experiment 6 to 10 datapoints were entered. For each inhibitor combination depicted per row, the following curves were fitted using Compusyn: (A) concentration-Fa curves plotting Fa values ranging from 0.01 to 0.99 against specified inhibitor concentrations. (B) Fa-CI curves plotting CI values ranging from 0 to 2 against Fa values ranging from 0.01 to 0.99. (C) Fa-Log 10 CI curves plotting logarithmic CI values ranging from 0.01 to 100 against Fa values ranging from 0.01 to 0.99. (B and C) Overall, CI values ≥1.1 suggest antagonism “A”, while CI values

    Techniques Used: Infection, Incubation, Immunostaining, Software, Concentration Assay

    Potency of a panel of HCV PI and an HCV NS4A inhibitor against SARS-CoV-2 in VeroE6 cells. VeroE6 cells were seeded in 96-well plates and the following day infected with SARS-CoV-2 followed by treatment with specified concentrations of the PI boceprevir, telaprevir, narlaprevir, simeprevir, paritaprevir, grazoprevir, glecaprevir, voxilaprevir, vaniprevir, danoprevir, deldeprevir, asunaprevir and faldaprevir, as well as HCV NS4A inhibitor ACH-806, as described in Materials and Methods. After 46-50 hours of incubation, SARS-CoV-2 infected cells were visualized by immunostaining for the SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Datapoints (red dots) are means of counts from 7 replicate cultures ± standard errors of the means (SEM) and represent % residual infectivity, determined as % SARS-CoV-2 positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration response curves (red lines) were fitted and EC50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay, as described in Materials and Methods. Datapoints (blue triangles) are means of 3 replicate cultures ± SEM and represent % cell viability relative to mean absorbance from 12 replicate nontreated control cultures. Sigmoidal concentration response curves were fitted and CC50 values were determined as shown in Supplementary Figure 3. The red / blue stippled line represents the drug concentrations at which DMSO is expected to induce antiviral effects with reduction of residual infectivity to
    Figure Legend Snippet: Potency of a panel of HCV PI and an HCV NS4A inhibitor against SARS-CoV-2 in VeroE6 cells. VeroE6 cells were seeded in 96-well plates and the following day infected with SARS-CoV-2 followed by treatment with specified concentrations of the PI boceprevir, telaprevir, narlaprevir, simeprevir, paritaprevir, grazoprevir, glecaprevir, voxilaprevir, vaniprevir, danoprevir, deldeprevir, asunaprevir and faldaprevir, as well as HCV NS4A inhibitor ACH-806, as described in Materials and Methods. After 46-50 hours of incubation, SARS-CoV-2 infected cells were visualized by immunostaining for the SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Datapoints (red dots) are means of counts from 7 replicate cultures ± standard errors of the means (SEM) and represent % residual infectivity, determined as % SARS-CoV-2 positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration response curves (red lines) were fitted and EC50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay, as described in Materials and Methods. Datapoints (blue triangles) are means of 3 replicate cultures ± SEM and represent % cell viability relative to mean absorbance from 12 replicate nontreated control cultures. Sigmoidal concentration response curves were fitted and CC50 values were determined as shown in Supplementary Figure 3. The red / blue stippled line represents the drug concentrations at which DMSO is expected to induce antiviral effects with reduction of residual infectivity to

    Techniques Used: Infection, Incubation, Immunostaining, Concentration Assay, Colorimetric Assay

    11) Product Images from "Serum Amyloid P Component Bound to Gram-Negative Bacteria Prevents Lipopolysaccharide-Mediated Classical Pathway Complement Activation"

    Article Title: Serum Amyloid P Component Bound to Gram-Negative Bacteria Prevents Lipopolysaccharide-Mediated Classical Pathway Complement Activation

    Journal: Infection and Immunity

    doi:

    SAP inhibits the lysis of serovar Copenhagen Re by serum. Log phase-grown serovar Copenhagen Re was incubated overnight in HBSS–0.2% albumin–10% Mueller-Hinton broth in different concentrations of SAP − serum in the absence or presence of SAP (20 μg/ml) at 37°C in 96-well flat-bottom plates. The increase in optical density at 665 nm (OD 665 ) was used to determine bacterial growth. Data are means for three separate experiments ± SEM.
    Figure Legend Snippet: SAP inhibits the lysis of serovar Copenhagen Re by serum. Log phase-grown serovar Copenhagen Re was incubated overnight in HBSS–0.2% albumin–10% Mueller-Hinton broth in different concentrations of SAP − serum in the absence or presence of SAP (20 μg/ml) at 37°C in 96-well flat-bottom plates. The increase in optical density at 665 nm (OD 665 ) was used to determine bacterial growth. Data are means for three separate experiments ± SEM.

    Techniques Used: Lysis, Incubation

    12) Product Images from "Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 in Vitro"

    Article Title: Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 in Vitro

    Journal: bioRxiv

    doi: 10.1101/2020.12.02.408112

    Potency of selected HCV PI against SARS-CoV-2 was confirmed in Huh7.5 cells. Huh7.5 cells were seeded in 96-well plates and the following day infected with SARS-CoV-2 followed by treatment with specified concentrations of the PI boceprevir, telaprevir and simeprevir, as described in Materials and Methods. After 70-74 hours incubation SARS-CoV-2 infected cells were visualized by immunostaining for the SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Datapoints (red dots) are means of 7 replicates ± SEM and represent % residual infectivity, determined as % SARS-CoV-2 positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration response curves (red lines) were fitted and EC50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay as described in Materials and Methods. Data points (blue triangles) are means of 3 replicate cultures ± SEM and represent % cell viability relative to mean absorbance of 12 nontreated controls. Sigmoidal concentration response curves were fitted and CC50 values were determined, as shown in Supplementary Figure 4. The blue stippled line represents the drug concentrations at which DMSO is expected to induce cytotoxicity with reduction of cell viability to
    Figure Legend Snippet: Potency of selected HCV PI against SARS-CoV-2 was confirmed in Huh7.5 cells. Huh7.5 cells were seeded in 96-well plates and the following day infected with SARS-CoV-2 followed by treatment with specified concentrations of the PI boceprevir, telaprevir and simeprevir, as described in Materials and Methods. After 70-74 hours incubation SARS-CoV-2 infected cells were visualized by immunostaining for the SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Datapoints (red dots) are means of 7 replicates ± SEM and represent % residual infectivity, determined as % SARS-CoV-2 positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration response curves (red lines) were fitted and EC50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay as described in Materials and Methods. Data points (blue triangles) are means of 3 replicate cultures ± SEM and represent % cell viability relative to mean absorbance of 12 nontreated controls. Sigmoidal concentration response curves were fitted and CC50 values were determined, as shown in Supplementary Figure 4. The blue stippled line represents the drug concentrations at which DMSO is expected to induce cytotoxicity with reduction of cell viability to

