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Promega celltiter glo atp assay
Sendai virus Y1/Y2 proteins are required for necroptosis. ( a ) Infection of L929 cells with ultraviolet-inactivated (UV) SeV rescues cell death as monitored by <t>CellTiter-Glo</t> (experiment repeated three times). Right panel: western blot of L929 cells infected with live or UV-inactivated SeV (10 HA U/ml) after 18 h (performed once) (41). ( b ) Schematic representation of SeV proteins. Six structural proteins (NP, P, M, F, HN, L) and accessory proteins (V, W, X, C, C', Y1, Y2). ( c ) L929 cells stably expressing a control (Ctl) shRNA or shRNA against P and accessory proteins (PVC) were treated with zVAD and infected with SeV (10 HA U/ml) and cell survival monitored. Right panel: knockdown was monitored by western blot using antibodies against the C proteins of SeV. ( d ) L929 cells were infected with Cantell strain of SeV, Z strain of SeV (SeV-Z), or mutants (d2Y-Z, V[-]-Z). 16–18 h post-infection, mRNA was isolated and used for quantitative RT-PCR using oligonucleotides specific for the SeV NP or γ -actin gene. Expression was plotted on a log scale relative to mock-infected samples after normalization to γ -actin. Standard error of the mean was calculated from three independent experiments. ( e ) Survival of cells infected with Cantell strain of SeV (SeV) or Z strain (SeV-Z, d2Y-Z, V[-]-Z) as measured by CellTiter-Glo assay. ( f ) Lysates from SeV-infected cells at various time points (M: mock-infected) were probed in a western blot analysis using SeV C protein or β-actin specific antibodies (experiment repeated twice). For 3A, 3C, 3D, 3E, data are averages of triplicates from a single experiment, which is representative of at least three independent experiments. *** P
Celltiter Glo Atp Assay, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Induction of necroptotic cell death by viral activation of the RIG-I or STING pathway"

Article Title: Induction of necroptotic cell death by viral activation of the RIG-I or STING pathway

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2016.153

Sendai virus Y1/Y2 proteins are required for necroptosis. ( a ) Infection of L929 cells with ultraviolet-inactivated (UV) SeV rescues cell death as monitored by CellTiter-Glo (experiment repeated three times). Right panel: western blot of L929 cells infected with live or UV-inactivated SeV (10 HA U/ml) after 18 h (performed once) (41). ( b ) Schematic representation of SeV proteins. Six structural proteins (NP, P, M, F, HN, L) and accessory proteins (V, W, X, C, C', Y1, Y2). ( c ) L929 cells stably expressing a control (Ctl) shRNA or shRNA against P and accessory proteins (PVC) were treated with zVAD and infected with SeV (10 HA U/ml) and cell survival monitored. Right panel: knockdown was monitored by western blot using antibodies against the C proteins of SeV. ( d ) L929 cells were infected with Cantell strain of SeV, Z strain of SeV (SeV-Z), or mutants (d2Y-Z, V[-]-Z). 16–18 h post-infection, mRNA was isolated and used for quantitative RT-PCR using oligonucleotides specific for the SeV NP or γ -actin gene. Expression was plotted on a log scale relative to mock-infected samples after normalization to γ -actin. Standard error of the mean was calculated from three independent experiments. ( e ) Survival of cells infected with Cantell strain of SeV (SeV) or Z strain (SeV-Z, d2Y-Z, V[-]-Z) as measured by CellTiter-Glo assay. ( f ) Lysates from SeV-infected cells at various time points (M: mock-infected) were probed in a western blot analysis using SeV C protein or β-actin specific antibodies (experiment repeated twice). For 3A, 3C, 3D, 3E, data are averages of triplicates from a single experiment, which is representative of at least three independent experiments. *** P
Figure Legend Snippet: Sendai virus Y1/Y2 proteins are required for necroptosis. ( a ) Infection of L929 cells with ultraviolet-inactivated (UV) SeV rescues cell death as monitored by CellTiter-Glo (experiment repeated three times). Right panel: western blot of L929 cells infected with live or UV-inactivated SeV (10 HA U/ml) after 18 h (performed once) (41). ( b ) Schematic representation of SeV proteins. Six structural proteins (NP, P, M, F, HN, L) and accessory proteins (V, W, X, C, C', Y1, Y2). ( c ) L929 cells stably expressing a control (Ctl) shRNA or shRNA against P and accessory proteins (PVC) were treated with zVAD and infected with SeV (10 HA U/ml) and cell survival monitored. Right panel: knockdown was monitored by western blot using antibodies against the C proteins of SeV. ( d ) L929 cells were infected with Cantell strain of SeV, Z strain of SeV (SeV-Z), or mutants (d2Y-Z, V[-]-Z). 16–18 h post-infection, mRNA was isolated and used for quantitative RT-PCR using oligonucleotides specific for the SeV NP or γ -actin gene. Expression was plotted on a log scale relative to mock-infected samples after normalization to γ -actin. Standard error of the mean was calculated from three independent experiments. ( e ) Survival of cells infected with Cantell strain of SeV (SeV) or Z strain (SeV-Z, d2Y-Z, V[-]-Z) as measured by CellTiter-Glo assay. ( f ) Lysates from SeV-infected cells at various time points (M: mock-infected) were probed in a western blot analysis using SeV C protein or β-actin specific antibodies (experiment repeated twice). For 3A, 3C, 3D, 3E, data are averages of triplicates from a single experiment, which is representative of at least three independent experiments. *** P