    Techniques Used: Infection, Incubation, Immunostaining, Concentration Assay, Colorimetric Assay

    Analysis of interactions of selected HCV PI with remdesivir. VeroE6 cells seeded in 96-well plates were infected the following day with SARS-CoV-2 followed by treatment with specified concentrations of the linear PI boceprevir (BOC) and narlaprevir (NAR), or the macrocyclic PI simeprevir (SIM), paritaprevir (PAR) and grazoprevir (GRA), or polymerase inhibitor remdesivir (REM), or a combination of these PI and remdesivir, as described in Materials and Methods. After 46-50 hours incubation SARS-CoV-2 infected cells were visualized by immunostaining for SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Fractional effect (Fa) values were calculated by relating counts from infected and treated cultures to the mean count from at least 21 infected nontreated cultures and were entered into CompuSyn software. Datapoints are means of 6 to 7 replicates, and for each treatment experiment 6 to 10 datapoints were entered. For each inhibitor combination depicted per row, the following curves were fitted using Compusyn: (A) concentration-Fa curves plotting Fa values ranging from 0.01 to 0.99 against specified inhibitor concentrations. (B) Fa-CI curves plotting CI values ranging from 0 to 2 against Fa values ranging from 0.01 to 0.99. (C) Fa-Log 10 CI curves plotting logarithmic CI values ranging from 0.01 to 100 against Fa values ranging from 0.01 to 0.99. (B and C) Overall, CI values ≥1.1 suggest antagonism “A”, while CI values
    Figure Legend Snippet: Analysis of interactions of selected HCV PI with remdesivir. VeroE6 cells seeded in 96-well plates were infected the following day with SARS-CoV-2 followed by treatment with specified concentrations of the linear PI boceprevir (BOC) and narlaprevir (NAR), or the macrocyclic PI simeprevir (SIM), paritaprevir (PAR) and grazoprevir (GRA), or polymerase inhibitor remdesivir (REM), or a combination of these PI and remdesivir, as described in Materials and Methods. After 46-50 hours incubation SARS-CoV-2 infected cells were visualized by immunostaining for SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Fractional effect (Fa) values were calculated by relating counts from infected and treated cultures to the mean count from at least 21 infected nontreated cultures and were entered into CompuSyn software. Datapoints are means of 6 to 7 replicates, and for each treatment experiment 6 to 10 datapoints were entered. For each inhibitor combination depicted per row, the following curves were fitted using Compusyn: (A) concentration-Fa curves plotting Fa values ranging from 0.01 to 0.99 against specified inhibitor concentrations. (B) Fa-CI curves plotting CI values ranging from 0 to 2 against Fa values ranging from 0.01 to 0.99. (C) Fa-Log 10 CI curves plotting logarithmic CI values ranging from 0.01 to 100 against Fa values ranging from 0.01 to 0.99. (B and C) Overall, CI values ≥1.1 suggest antagonism “A”, while CI values

    Techniques Used: Infection, Incubation, Immunostaining, Software, Concentration Assay

    Potency of a panel of HCV PI and an HCV NS4A inhibitor against SARS-CoV-2 in VeroE6 cells. VeroE6 cells were seeded in 96-well plates and the following day infected with SARS-CoV-2 followed by treatment with specified concentrations of the PI boceprevir, telaprevir, narlaprevir, simeprevir, paritaprevir, grazoprevir, glecaprevir, voxilaprevir, vaniprevir, danoprevir, deldeprevir, asunaprevir and faldaprevir, as well as HCV NS4A inhibitor ACH-806, as described in Materials and Methods. After 46-50 hours of incubation, SARS-CoV-2 infected cells were visualized by immunostaining for the SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Datapoints (red dots) are means of counts from 7 replicate cultures ± standard errors of the means (SEM) and represent % residual infectivity, determined as % SARS-CoV-2 positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration response curves (red lines) were fitted and EC50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay, as described in Materials and Methods. Datapoints (blue triangles) are means of 3 replicate cultures ± SEM and represent % cell viability relative to mean absorbance from 12 replicate nontreated control cultures. Sigmoidal concentration response curves were fitted and CC50 values were determined as shown in Supplementary Figure 3. The red / blue stippled line represents the drug concentrations at which DMSO is expected to induce antiviral effects with reduction of residual infectivity to
    Figure Legend Snippet: Potency of a panel of HCV PI and an HCV NS4A inhibitor against SARS-CoV-2 in VeroE6 cells. VeroE6 cells were seeded in 96-well plates and the following day infected with SARS-CoV-2 followed by treatment with specified concentrations of the PI boceprevir, telaprevir, narlaprevir, simeprevir, paritaprevir, grazoprevir, glecaprevir, voxilaprevir, vaniprevir, danoprevir, deldeprevir, asunaprevir and faldaprevir, as well as HCV NS4A inhibitor ACH-806, as described in Materials and Methods. After 46-50 hours of incubation, SARS-CoV-2 infected cells were visualized by immunostaining for the SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Datapoints (red dots) are means of counts from 7 replicate cultures ± standard errors of the means (SEM) and represent % residual infectivity, determined as % SARS-CoV-2 positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration response curves (red lines) were fitted and EC50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay, as described in Materials and Methods. Datapoints (blue triangles) are means of 3 replicate cultures ± SEM and represent % cell viability relative to mean absorbance from 12 replicate nontreated control cultures. Sigmoidal concentration response curves were fitted and CC50 values were determined as shown in Supplementary Figure 3. The red / blue stippled line represents the drug concentrations at which DMSO is expected to induce antiviral effects with reduction of residual infectivity to

    Techniques Used: Infection, Incubation, Immunostaining, Concentration Assay, Colorimetric Assay

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    Purification:

    Article Title: Triptolide, a constituent of immunosuppressive Chinese herbal medicine, is a potent suppressor of dendritic-cell maturation and trafficking
    Article Snippet: .. Purified CD4+ T cells (105 /well) were cultured in 96-well flat-bottom plates with CD3/CD28 T-cell expansion Dynabeads (5 × 103 /well; Dynal ASA, Oslo, Norway). ..

    Enzyme-linked Immunosorbent Assay:

    Article Title: Role for Bcl-xL as an inhibitor of cytosolic cytochrome C accumulation in DNA damage-induced apoptosis
    Article Snippet: .. Cytochrome C was adsorbed onto the plastic surface of a 96-well ELISA plate, followed by addition of blocking buffer (Superblock, Pierce) to reduce nonspecific binding. ..

    Article Title: Intranasal Delivery of Cationic PLGA Nano/Microparticles- Loaded FMDV DNA Vaccine Encoding IL-6 Elicited Protective Immunity against FMDV Challenge
    Article Snippet: .. Lysed cells normalized to the total protein content were added to 96-well flat-bottom plates and detection of IL-6 by ELISA carried out as described by the bovine IL-6 kit instruction (Thermo Fisher) just expressing the IL-6 titer from transfected cells in optical density (OD). .. Preparation of plasmid DNA (pDNA)-adsorbed CHT-PLGA Nanoparticles were prepared using the emulsion-diffusion-evaporation technique .