Techniques Used: Infection, Western Blot, Stable Transfection, Expressing, CTL Assay, shRNA, Isolation, Quantitative RT-PCR, Glo Assay

Sendai virus and MHV68 cause RIP1- and RIP3-dependent necroptosis. ( a ) L929 cells were treated with zVAD (10 μ M) and infected with MCMV (MOI 5), HSV-1 (MOI 5), LCMV (MOI 5), MHV68 (MOI 5), SeV (10 HA U/ml), KSHV (titre giving 70% infection) or WSN (MOI 5) and % cell survival was determined by the CellTiter-Glo assay. ( b ) Cell death of L929 cells treated with zVAD (10 μ M), Necrostatin-1 (20 μM) and infected with SeV (10 HA U/ml) or MHV68 (MOI 5). ( c ) L929 cells stably expressing mouse shRIP3 or control shRNA were treated with or without zVAD (5 μ M) in the absence or presence of SeV (10 HA U/ml) or MHV68 (MOI 5). Right panel: cell lysates of the shRNA retrovirally infected cells were probed by RIP3- or actin-specific antibodies. ( d ) L929 cells were mock-treated or treated with Necrostatin-1 (Nec-1) at 20 μM and infected with indicated titres of SeV. Cell death was monitored 18–20 h post-infection by CellTiter-Glo. (e ) Increased relative expression of viral NP protein from RIP3 shRNA knockdown cells as compared to control shRNA knockdown cells by quantitative RT-PCR. Expression is relative to mock-infected samples after normalization to γ -actin. ( f ) TNF ELISA from supernatants of L929 cells treated with zVAD and infected with SeV (10 or 100 HA U/ml) or MHV68 (MOI 5 or 10). ( g ) Death of cells treated with control or anti-TNF neutralizing antibody, zVAD (10 μ M and 100 μ M), TNF (60 pg/ml), SeV (100 HA U/ml) or MHV68 (MOI 5). ( h ) LA-4 cells were treated with zVAD (20 μ M), TNF (100 ng/ml) or Nec-1 (20 μ M) and infected with SeV (100 HA U/ml) or MHV68 (MOI 10). Cell survival was monitored 24 h post-infection. ( i ) LA-4 cells were treated with anti-control or anti-TNF neutralizing antibodies and treated with zVAD, TNF and infected with SeV (100 HA U/ml) before cell survival was assayed. Experiment repeated twice. Data are averages of triplicates from a single experiment, which is representative of at least three independent experiments (unless otherwise stated). *** P
Figure Legend Snippet: Sendai virus and MHV68 cause RIP1- and RIP3-dependent necroptosis. ( a ) L929 cells were treated with zVAD (10 μ M) and infected with MCMV (MOI 5), HSV-1 (MOI 5), LCMV (MOI 5), MHV68 (MOI 5), SeV (10 HA U/ml), KSHV (titre giving 70% infection) or WSN (MOI 5) and % cell survival was determined by the CellTiter-Glo assay. ( b ) Cell death of L929 cells treated with zVAD (10 μ M), Necrostatin-1 (20 μM) and infected with SeV (10 HA U/ml) or MHV68 (MOI 5). ( c ) L929 cells stably expressing mouse shRIP3 or control shRNA were treated with or without zVAD (5 μ M) in the absence or presence of SeV (10 HA U/ml) or MHV68 (MOI 5). Right panel: cell lysates of the shRNA retrovirally infected cells were probed by RIP3- or actin-specific antibodies. ( d ) L929 cells were mock-treated or treated with Necrostatin-1 (Nec-1) at 20 μM and infected with indicated titres of SeV. Cell death was monitored 18–20 h post-infection by CellTiter-Glo. (e ) Increased relative expression of viral NP protein from RIP3 shRNA knockdown cells as compared to control shRNA knockdown cells by quantitative RT-PCR. Expression is relative to mock-infected samples after normalization to γ -actin. ( f ) TNF ELISA from supernatants of L929 cells treated with zVAD and infected with SeV (10 or 100 HA U/ml) or MHV68 (MOI 5 or 10). ( g ) Death of cells treated with control or anti-TNF neutralizing antibody, zVAD (10 μ M and 100 μ M), TNF (60 pg/ml), SeV (100 HA U/ml) or MHV68 (MOI 5). ( h ) LA-4 cells were treated with zVAD (20 μ M), TNF (100 ng/ml) or Nec-1 (20 μ M) and infected with SeV (100 HA U/ml) or MHV68 (MOI 10). Cell survival was monitored 24 h post-infection. ( i ) LA-4 cells were treated with anti-control or anti-TNF neutralizing antibodies and treated with zVAD, TNF and infected with SeV (100 HA U/ml) before cell survival was assayed. Experiment repeated twice. Data are averages of triplicates from a single experiment, which is representative of at least three independent experiments (unless otherwise stated). *** P

Techniques Used: Infection, Glo Assay, Stable Transfection, Expressing, shRNA, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Related Articles

Clone Assay:

Article Title: Isolation of Cell Lines That Show Novel, Murine Leukemia Virus-Specific Blocks to Early Steps of Retroviral Replication
Article Snippet: Paragraph title: Screening MLV-resistant clones. ... At 48 h postinfection, one plate was assayed for β-galactosidase activity with a Galacto-Star chemiluminescent kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions, and the other plate was assayed for cell number and cell viability by using CellTiter-Glo reagent (Promega, Madison, WI) following the manufacturer's instructions.

Cell Cycle Assay:

Article Title: Palbociclib resistance confers dependence on an FGFR-MAP kinase-mTOR-driven pathway in KRAS-mutant non-small cell lung cancer
Article Snippet: Resistance was confirmed by assessment of proliferation and cell cycle analysis in the presence of palbociclib. .. Cell viability assays were performed using CellTiter-Glo (Promega) starting with 1 × 103 cells in 96-well plates.

Construct:

Article Title: Palbociclib resistance confers dependence on an FGFR-MAP kinase-mTOR-driven pathway in KRAS-mutant non-small cell lung cancer
Article Snippet: For depletion experiments, pLKO.1 shRNA constructs targeting CDK1, CDK2, CDK4, CDK6, cyclin D1, cyclin D3 and cyclin E (Harvard PlasmID Database) were expressed by lentiviral infection. .. Cell viability assays were performed using CellTiter-Glo (Promega) starting with 1 × 103 cells in 96-well plates.

Proliferation Assay:

Article Title: Comprehensive identification of microRNA arm selection preference in lung cancer: miR-324-5p and −3p serve oncogenic functions in lung cancer
Article Snippet: For the cell proliferation assay, 1×103 A549 cells transfected with 10 nM miR-324-5p mimics, miR-324-3p mimics, or a scramble control were seeded in a 96-well plate. .. Proliferation was determined at 0, 1, 2, 3, and 4 days using the CellTiter-Glo One Solution assay, according to the manufacturer's instructions (Promega Corporation, Madison, WI, USA).

Article Title: Linc00659, a long noncoding RNA, acts as novel oncogene in regulating cancer cell growth in colorectal cancer
Article Snippet: .. Cell viability was determined using a CellTiter-Glo One Solution cell proliferation assay (Promega Corporation, Madison, WI, USA), which was performed according to the manufacturer’s instructions. .. Soft agar assay In brief, this assay was performed as follows: The base agar layer was prepared with 1% agar (Laboratorios CONDA, Torrejón de Ardoz, Madrid, Spain) dissolved in 1.5 mL phosphate-buffered saline on 6-well plates; subsequently, 1.5 mL of 0.7% agarose (Invitrogen, Grand Island, NY, USA) solution containing the cells (10,000 cells per well) was inoculated on top of the base agar layer.

Article Title: NOXA genetic amplification or pharmacologic induction primes lymphoma cells to BCL2 inhibitor-induced cell death
Article Snippet: Paragraph title: In Vitro Proliferation Assay. ... Cells were seeded in 96-well plates at 25,000 cells per 100 μL per well or in 24-well plates at 250,000 cells per 1 mL per well with either vehicle (DMSO 0.1%) or increasing concentrations of drugs for 24, 48, and 72 h. Cell viability was assessed by adding MTS reagent or CellTiter-Glo reagent (Promega) to the culture medium at 1:5 ratios, according to the manufacturer’s instructions.

Two Tailed Test:

Article Title: NOXA genetic amplification or pharmacologic induction primes lymphoma cells to BCL2 inhibitor-induced cell death
Article Snippet: Cells were seeded in 96-well plates at 25,000 cells per 100 μL per well or in 24-well plates at 250,000 cells per 1 mL per well with either vehicle (DMSO 0.1%) or increasing concentrations of drugs for 24, 48, and 72 h. Cell viability was assessed by adding MTS reagent or CellTiter-Glo reagent (Promega) to the culture medium at 1:5 ratios, according to the manufacturer’s instructions. .. The two-tailed Student t test and Wilcoxon rank test were used to estimate the statistical significance of differences between results from the three experiments.