    Incubation:

    Article Title: Identification and characterization of noncalcemic, tissue-selective, nonsecosteroidal vitamin D receptor modulators
    Article Snippet: .. KerTr cells (human transformed skin keratinocyte, obtained from ATCC) were plated in 96-well flat-bottom plates (3000 cells per well) in 100 μl keratinocyte serum-free medium supplemented with bovine pituitary extract in the absence of EGF (Invitrogen Corp.) and incubated at 37°C for 2 days. .. The cells were treated with various concentrations of VDR ligands in triplicate, dissolved in 100 μl keratinocyte serum-free medium supplemented with bovine pituitary extract in the absence of EGF, and incubated at 37°C for 72 hours.

    other:

    Article Title: Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors
    Article Snippet: Cells were pretreated with DMSO or MB or 25 μg/ml pembrolizumab or 20 μg/ml nivolumab for 1 h and then seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28, with media supplemented with 10 μg/ml of hPD‐L1 protein.

    Blocking Assay:

    Article Title: Role for Bcl-xL as an inhibitor of cytosolic cytochrome C accumulation in DNA damage-induced apoptosis
    Article Snippet: .. Cytochrome C was adsorbed onto the plastic surface of a 96-well ELISA plate, followed by addition of blocking buffer (Superblock, Pierce) to reduce nonspecific binding. ..

    Expressing:

    Article Title: Intranasal Delivery of Cationic PLGA Nano/Microparticles- Loaded FMDV DNA Vaccine Encoding IL-6 Elicited Protective Immunity against FMDV Challenge
    Article Snippet: .. Lysed cells normalized to the total protein content were added to 96-well flat-bottom plates and detection of IL-6 by ELISA carried out as described by the bovine IL-6 kit instruction (Thermo Fisher) just expressing the IL-6 titer from transfected cells in optical density (OD). .. Preparation of plasmid DNA (pDNA)-adsorbed CHT-PLGA Nanoparticles were prepared using the emulsion-diffusion-evaporation technique .

    Transformation Assay:

    Article Title: Identification and characterization of noncalcemic, tissue-selective, nonsecosteroidal vitamin D receptor modulators
    Article Snippet: .. KerTr cells (human transformed skin keratinocyte, obtained from ATCC) were plated in 96-well flat-bottom plates (3000 cells per well) in 100 μl keratinocyte serum-free medium supplemented with bovine pituitary extract in the absence of EGF (Invitrogen Corp.) and incubated at 37°C for 2 days. .. The cells were treated with various concentrations of VDR ligands in triplicate, dissolved in 100 μl keratinocyte serum-free medium supplemented with bovine pituitary extract in the absence of EGF, and incubated at 37°C for 72 hours.

    Recombinant:

    Article Title: Generation and characterization of monoclonal antibodies against the N-terminus of alpha-2-antiplasmin
    Article Snippet: .. 96-well microtiter plates (Nunc A/S, Roskilde, Denmark) were coated with 2 μg/ml (200 μl per well in PBS containing 137 mM NaCl, 2.7 mM KCL, 6.5 mM Na2 HPO4 and 1 mM KH2 PO4 (pH 7.4)) of recombinant Met(R6)-α2AP, Met(W6)-α2AP or Asn-α2AP for 24 hours at 4°C. .. Non-specific sites were blocked with 200 μl 1% bovine serum albumin (Sigma, Steinheim, Germany) in PBS and incubated for two hours at RT.

    Plasmid Preparation:

    Article Title: Inhibition of Pyrimidine Biosynthesis Pathway Suppresses Viral Growth through Innate Immunity
    Article Snippet: .. For one 96-well culture plate, 17 µg of plasmid were diluted in 500 µl of DMEM (Gibco-Invitrogen). .. In parallel, 53 µg of poly(ethyleneimine) from Sigma-Aldrich (PEI) were diluted in 500 µl of DMEM (Gibco-BRL).

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  • 99
    Thermo Fisher 96 well flat bottomed microtiter plates
    Carvacrol influences the ATP-recovery effect on Vac-induced cell death Semi-confluent #12537-GB cells (seeded in <t>96-well</t> flat-bottomed <t>microtiter</t> plates) were treated with DMSO diluted in medium at 10 −4 served as control, Vac (7 μM), Vac+ATP or ATP without carvacrol or with 50 μM and 100 μM carvacrol. PI-positive (dead) cells are given as RCU (y-axis, RCU×μM 2 /image). Glioma cells were followed for 24 h (x-axis) (IncuCyteZOOM®) (A, B) . Quantitative analysis at 8 h for glioma cell lines (* in A and B) (C) . Vac+ATP vs. Vac+ATP+carvacrol 100 μM (multiple comparison two way ANOVA, p=0.02). All values are given as means ± SD (6× replicates). All imaging was performed using IncuCyteZOOM® at 10× objective. Results are representative of 3 independent experiments.
    96 Well Flat Bottomed Microtiter Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/96 well flat bottomed microtiter plates/product/Thermo Fisher
    Average 99 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    96 well flat bottomed microtiter plates - by Bioz Stars, 2021-01
    99/100 stars
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    91
    Thermo Fisher 96 well flat bottom uv transparent plate
    Changes in PANC-1 cell aggregate volume following exposure to 3 μM empty and curcumin loaded SMA and FA-DABA-SMA and 3 μM curcumin only. ( A ) Phase contrast images at 4× objective of changes in spheroid volume over the course of three days following exposure to 3 μM empty and curcumin loaded SMA and FA-DABA-SMA and 3 μM curcumin only. A total of 10,000 cells were plated per well in a 96 well plate for a total of 7 days (4 days for cell aggregate formation followed by 3 days of treatment). ( B ) Cell aggregate volume was measured daily following treatment using V = (4/3) πr 3  where π = 3.1415 and r = average radius (μm). Radius was measured using a scale bar. Cell aggregate volume shown in the graph represents a cell aggregate volume ± standard error (error bars). Results were compared by a one-way ANOVA at 95% confidence using Fisher’s LSD test. The data presented in the graph are the combined results of two independent experiments that showed similar results.  Notes:  The control is represented by the spheroids that were untreated. Indicated  p -values are a comparison between treated vs. untreated cells ( n  = 26 cell aggregates). The image scale bar represents 100 μm. The insets are magnified 300%. Phase contrast images are not a complete representation of all the spheroids that were measured and plotted in the bar graph.  Abbreviations:  SMA, poly(styrene- alt -maleic anhydride); FA, folic acid; DABA, 2,4-diaminobutyric acid; Cur, curcumin; CA, cell aggregate.
    96 Well Flat Bottom Uv Transparent Plate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/96 well flat bottom uv transparent plate/product/Thermo Fisher
    Average 91 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    96 well flat bottom uv transparent plate - by Bioz Stars, 2021-01
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    92
    Thermo Fisher coomassie protein assay kit
    MPK1 and MPK7 activations by wounding require protein synthesis A and B. Kinase activity of MPK1 (A) and MPK7 (B) after immunoprecipitation with an anti-HA antibody from leaves of indicated genetic background following 100µM CHX and MOCK (DMSO) spraying prior to wounding. Protein amount is monitored by western-blot using an anti-HA antibody. Equal loading is controlled by <t>Coomassie</t> staining.
    Coomassie Protein Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/coomassie protein assay kit/product/Thermo Fisher
    Average 92 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    coomassie protein assay kit - by Bioz Stars, 2021-01
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    Image Search Results