Activity Assay:

Article Title: Isolation of Cell Lines That Show Novel, Murine Leukemia Virus-Specific Blocks to Early Steps of Retroviral Replication
Article Snippet: .. At 48 h postinfection, one plate was assayed for β-galactosidase activity with a Galacto-Star chemiluminescent kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions, and the other plate was assayed for cell number and cell viability by using CellTiter-Glo reagent (Promega, Madison, WI) following the manufacturer's instructions. ..

Cell Culture:

Article Title: Recurrent activating mutations of G-protein-coupled receptor CYSLTR2 in uveal melanoma
Article Snippet: All cell culture media contained 10% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), and l-glutamine (2 mM). .. Cell growth was assessed using CellTiter-Glo (Promega), and the fold change in growth at day 3 was calculated relative to the day 1 luminescence values.

Article Title: Palbociclib resistance confers dependence on an FGFR-MAP kinase-mTOR-driven pathway in KRAS-mutant non-small cell lung cancer
Article Snippet: Paragraph title: Cell culture and drug treatments ... Cell viability assays were performed using CellTiter-Glo (Promega) starting with 1 × 103 cells in 96-well plates.

Modification:

Article Title: Three-Dimensional Lung Tumor Microenvironment Modulates Therapeutic Compound Responsiveness In Vitro - Implication for Drug Development
Article Snippet: .. Cell viability at day one and three using CellTiter-Glo Assay (Promega) was performed according to a modified manufacturer's instructions for tumor spheroids . .. EGFR and cMET Receptor Expression

Transfection:

Article Title: Comprehensive identification of microRNA arm selection preference in lung cancer: miR-324-5p and −3p serve oncogenic functions in lung cancer
Article Snippet: For the cell proliferation assay, 1×103 A549 cells transfected with 10 nM miR-324-5p mimics, miR-324-3p mimics, or a scramble control were seeded in a 96-well plate. .. Proliferation was determined at 0, 1, 2, 3, and 4 days using the CellTiter-Glo One Solution assay, according to the manufacturer's instructions (Promega Corporation, Madison, WI, USA).

Article Title: Linc00659, a long noncoding RNA, acts as novel oncogene in regulating cancer cell growth in colorectal cancer
Article Snippet: Proliferation For cell proliferation analysis, 2500 living cells were transfected with either siLinc00659 or a scrambled control and plated onto 96-well plates. .. Cell viability was determined using a CellTiter-Glo One Solution cell proliferation assay (Promega Corporation, Madison, WI, USA), which was performed according to the manufacturer’s instructions.

Infection:

Article Title: Palbociclib resistance confers dependence on an FGFR-MAP kinase-mTOR-driven pathway in KRAS-mutant non-small cell lung cancer
Article Snippet: For depletion experiments, pLKO.1 shRNA constructs targeting CDK1, CDK2, CDK4, CDK6, cyclin D1, cyclin D3 and cyclin E (Harvard PlasmID Database) were expressed by lentiviral infection. .. Cell viability assays were performed using CellTiter-Glo (Promega) starting with 1 × 103 cells in 96-well plates.

Light Microscopy:

Article Title: Comprehensive identification of microRNA arm selection preference in lung cancer: miR-324-5p and −3p serve oncogenic functions in lung cancer
Article Snippet: Then, the images of a representative colony formation were examined using a light microscope (magnification, ×100) (CKX41; Olympus Corporation, Tokyo, Japan). .. Proliferation was determined at 0, 1, 2, 3, and 4 days using the CellTiter-Glo One Solution assay, according to the manufacturer's instructions (Promega Corporation, Madison, WI, USA).

Generated:

Article Title: Three-Dimensional Lung Tumor Microenvironment Modulates Therapeutic Compound Responsiveness In Vitro - Implication for Drug Development
Article Snippet: Generation of Spheroids Spheroids were generated by plating lung tumor cells at 1×104 cells/well into “U” bottom Ultra Low Adherence (ULA) 96-well plates (Corning, Tewksbury, USA) at 200 μl/well. .. Cell viability at day one and three using CellTiter-Glo Assay (Promega) was performed according to a modified manufacturer's instructions for tumor spheroids .

other:

Article Title: Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition
Article Snippet: Cells were treated with vehicle or varying concentrations of screen drugs (10,000-fold range) in the absence or presence of 1 μM WZ4002 for 72 hours and cell viability determined by CellTiter-Glo assay.