    Carvacrol influences the ATP-recovery effect on Vac-induced cell death Semi-confluent #12537-GB cells (seeded in 96-well flat-bottomed microtiter plates) were treated with DMSO diluted in medium at 10 −4 served as control, Vac (7 μM), Vac+ATP or ATP without carvacrol or with 50 μM and 100 μM carvacrol. PI-positive (dead) cells are given as RCU (y-axis, RCU×μM 2 /image). Glioma cells were followed for 24 h (x-axis) (IncuCyteZOOM®) (A, B) . Quantitative analysis at 8 h for glioma cell lines (* in A and B) (C) . Vac+ATP vs. Vac+ATP+carvacrol 100 μM (multiple comparison two way ANOVA, p=0.02). All values are given as means ± SD (6× replicates). All imaging was performed using IncuCyteZOOM® at 10× objective. Results are representative of 3 independent experiments.

    Journal: Oncotarget

    Article Title: Vacquinol-1 inducible cell death in glioblastoma multiforme is counter regulated by TRPM7 activity induced by exogenous ATP

    doi: 10.18632/oncotarget.16703

    Figure Lengend Snippet: Carvacrol influences the ATP-recovery effect on Vac-induced cell death Semi-confluent #12537-GB cells (seeded in 96-well flat-bottomed microtiter plates) were treated with DMSO diluted in medium at 10 −4 served as control, Vac (7 μM), Vac+ATP or ATP without carvacrol or with 50 μM and 100 μM carvacrol. PI-positive (dead) cells are given as RCU (y-axis, RCU×μM 2 /image). Glioma cells were followed for 24 h (x-axis) (IncuCyteZOOM®) (A, B) . Quantitative analysis at 8 h for glioma cell lines (* in A and B) (C) . Vac+ATP vs. Vac+ATP+carvacrol 100 μM (multiple comparison two way ANOVA, p=0.02). All values are given as means ± SD (6× replicates). All imaging was performed using IncuCyteZOOM® at 10× objective. Results are representative of 3 independent experiments.

    Article Snippet: Semi-confluent cell layers seeded into 96-well flat-bottomed microtiter plates ( ThermoFisher.com ) were treated with a final concentration of 7 μM Vac in the presence of 10 μg/ml PI ( Sigma.com ) with and without ATP, carvacrol ( Sigma.com ), suramin ( Tocris/Biotechné.com , UK) or A-438079 ( Sellekchem.com ).

    Techniques: Imaging

    Vac leads to caspase 3/7 activation in glioma cells, counter regulated by ATP Caspase 3/7 activity given as relative color units (RCU) per μM 2 /image. Semi-confluent glioma cells (seeded in 96-well flat-bottomed microtiter plates) were treated with DMSO diluted in medium at 10 −4 served as control; 7 μM Vac; Vac+ATP 1 mM; and 1 mM ATP alone. Caspase 3/7 activity was monitored over 16 h (x-axis) (IncuCyteZOOM®) by adding IncuCyte® Caspase-3/7 reagent (y-axis, RCU×μM 2 /image) (A) . Quantitative analysis at 2 h for glioma cells (* in A). Vac vs. control and Vac vs. Vac+ATP (both p

    Journal: Oncotarget

    Article Title: Vacquinol-1 inducible cell death in glioblastoma multiforme is counter regulated by TRPM7 activity induced by exogenous ATP

    doi: 10.18632/oncotarget.16703

    Figure Lengend Snippet: Vac leads to caspase 3/7 activation in glioma cells, counter regulated by ATP Caspase 3/7 activity given as relative color units (RCU) per μM 2 /image. Semi-confluent glioma cells (seeded in 96-well flat-bottomed microtiter plates) were treated with DMSO diluted in medium at 10 −4 served as control; 7 μM Vac; Vac+ATP 1 mM; and 1 mM ATP alone. Caspase 3/7 activity was monitored over 16 h (x-axis) (IncuCyteZOOM®) by adding IncuCyte® Caspase-3/7 reagent (y-axis, RCU×μM 2 /image) (A) . Quantitative analysis at 2 h for glioma cells (* in A). Vac vs. control and Vac vs. Vac+ATP (both p

    Article Snippet: Semi-confluent cell layers seeded into 96-well flat-bottomed microtiter plates ( ThermoFisher.com ) were treated with a final concentration of 7 μM Vac in the presence of 10 μg/ml PI ( Sigma.com ) with and without ATP, carvacrol ( Sigma.com ), suramin ( Tocris/Biotechné.com , UK) or A-438079 ( Sellekchem.com ).

    Techniques: Activation Assay, Activity Assay

    Vac- vs STS-mediated cell death Semi-confluent glioma cells (seeded in 96-well flat-bottomed microtiter plates) were treated with 2×10 −3 DMSO diluted in medium served as control, 7 μM Vac or 1 μM STS. Cell death was monitored over 20 h (x-axis) by PI staining (y-axis, RCU×μM 2 /image). All values are given as means ± SD (8× replicates) (A) . All imaging was performed using IncuCyteZOOM® at 10× objective. Caspase 3/7 activity was determined by luminescence assay (RLU, y-axis by Caspase Glo 3/7 assay, Promega.com ) after 4 h (*, A); control vs. Vac (t-test, p=0.0002); control vs. STS (t-test, p

    Journal: Oncotarget

    Article Title: Vacquinol-1 inducible cell death in glioblastoma multiforme is counter regulated by TRPM7 activity induced by exogenous ATP

    doi: 10.18632/oncotarget.16703

    Figure Lengend Snippet: Vac- vs STS-mediated cell death Semi-confluent glioma cells (seeded in 96-well flat-bottomed microtiter plates) were treated with 2×10 −3 DMSO diluted in medium served as control, 7 μM Vac or 1 μM STS. Cell death was monitored over 20 h (x-axis) by PI staining (y-axis, RCU×μM 2 /image). All values are given as means ± SD (8× replicates) (A) . All imaging was performed using IncuCyteZOOM® at 10× objective. Caspase 3/7 activity was determined by luminescence assay (RLU, y-axis by Caspase Glo 3/7 assay, Promega.com ) after 4 h (*, A); control vs. Vac (t-test, p=0.0002); control vs. STS (t-test, p

    Article Snippet: Semi-confluent cell layers seeded into 96-well flat-bottomed microtiter plates ( ThermoFisher.com ) were treated with a final concentration of 7 μM Vac in the presence of 10 μg/ml PI ( Sigma.com ) with and without ATP, carvacrol ( Sigma.com ), suramin ( Tocris/Biotechné.com , UK) or A-438079 ( Sellekchem.com ).

    Techniques: Staining, Imaging, Activity Assay, Luminescence Assay, Caspase-Glo Assay, T-Test

    ATP concentrations interfering with Vac-induced cell death, inhibition by purinergic receptor inhibitors Semi-confluent #12537-GB cells (seeded in 96-well flat-bottomed microtiter plates) were treated with 7 μM Vac with or without 10 nM, 100 nM, 1 μM, 10 μM, 100 μM and 1 mM ATP, or DMSO diluted in medium at 10 −4 served as control. PI-positive (dead) cells are given as RCU (y-axis, RCU×μM 2 /image). Glioma cells were followed for 17 h (x-axis). All values are means of PI fluorescence ± SD (triplicate values) (A) . Vac-induced cell death (7 μM) (y-axis, RCU×μM 2 /image) recorded for 24 h (x-axis) was attenuated by ATP (1 mM) but was further increased by ATP in the presence of the universal purinergic inhibitor Suramin (30 μM). All values are means of PI fluorescence ± SD (triplicate values) (B) . Vac-induced cell death (7 μM) (y-axis: RCU×μM 2 /image) recorded for 24 h (x-axis) was attenuated by ATP (1 mM) but was further increased by ATP in the presence of the selective P2×7 inhibitor A-438079 (100 μM). All values are means of PI fluorescence ± SD (6× replicates) (C) . All imaging was performed using IncuCyteZOOM® at 10× objective. Results are representative of 2 independent experiments.

    Journal: Oncotarget

    Article Title: Vacquinol-1 inducible cell death in glioblastoma multiforme is counter regulated by TRPM7 activity induced by exogenous ATP

    doi: 10.18632/oncotarget.16703

    Figure Lengend Snippet: ATP concentrations interfering with Vac-induced cell death, inhibition by purinergic receptor inhibitors Semi-confluent #12537-GB cells (seeded in 96-well flat-bottomed microtiter plates) were treated with 7 μM Vac with or without 10 nM, 100 nM, 1 μM, 10 μM, 100 μM and 1 mM ATP, or DMSO diluted in medium at 10 −4 served as control. PI-positive (dead) cells are given as RCU (y-axis, RCU×μM 2 /image). Glioma cells were followed for 17 h (x-axis). All values are means of PI fluorescence ± SD (triplicate values) (A) . Vac-induced cell death (7 μM) (y-axis, RCU×μM 2 /image) recorded for 24 h (x-axis) was attenuated by ATP (1 mM) but was further increased by ATP in the presence of the universal purinergic inhibitor Suramin (30 μM). All values are means of PI fluorescence ± SD (triplicate values) (B) . Vac-induced cell death (7 μM) (y-axis: RCU×μM 2 /image) recorded for 24 h (x-axis) was attenuated by ATP (1 mM) but was further increased by ATP in the presence of the selective P2×7 inhibitor A-438079 (100 μM). All values are means of PI fluorescence ± SD (6× replicates) (C) . All imaging was performed using IncuCyteZOOM® at 10× objective. Results are representative of 2 independent experiments.

    Article Snippet: Semi-confluent cell layers seeded into 96-well flat-bottomed microtiter plates ( ThermoFisher.com ) were treated with a final concentration of 7 μM Vac in the presence of 10 μg/ml PI ( Sigma.com ) with and without ATP, carvacrol ( Sigma.com ), suramin ( Tocris/Biotechné.com , UK) or A-438079 ( Sellekchem.com ).

    Techniques: Inhibition, Fluorescence, Imaging

    Changes in PANC-1 cell aggregate volume following exposure to 3 μM empty and curcumin loaded SMA and FA-DABA-SMA and 3 μM curcumin only. ( A ) Phase contrast images at 4× objective of changes in spheroid volume over the course of three days following exposure to 3 μM empty and curcumin loaded SMA and FA-DABA-SMA and 3 μM curcumin only. A total of 10,000 cells were plated per well in a 96 well plate for a total of 7 days (4 days for cell aggregate formation followed by 3 days of treatment). ( B ) Cell aggregate volume was measured daily following treatment using V = (4/3) πr 3  where π = 3.1415 and r = average radius (μm). Radius was measured using a scale bar. Cell aggregate volume shown in the graph represents a cell aggregate volume ± standard error (error bars). Results were compared by a one-way ANOVA at 95% confidence using Fisher’s LSD test. The data presented in the graph are the combined results of two independent experiments that showed similar results.  Notes:  The control is represented by the spheroids that were untreated. Indicated  p -values are a comparison between treated vs. untreated cells ( n  = 26 cell aggregates). The image scale bar represents 100 μm. The insets are magnified 300%. Phase contrast images are not a complete representation of all the spheroids that were measured and plotted in the bar graph.  Abbreviations:  SMA, poly(styrene- alt -maleic anhydride); FA, folic acid; DABA, 2,4-diaminobutyric acid; Cur, curcumin; CA, cell aggregate.

    Journal: Nanomaterials

    Article Title: Functionalized Folic Acid-Conjugated Amphiphilic Alternating Copolymer Actively Targets 3D Multicellular Tumour Spheroids and Delivers the Hydrophobic Drug to the Inner Core

    doi: 10.3390/nano8080588

    Figure Lengend Snippet: Changes in PANC-1 cell aggregate volume following exposure to 3 μM empty and curcumin loaded SMA and FA-DABA-SMA and 3 μM curcumin only. ( A ) Phase contrast images at 4× objective of changes in spheroid volume over the course of three days following exposure to 3 μM empty and curcumin loaded SMA and FA-DABA-SMA and 3 μM curcumin only. A total of 10,000 cells were plated per well in a 96 well plate for a total of 7 days (4 days for cell aggregate formation followed by 3 days of treatment). ( B ) Cell aggregate volume was measured daily following treatment using V = (4/3) πr 3 where π = 3.1415 and r = average radius (μm). Radius was measured using a scale bar. Cell aggregate volume shown in the graph represents a cell aggregate volume ± standard error (error bars). Results were compared by a one-way ANOVA at 95% confidence using Fisher’s LSD test. The data presented in the graph are the combined results of two independent experiments that showed similar results. Notes: The control is represented by the spheroids that were untreated. Indicated p -values are a comparison between treated vs. untreated cells ( n = 26 cell aggregates). The image scale bar represents 100 μm. The insets are magnified 300%. Phase contrast images are not a complete representation of all the spheroids that were measured and plotted in the bar graph. Abbreviations: SMA, poly(styrene- alt -maleic anhydride); FA, folic acid; DABA, 2,4-diaminobutyric acid; Cur, curcumin; CA, cell aggregate.