Imaging:

Article Title: Three-Dimensional Lung Tumor Microenvironment Modulates Therapeutic Compound Responsiveness In Vitro - Implication for Drug Development
Article Snippet: Images were captured by Operetta High Content Imaging System (Perkin Elmer, Waltham, USA) using a 2x objective lens and the size was determined using Harmony software (Perkin Elmer). .. Cell viability at day one and three using CellTiter-Glo Assay (Promega) was performed according to a modified manufacturer's instructions for tumor spheroids .

Isolation:

Article Title: Palbociclib resistance confers dependence on an FGFR-MAP kinase-mTOR-driven pathway in KRAS-mutant non-small cell lung cancer
Article Snippet: Palbociclib-resistant cells H358-PR100, H358-PR250, H460-PR500 and H441-PR500 were isolated after the serial addition of increasing concentrations of palbociclib to culture media. .. Cell viability assays were performed using CellTiter-Glo (Promega) starting with 1 × 103 cells in 96-well plates.

Plasmid Preparation:

Article Title: Palbociclib resistance confers dependence on an FGFR-MAP kinase-mTOR-driven pathway in KRAS-mutant non-small cell lung cancer
Article Snippet: For depletion experiments, pLKO.1 shRNA constructs targeting CDK1, CDK2, CDK4, CDK6, cyclin D1, cyclin D3 and cyclin E (Harvard PlasmID Database) were expressed by lentiviral infection. .. Cell viability assays were performed using CellTiter-Glo (Promega) starting with 1 × 103 cells in 96-well plates.

Software:

Article Title: Three-Dimensional Lung Tumor Microenvironment Modulates Therapeutic Compound Responsiveness In Vitro - Implication for Drug Development
Article Snippet: Images were captured by Operetta High Content Imaging System (Perkin Elmer, Waltham, USA) using a 2x objective lens and the size was determined using Harmony software (Perkin Elmer). .. Cell viability at day one and three using CellTiter-Glo Assay (Promega) was performed according to a modified manufacturer's instructions for tumor spheroids .

Article Title: NOXA genetic amplification or pharmacologic induction primes lymphoma cells to BCL2 inhibitor-induced cell death
Article Snippet: Cells were seeded in 96-well plates at 25,000 cells per 100 μL per well or in 24-well plates at 250,000 cells per 1 mL per well with either vehicle (DMSO 0.1%) or increasing concentrations of drugs for 24, 48, and 72 h. Cell viability was assessed by adding MTS reagent or CellTiter-Glo reagent (Promega) to the culture medium at 1:5 ratios, according to the manufacturer’s instructions. .. The PRISM software was used for the statistical analyses.

shRNA:

Article Title: Palbociclib resistance confers dependence on an FGFR-MAP kinase-mTOR-driven pathway in KRAS-mutant non-small cell lung cancer
Article Snippet: For depletion experiments, pLKO.1 shRNA constructs targeting CDK1, CDK2, CDK4, CDK6, cyclin D1, cyclin D3 and cyclin E (Harvard PlasmID Database) were expressed by lentiviral infection. .. Cell viability assays were performed using CellTiter-Glo (Promega) starting with 1 × 103 cells in 96-well plates.

In Vitro:

Article Title: Recurrent activating mutations of G-protein-coupled receptor CYSLTR2 in uveal melanoma
Article Snippet: Paragraph title: Cell lines and in vitro analysis ... Cell growth was assessed using CellTiter-Glo (Promega), and the fold change in growth at day 3 was calculated relative to the day 1 luminescence values.

Article Title: NOXA genetic amplification or pharmacologic induction primes lymphoma cells to BCL2 inhibitor-induced cell death
Article Snippet: Paragraph title: In Vitro Proliferation Assay. ... Cells were seeded in 96-well plates at 25,000 cells per 100 μL per well or in 24-well plates at 250,000 cells per 1 mL per well with either vehicle (DMSO 0.1%) or increasing concentrations of drugs for 24, 48, and 72 h. Cell viability was assessed by adding MTS reagent or CellTiter-Glo reagent (Promega) to the culture medium at 1:5 ratios, according to the manufacturer’s instructions.

Incubation:

Article Title: Isolation of Cell Lines That Show Novel, Murine Leukemia Virus-Specific Blocks to Early Steps of Retroviral Replication
Article Snippet: Single-cell clones from pools 2, 3, and 5 to 10 were plated on duplicate plates and incubated for 2 h with pMMP-nls-LacZ [VSV-G] at an approximate MOI of 1 LacZ transducing unit (LTU) in the presence of 4 μg/ml Polybrene. .. At 48 h postinfection, one plate was assayed for β-galactosidase activity with a Galacto-Star chemiluminescent kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions, and the other plate was assayed for cell number and cell viability by using CellTiter-Glo reagent (Promega, Madison, WI) following the manufacturer's instructions.