    Article Snippet: An amount of 100 μL of the dissolved precipitate was transferred in triplicate to a 96-well flat bottom UV-transparent plate and was measured using Thermo Scientific Varioskan Flash Microplate Reader.

    Techniques:

    Changes in MDA-MB231 spheroid volume following exposure to 3 μM empty and curcumin loaded SMA and FA-DABA-SMA and 3 μM curcumin only. ( A ) Phase contrast images at 4× objective of changes in spheroid volume over the course of three days following exposure to 3 μM unloaded and curcumin loaded SMA and FA-DABA-SMA and 3 μM curcumin only. A total of 10,000 cells were plated per well in a 96 well plate for a total of 7 days (4 days for spheroid formation followed by 3 days of treatment). ( B ) Spheroid volume was measured daily following treatment using V = (4/3) πr 3  where π = 3.1415 and r = average radius (μm). Radius was measured using a scale bar. Spheroid volume shown in the graph represent spheroid volume ± standard error (error bars). Results were compared by a one-way ANOVA at 95% confidence using Fisher’s LSD test. The data presented in the graph are the combined results of two independent experiments that showed similar results.  Notes:  The control is represented by the spheroids that were untreated. Indicated  p -values vs. untreated cells ( n  = 38). The image scale bar represents 100 μm. The insets are magnified 300%. Phase contrast images are not the complete representation of all the spheroids that were measured.  Abbreviations : SMA, poly(styrene- alt -maleic anhydride); FA, folic acid; DABA, 2,4-diaminobutyric acid; Cur, curcumin.

    Journal: Nanomaterials

    Article Title: Functionalized Folic Acid-Conjugated Amphiphilic Alternating Copolymer Actively Targets 3D Multicellular Tumour Spheroids and Delivers the Hydrophobic Drug to the Inner Core

    doi: 10.3390/nano8080588

    Figure Lengend Snippet: Changes in MDA-MB231 spheroid volume following exposure to 3 μM empty and curcumin loaded SMA and FA-DABA-SMA and 3 μM curcumin only. ( A ) Phase contrast images at 4× objective of changes in spheroid volume over the course of three days following exposure to 3 μM unloaded and curcumin loaded SMA and FA-DABA-SMA and 3 μM curcumin only. A total of 10,000 cells were plated per well in a 96 well plate for a total of 7 days (4 days for spheroid formation followed by 3 days of treatment). ( B ) Spheroid volume was measured daily following treatment using V = (4/3) πr 3 where π = 3.1415 and r = average radius (μm). Radius was measured using a scale bar. Spheroid volume shown in the graph represent spheroid volume ± standard error (error bars). Results were compared by a one-way ANOVA at 95% confidence using Fisher’s LSD test. The data presented in the graph are the combined results of two independent experiments that showed similar results. Notes: The control is represented by the spheroids that were untreated. Indicated p -values vs. untreated cells ( n = 38). The image scale bar represents 100 μm. The insets are magnified 300%. Phase contrast images are not the complete representation of all the spheroids that were measured. Abbreviations : SMA, poly(styrene- alt -maleic anhydride); FA, folic acid; DABA, 2,4-diaminobutyric acid; Cur, curcumin.

    Article Snippet: An amount of 100 μL of the dissolved precipitate was transferred in triplicate to a 96-well flat bottom UV-transparent plate and was measured using Thermo Scientific Varioskan Flash Microplate Reader.

    Techniques: Multiple Displacement Amplification

    MPK1 and MPK7 activations by wounding require protein synthesis A and B. Kinase activity of MPK1 (A) and MPK7 (B) after immunoprecipitation with an anti-HA antibody from leaves of indicated genetic background following 100µM CHX and MOCK (DMSO) spraying prior to wounding. Protein amount is monitored by western-blot using an anti-HA antibody. Equal loading is controlled by Coomassie staining.

    Journal: bioRxiv

    Article Title: Wounding and insect feeding trigger two independent MAPK pathways with distinct regulation and kinetics

    doi: 10.1101/855098

    Figure Lengend Snippet: MPK1 and MPK7 activations by wounding require protein synthesis A and B. Kinase activity of MPK1 (A) and MPK7 (B) after immunoprecipitation with an anti-HA antibody from leaves of indicated genetic background following 100µM CHX and MOCK (DMSO) spraying prior to wounding. Protein amount is monitored by western-blot using an anti-HA antibody. Equal loading is controlled by Coomassie staining.

    Article Snippet: -Fill the necessary wells of a 96 wells flat-bottom transparent plate with 250 µL Bradford reagent (Coomassie Protein Assay Kit, Thermo Scientific).

    Techniques: Activity Assay, Immunoprecipitation, Western Blot, Staining

    MPK1, MPK2 and MPK7 are activated by JA A to C. Kinase activity of MPK2 (A), MPK1 (B) and MPK7 (C) after immunoprecipitation with an anti-HA antibody from leaves of WT (A, B, C) and mkk3-1 (A, C) plants expressing a HA-tagged version of MPK2 (A), MPK1 (B) and MPK7 (C) following 50µM JA or MOCK (EtOH) treatments for indicated times. Protein amount is monitored by western-blot using anti-HA antibody. Equal loading is controlled by Coomassie staining.

    Journal: bioRxiv

    Article Title: Wounding and insect feeding trigger two independent MAPK pathways with distinct regulation and kinetics

    doi: 10.1101/855098

    Figure Lengend Snippet: MPK1, MPK2 and MPK7 are activated by JA A to C. Kinase activity of MPK2 (A), MPK1 (B) and MPK7 (C) after immunoprecipitation with an anti-HA antibody from leaves of WT (A, B, C) and mkk3-1 (A, C) plants expressing a HA-tagged version of MPK2 (A), MPK1 (B) and MPK7 (C) following 50µM JA or MOCK (EtOH) treatments for indicated times. Protein amount is monitored by western-blot using anti-HA antibody. Equal loading is controlled by Coomassie staining.

    Article Snippet: -Fill the necessary wells of a 96 wells flat-bottom transparent plate with 250 µL Bradford reagent (Coomassie Protein Assay Kit, Thermo Scientific).

    Techniques: Activity Assay, Immunoprecipitation, Expressing, Western Blot, Staining

    MKK4/5-MPK3/6 are not involved in wound-induced JA production A and B. Kinase activity of MPK2 after immunoprecipitation with an anti-MPK2 specific antibody from Col-0 (A, B) and mkk3-1 (A) and mkk4mkk5 (B) leaves following wounding at indicated times. MPK3/6 activation was monitored by western-blot using antibody raised against the phosphorylated form of ERK2 (anti-pTpY). Equal loading is controlled by Coomassie staining. C. JA, JA-Isoleucine and Cis-OPDA contents in wounded leaves of Col-0 and mkk4mkk5 . Values are mean±SE of 3 biological replicates.