Colony Assay:

Article Title: Comprehensive identification of microRNA arm selection preference in lung cancer: miR-324-5p and −3p serve oncogenic functions in lung cancer
Article Snippet: Paragraph title: Cell proliferation and colony formation assay ... Proliferation was determined at 0, 1, 2, 3, and 4 days using the CellTiter-Glo One Solution assay, according to the manufacturer's instructions (Promega Corporation, Madison, WI, USA).

Concentration Assay:

Article Title: Macroautophagy is dispensable for growth of KRAS mutant tumors and chloroquine efficacy
Article Snippet: .. After 72 h, cell proliferation was assessed relative to 0.4% DMSO (0%) and 1 μM MG132 (100%) using CellTiter-Glo (Promega), and IC50 values were determined from a curve fitted to duplicate 11-point concentration-response datasets from two independent assay plates. .. For FACS-based GFP-p62 pooled shRNA screening, 20 million H4 Tet-Off GFP-p62 cells were infected with the DECODER shRNA library using lentiviral particles at a multiplicity of infection of 0.8.

Staining:

Article Title: Comprehensive identification of microRNA arm selection preference in lung cancer: miR-324-5p and −3p serve oncogenic functions in lung cancer
Article Snippet: The cells were then fixed with 4% formaldehyde (Sigma-Aldrich; Merck KGaA) for 10 min at room temperature and stained with crystal violet solution (0.05% crystal violet, 1% formaldehyde and 1% methanol; all from Sigma-Aldrich; Merck KGaA) for 20 min at room temperature. .. Proliferation was determined at 0, 1, 2, 3, and 4 days using the CellTiter-Glo One Solution assay, according to the manufacturer's instructions (Promega Corporation, Madison, WI, USA).

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    Kaempferol rescues cells against BFA induced cell death. ( a ) Screening for cytoprotective compounds (50 µM) against BFA (1 µg/ml) induced ER stress in IMR32 cell line. Cells were plated and pre-incubated with the test compounds (90 min) and treated with ER stress inducer BFA, incubated for 24 h. Cell viability was estimated by MTT assay. BFA: Brefeldin A; Withan: Withanone; With A: Withanaloide A; With B: Withanaloide B; kaemp: kaempferol; kaempG: Kaempfeol-3-O-robinbioside-7-O-glucoside; Bacop I: Bacopaside I; PEA: Palmitoylethanolamide; PBA: Phenyl Butyric acid; Sal: Salubrinal. ( b ) Confirmation of cytoprotective activity of kaempferol by measuring cellular <t>ATP</t> level using CellTitre <t>Glo</t> reagent. ( c ) Phase contrast images of IMR32 cells after BFA treatment in presence or absence of kaempferol pre-incubation. ( d–f ) Cytoprotective activity of kaempferol against BFA induced cell death in multiple cell lines with incubation time of 24 hours. Cell viability was assessed using MTT assay. ( g ) Inhibition of caspase 3/7 activation by kaempferol against BFA and CDDO-Me induced caspase 3/7 activation in IMR32 cells. Data represents mean ± SEM of experiment performed in triplicate (n = 3). Note: *Represents the significance between cell death inducer alone treated condition compared to kaempferol pre-treated condition, at p ≤ 0.05 (one way ANOVA).
    Atp Level, supplied by Promega, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega atp based luminescence assay kit
    BNTX sensitizes human pancreatic cancer cells to TRAIL-induced apoptosis (A) Assay data for a screening analysis of AsPC-1 cells pre-incubated with various test compounds (5 μM) and were then exposed to TRAIL (50 ng/ml). Cell survival was measured as a percentage of the control sample. The arrow indicates BNTX (5 μM). (B) AsPC-1 cells were exposed to various concentrations of BNTX with or without TRAIL (25 ng/ml) for 24 h. Cell viability was measured by a luminescent <t>CellTiter-Glo</t> assay, and relative cell survival was determined by assaying <t>ATP</t> levels. (C) Relative survival of three pancreatic cancer cell lines (AsPC-1, PANC-1, and MIA PaCa-2) exposed to TRAIL (25 ng/ml) with or without BNTX (2.5 μM) for 24 h. Cell viability was measured by a luminescent CellTiter-Glo assay. (D) Apoptotic activity in AsPC-1 cells exposed to TRAIL (25 ng/ml) with or without BNTX (2.5 μM) for 24 h. Cells were analyzed by annexin V/PI double-stained flow cytometry. (E) The percentage of apoptotic cells. Values are the mean ± SD from two independent experiments performed in duplicate. Asterisks indicate significant differences compared with the control ( *** P
    Atp Based Luminescence Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    AQP1 functions as a genetically encoded reporter for diffusion-weighted MRI. ( a ) Illustration of the impact of aquaporin expression on water diffusion across the cell membrane and the resulting decrease in diffusion-weighted signal intensity. ( b ) Diffusion-weighted images of CHO, U87 and Neuro 2a cell pellets expressing AQP1 or GFP, acquired using a b -value of ∼1,000 s mm −2 . Scale bars, 3 mm. ( c ) ADC of water in CHO, U87 and Neuro 2a cells expressing AQP1 relative to GFP controls, measured at Δ eff =398 ms. Transgene expression in CHO cells was induced with 1 μg ml −1 doxycycline, whereas U87 and Neuro 2a cells express AQP1 from a constitutive promoter. n =4 (U87, Neuro 2a) and 5 (CHO) biological replicates. ( d ) Longitudinal ( T 1 ) and ( e ) transverse ( T 2 ) relaxation rates in cells expressing AQP1 or GFP. n =3 (Neuro 2a, CHO) or 4 (U87) biological replicates. ( f ) Cell viability on AQP1 or GFP expression. n =12 (resazurin assay), 6 <t>(ATP</t> content), 4 (LDH release) and 3 (ethidium staining) biological replicates. Error bars±s.e.m. ( g ) Phase-contrast images of CHO, U87 and Neuro 2a cells expressing AQP1 or GFP. Scale bars, 10 μm.
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    Kaempferol rescues cells against BFA induced cell death. ( a ) Screening for cytoprotective compounds (50 µM) against BFA (1 µg/ml) induced ER stress in IMR32 cell line. Cells were plated and pre-incubated with the test compounds (90 min) and treated with ER stress inducer BFA, incubated for 24 h. Cell viability was estimated by MTT assay. BFA: Brefeldin A; Withan: Withanone; With A: Withanaloide A; With B: Withanaloide B; kaemp: kaempferol; kaempG: Kaempfeol-3-O-robinbioside-7-O-glucoside; Bacop I: Bacopaside I; PEA: Palmitoylethanolamide; PBA: Phenyl Butyric acid; Sal: Salubrinal. ( b ) Confirmation of cytoprotective activity of kaempferol by measuring cellular ATP level using CellTitre Glo reagent. ( c ) Phase contrast images of IMR32 cells after BFA treatment in presence or absence of kaempferol pre-incubation. ( d–f ) Cytoprotective activity of kaempferol against BFA induced cell death in multiple cell lines with incubation time of 24 hours. Cell viability was assessed using MTT assay. ( g ) Inhibition of caspase 3/7 activation by kaempferol against BFA and CDDO-Me induced caspase 3/7 activation in IMR32 cells. Data represents mean ± SEM of experiment performed in triplicate (n = 3). Note: *Represents the significance between cell death inducer alone treated condition compared to kaempferol pre-treated condition, at p ≤ 0.05 (one way ANOVA).

    Journal: Scientific Reports

    Article Title: Kaempferol mitigates Endoplasmic Reticulum Stress Induced Cell Death by targeting caspase 3/7

    doi: 10.1038/s41598-018-20499-7

    Figure Lengend Snippet: Kaempferol rescues cells against BFA induced cell death. ( a ) Screening for cytoprotective compounds (50 µM) against BFA (1 µg/ml) induced ER stress in IMR32 cell line. Cells were plated and pre-incubated with the test compounds (90 min) and treated with ER stress inducer BFA, incubated for 24 h. Cell viability was estimated by MTT assay. BFA: Brefeldin A; Withan: Withanone; With A: Withanaloide A; With B: Withanaloide B; kaemp: kaempferol; kaempG: Kaempfeol-3-O-robinbioside-7-O-glucoside; Bacop I: Bacopaside I; PEA: Palmitoylethanolamide; PBA: Phenyl Butyric acid; Sal: Salubrinal. ( b ) Confirmation of cytoprotective activity of kaempferol by measuring cellular ATP level using CellTitre Glo reagent. ( c ) Phase contrast images of IMR32 cells after BFA treatment in presence or absence of kaempferol pre-incubation. ( d–f ) Cytoprotective activity of kaempferol against BFA induced cell death in multiple cell lines with incubation time of 24 hours. Cell viability was assessed using MTT assay. ( g ) Inhibition of caspase 3/7 activation by kaempferol against BFA and CDDO-Me induced caspase 3/7 activation in IMR32 cells. Data represents mean ± SEM of experiment performed in triplicate (n = 3). Note: *Represents the significance between cell death inducer alone treated condition compared to kaempferol pre-treated condition, at p ≤ 0.05 (one way ANOVA).

    Article Snippet: The cytoprotective activity of kaempferol was confirmed by an orthogonal assay that measures the ATP level (CellTiter-Glo luminescent cell viability assay, Promega, India) and trypan blue dye exclusion assay as a surrogate for the cell viability (Fig. and Supplementary Figure 4a).