    Journal: bioRxiv

    Article Title: Wounding and insect feeding trigger two independent MAPK pathways with distinct regulation and kinetics

    doi: 10.1101/855098

    Figure Lengend Snippet: MKK4/5-MPK3/6 are not involved in wound-induced JA production A and B. Kinase activity of MPK2 after immunoprecipitation with an anti-MPK2 specific antibody from Col-0 (A, B) and mkk3-1 (A) and mkk4mkk5 (B) leaves following wounding at indicated times. MPK3/6 activation was monitored by western-blot using antibody raised against the phosphorylated form of ERK2 (anti-pTpY). Equal loading is controlled by Coomassie staining. C. JA, JA-Isoleucine and Cis-OPDA contents in wounded leaves of Col-0 and mkk4mkk5 . Values are mean±SE of 3 biological replicates.

    Article Snippet: -Fill the necessary wells of a 96 wells flat-bottom transparent plate with 250 µL Bradford reagent (Coomassie Protein Assay Kit, Thermo Scientific).

    Techniques: Activity Assay, Immunoprecipitation, Activation Assay, Western Blot, Staining

    MPK3 and MPK6 are activated by cycloheximide A. Western-blots using antibody raised against the phosphorylated form of ERK2 (anti-pTpY) from leaves of indicated genetic backgrounds following 100µM CHX and MOCK (DMSO) spraying prior to wounding. Equal loading is controlled by Coomassie staining. B. Kinase activity of MPK3 and MPK6 after immunoprecipitation with specific antibodies from Col-0 leaves following 100µM CHX and MOCK (DMSO) spraying prior to wounding. Western-blots show MAPK amount.

    Journal: bioRxiv

    Article Title: Wounding and insect feeding trigger two independent MAPK pathways with distinct regulation and kinetics

    doi: 10.1101/855098

    Figure Lengend Snippet: MPK3 and MPK6 are activated by cycloheximide A. Western-blots using antibody raised against the phosphorylated form of ERK2 (anti-pTpY) from leaves of indicated genetic backgrounds following 100µM CHX and MOCK (DMSO) spraying prior to wounding. Equal loading is controlled by Coomassie staining. B. Kinase activity of MPK3 and MPK6 after immunoprecipitation with specific antibodies from Col-0 leaves following 100µM CHX and MOCK (DMSO) spraying prior to wounding. Western-blots show MAPK amount.

    Article Snippet: -Fill the necessary wells of a 96 wells flat-bottom transparent plate with 250 µL Bradford reagent (Coomassie Protein Assay Kit, Thermo Scientific).

    Techniques: Western Blot, Staining, Activity Assay, Immunoprecipitation

    MKK3-MPK2 module is activated by Spodoptera littoralis feeding A. Kinase activity of MPK2 after immunoprecipitation with an anti-MPK2 specific antibody from leaves on which S. littoralis fed during 15 minutes before to be removed (at t=15’). MPK3/6 activation was monitored by western-blot using antibody raised against the phosphorylated form of ERK2 (anti-pTpY). Equal loading is controlled by Coomassie staining. B and C. Kinase activity of MPK2 after immunoprecipitation with an anti-MPK2 specific antibody from leaves on which S. littoralis fed for 1 and 3 hours in Col-0 and coi1-34 . D. Weight of S. littoralis caterpillars after feeding for 8 days on Col-0 and mkk3-1 rosettes. Box plot shows distribution of caterpillar weight (n > 120 in 5 biological replicates). Crosses show averages of biological replicates (39.5±5.2 and 54.4±6.6 mg for Col-0 and mkk3-1 , respectively [n=5]; statistical difference based on the Mann-Whitney test with p

    Journal: bioRxiv

    Article Title: Wounding and insect feeding trigger two independent MAPK pathways with distinct regulation and kinetics

    doi: 10.1101/855098

    Figure Lengend Snippet: MKK3-MPK2 module is activated by Spodoptera littoralis feeding A. Kinase activity of MPK2 after immunoprecipitation with an anti-MPK2 specific antibody from leaves on which S. littoralis fed during 15 minutes before to be removed (at t=15’). MPK3/6 activation was monitored by western-blot using antibody raised against the phosphorylated form of ERK2 (anti-pTpY). Equal loading is controlled by Coomassie staining. B and C. Kinase activity of MPK2 after immunoprecipitation with an anti-MPK2 specific antibody from leaves on which S. littoralis fed for 1 and 3 hours in Col-0 and coi1-34 . D. Weight of S. littoralis caterpillars after feeding for 8 days on Col-0 and mkk3-1 rosettes. Box plot shows distribution of caterpillar weight (n > 120 in 5 biological replicates). Crosses show averages of biological replicates (39.5±5.2 and 54.4±6.6 mg for Col-0 and mkk3-1 , respectively [n=5]; statistical difference based on the Mann-Whitney test with p

    Article Snippet: -Fill the necessary wells of a 96 wells flat-bottom transparent plate with 250 µL Bradford reagent (Coomassie Protein Assay Kit, Thermo Scientific).

    Techniques: Activity Assay, Immunoprecipitation, Activation Assay, Western Blot, Staining, MANN-WHITNEY

    MPK1 and MPK7 are activated by wounding A and B. Kinase activity of MPK1 (A) and MPK7 (B) after immunoprecipitation with an anti-HA antibody from leaves of plants expressing a HA-tagged version of MPK1/7 following wounding at the indicated times. Protein amount is monitored by western-blot using anti-HA antibody. Equal loading is controlled by Coomassie staining.

    Journal: bioRxiv

    Article Title: Wounding and insect feeding trigger two independent MAPK pathways with distinct regulation and kinetics

    doi: 10.1101/855098

    Figure Lengend Snippet: MPK1 and MPK7 are activated by wounding A and B. Kinase activity of MPK1 (A) and MPK7 (B) after immunoprecipitation with an anti-HA antibody from leaves of plants expressing a HA-tagged version of MPK1/7 following wounding at the indicated times. Protein amount is monitored by western-blot using anti-HA antibody. Equal loading is controlled by Coomassie staining.

    Article Snippet: -Fill the necessary wells of a 96 wells flat-bottom transparent plate with 250 µL Bradford reagent (Coomassie Protein Assay Kit, Thermo Scientific).

    Techniques: Activity Assay, Immunoprecipitation, Expressing, Western Blot, Staining

    MPK2 activation by wounding depends on MKK3 A. Kinase activity of MPK2 after immunoprecipitation with an anti-MPK2 specific antibody from Col-0 and mkk3-1 leaves following wounding at the indicated times. B. Kinase activity of MPK2 after immunoprecipitation with an anti-HA antibody from leaves of plants expressing an HA-tagged version of MPK2 in Col-0 and mkk3-1 leaves following wounding at the indicated times. Protein amount is monitored by western-blot using anti-HA antibody. Equal loading is controlled by Coomassie staining.

    Journal: bioRxiv

    Article Title: Wounding and insect feeding trigger two independent MAPK pathways with distinct regulation and kinetics

    doi: 10.1101/855098

    Figure Lengend Snippet: MPK2 activation by wounding depends on MKK3 A. Kinase activity of MPK2 after immunoprecipitation with an anti-MPK2 specific antibody from Col-0 and mkk3-1 leaves following wounding at the indicated times. B. Kinase activity of MPK2 after immunoprecipitation with an anti-HA antibody from leaves of plants expressing an HA-tagged version of MPK2 in Col-0 and mkk3-1 leaves following wounding at the indicated times. Protein amount is monitored by western-blot using anti-HA antibody. Equal loading is controlled by Coomassie staining.

    Article Snippet: -Fill the necessary wells of a 96 wells flat-bottom transparent plate with 250 µL Bradford reagent (Coomassie Protein Assay Kit, Thermo Scientific).

    Techniques: Activation Assay, Activity Assay, Immunoprecipitation, Expressing, Western Blot, Staining

    Flg22 rapidly and transiently activates MPK3 and MPK6 in leaf punches Punches from Col-0 leaves were equilibrated over night in water and then treated for the indicated time with 500 nM flg22 or water (MOCK). MPK3 and MPK6 phosphorylation is monitored by western-blot using an antibody raised against the phosphorylated form of ERK2 (anti-pTpY). Equal loading is controlled by Coomassie staining.

    Journal: bioRxiv

    Article Title: Wounding and insect feeding trigger two independent MAPK pathways with distinct regulation and kinetics

    doi: 10.1101/855098

    Figure Lengend Snippet: Flg22 rapidly and transiently activates MPK3 and MPK6 in leaf punches Punches from Col-0 leaves were equilibrated over night in water and then treated for the indicated time with 500 nM flg22 or water (MOCK). MPK3 and MPK6 phosphorylation is monitored by western-blot using an antibody raised against the phosphorylated form of ERK2 (anti-pTpY). Equal loading is controlled by Coomassie staining.

    Article Snippet: -Fill the necessary wells of a 96 wells flat-bottom transparent plate with 250 µL Bradford reagent (Coomassie Protein Assay Kit, Thermo Scientific).

    Techniques: Western Blot, Staining

    MPK3 and MPK6 activity is independent of JA A. Western-blots using antibody raised against the phosphorylated form of ERK2 (anti-pTpY) in in vitro Col-0 and mkk3-1 plantlets treated with 50µM JA, 1µM flg22 and MOCK (EtOH). Equal loading is controlled by Coomassie staining. B. Western-blots using antibody raised against the phosphorylated form of ERK2 (anti-pTpY) in Col-0 and coi1-34 in response to wounding. Equal loading is controlled by Coomassie staining.

    Journal: bioRxiv

    Article Title: Wounding and insect feeding trigger two independent MAPK pathways with distinct regulation and kinetics

    doi: 10.1101/855098

    Figure Lengend Snippet: MPK3 and MPK6 activity is independent of JA A. Western-blots using antibody raised against the phosphorylated form of ERK2 (anti-pTpY) in in vitro Col-0 and mkk3-1 plantlets treated with 50µM JA, 1µM flg22 and MOCK (EtOH). Equal loading is controlled by Coomassie staining. B. Western-blots using antibody raised against the phosphorylated form of ERK2 (anti-pTpY) in Col-0 and coi1-34 in response to wounding. Equal loading is controlled by Coomassie staining.

    Article Snippet: -Fill the necessary wells of a 96 wells flat-bottom transparent plate with 250 µL Bradford reagent (Coomassie Protein Assay Kit, Thermo Scientific).

    Techniques: Activity Assay, Western Blot, In Vitro, Staining

    MPK3 and MPK6 activation depends on MKK4/MKK5 but not on MKK3 A and B. Western-blot using antibody raised against the phosphorylated form of ERK2 (anti-pTpY) in indicated genetic backgrounds after wounding. Equal loading is controlled by Coomassie staining. Figure S13A is an un-cropped version of western blot shown in figure 6A . C. Kinase activity of MPK2, MPK3, MPK6 after immunoprecipitation with an appropriate specific antibody from wounded leaves of Col-0 and mkk4mkk5 plants. MPK3/6 activation was monitored by western-blot using antibody raised against the phosphorylated form of ERK2 (anti-pTpY). Equal loading is controlled by Coomassie staining. D to F. Western-blots using antibody raised against the phosphorylated form of ERK2 (anti-pTpY) in indicated genetic backgrounds. “?” (D) referred to a map3k14-1 specific band which could correspond to the over activation of clade-C MAPKs. Equal loading is controlled by Coomassie staining.

    Journal: bioRxiv

    Article Title: Wounding and insect feeding trigger two independent MAPK pathways with distinct regulation and kinetics

    doi: 10.1101/855098

    Figure Lengend Snippet: MPK3 and MPK6 activation depends on MKK4/MKK5 but not on MKK3 A and B. Western-blot using antibody raised against the phosphorylated form of ERK2 (anti-pTpY) in indicated genetic backgrounds after wounding. Equal loading is controlled by Coomassie staining. Figure S13A is an un-cropped version of western blot shown in figure 6A . C. Kinase activity of MPK2, MPK3, MPK6 after immunoprecipitation with an appropriate specific antibody from wounded leaves of Col-0 and mkk4mkk5 plants. MPK3/6 activation was monitored by western-blot using antibody raised against the phosphorylated form of ERK2 (anti-pTpY). Equal loading is controlled by Coomassie staining. D to F. Western-blots using antibody raised against the phosphorylated form of ERK2 (anti-pTpY) in indicated genetic backgrounds. “?” (D) referred to a map3k14-1 specific band which could correspond to the over activation of clade-C MAPKs. Equal loading is controlled by Coomassie staining.

    Article Snippet: -Fill the necessary wells of a 96 wells flat-bottom transparent plate with 250 µL Bradford reagent (Coomassie Protein Assay Kit, Thermo Scientific).

    Techniques: Activation Assay, Western Blot, Staining, Activity Assay, Immunoprecipitation

    MAP3K18 accumulates in response to wounding Western-blot using anti-GFP antibody showing MAP3K18-YFP protein level expressed under its own promoter in wounded leaves. Equal loading is controlled by Coomassie staining.

    Journal: bioRxiv

    Article Title: Wounding and insect feeding trigger two independent MAPK pathways with distinct regulation and kinetics

    doi: 10.1101/855098

    Figure Lengend Snippet: MAP3K18 accumulates in response to wounding Western-blot using anti-GFP antibody showing MAP3K18-YFP protein level expressed under its own promoter in wounded leaves. Equal loading is controlled by Coomassie staining.

    Article Snippet: -Fill the necessary wells of a 96 wells flat-bottom transparent plate with 250 µL Bradford reagent (Coomassie Protein Assay Kit, Thermo Scientific).

    Techniques: Western Blot, Staining