    Techniques: Incubation, MTT Assay, Activity Assay, Inhibition, Activation Assay

    BNTX sensitizes human pancreatic cancer cells to TRAIL-induced apoptosis (A) Assay data for a screening analysis of AsPC-1 cells pre-incubated with various test compounds (5 μM) and were then exposed to TRAIL (50 ng/ml). Cell survival was measured as a percentage of the control sample. The arrow indicates BNTX (5 μM). (B) AsPC-1 cells were exposed to various concentrations of BNTX with or without TRAIL (25 ng/ml) for 24 h. Cell viability was measured by a luminescent CellTiter-Glo assay, and relative cell survival was determined by assaying ATP levels. (C) Relative survival of three pancreatic cancer cell lines (AsPC-1, PANC-1, and MIA PaCa-2) exposed to TRAIL (25 ng/ml) with or without BNTX (2.5 μM) for 24 h. Cell viability was measured by a luminescent CellTiter-Glo assay. (D) Apoptotic activity in AsPC-1 cells exposed to TRAIL (25 ng/ml) with or without BNTX (2.5 μM) for 24 h. Cells were analyzed by annexin V/PI double-stained flow cytometry. (E) The percentage of apoptotic cells. Values are the mean ± SD from two independent experiments performed in duplicate. Asterisks indicate significant differences compared with the control ( *** P

    Journal: Oncotarget

    Article Title: Downregulation of X-linked inhibitor of apoptosis protein by ‘7-Benzylidenenaltrexone maleate’ sensitizes pancreatic cancer cells to TRAIL-induced apoptosis

    doi: 10.18632/oncotarget.17841

    Figure Lengend Snippet: BNTX sensitizes human pancreatic cancer cells to TRAIL-induced apoptosis (A) Assay data for a screening analysis of AsPC-1 cells pre-incubated with various test compounds (5 μM) and were then exposed to TRAIL (50 ng/ml). Cell survival was measured as a percentage of the control sample. The arrow indicates BNTX (5 μM). (B) AsPC-1 cells were exposed to various concentrations of BNTX with or without TRAIL (25 ng/ml) for 24 h. Cell viability was measured by a luminescent CellTiter-Glo assay, and relative cell survival was determined by assaying ATP levels. (C) Relative survival of three pancreatic cancer cell lines (AsPC-1, PANC-1, and MIA PaCa-2) exposed to TRAIL (25 ng/ml) with or without BNTX (2.5 μM) for 24 h. Cell viability was measured by a luminescent CellTiter-Glo assay. (D) Apoptotic activity in AsPC-1 cells exposed to TRAIL (25 ng/ml) with or without BNTX (2.5 μM) for 24 h. Cells were analyzed by annexin V/PI double-stained flow cytometry. (E) The percentage of apoptotic cells. Values are the mean ± SD from two independent experiments performed in duplicate. Asterisks indicate significant differences compared with the control ( *** P

    Article Snippet: Cell viability assay and flow cytometry Cells were seeded at 2∼3 × 103 cells/well in 96-well plates and were pre-treated with BNTX at indicated concentrations for 0.5 h followed by TRAIL (25 ng/ml) for an additional 24 h. Cell viability assays were performed using a cellular ATP-based luminescence assay kit (CellTiter-Glo; Promega, Madison, WI).

    Techniques: Incubation, Glo Assay, Activity Assay, Staining, Flow Cytometry, Cytometry

    Intracellular ATP levels in wild-type and CatE −/− macrophages. Macrophages (1 × 10 5 cells) were cultured on 96-well plates at 37°C for 24 h. After replacement with fresh media, the culture plate was incubated at room temperature (25°C) for 30 min. ATP levels were determined by the manufacturer’s protocol of the CellTiter-Glo assay kit according to the manufacturer’s protocol. The concentration of intracellular ATP was determined by the titration of the control medium without cells plus 0.1−1.0 µM ATP.

    Journal: PLoS ONE

    Article Title: Cathepsin E Deficiency Impairs Autophagic Proteolysis in Macrophages

    doi: 10.1371/journal.pone.0082415

    Figure Lengend Snippet: Intracellular ATP levels in wild-type and CatE −/− macrophages. Macrophages (1 × 10 5 cells) were cultured on 96-well plates at 37°C for 24 h. After replacement with fresh media, the culture plate was incubated at room temperature (25°C) for 30 min. ATP levels were determined by the manufacturer’s protocol of the CellTiter-Glo assay kit according to the manufacturer’s protocol. The concentration of intracellular ATP was determined by the titration of the control medium without cells plus 0.1−1.0 µM ATP.

    Article Snippet: The ATP assay kit (CellTiter-Gio Luminescent cell viability assay) was purchased from Promega (Madison, WI, USA).

    Techniques: Cell Culture, Incubation, Glo Assay, Concentration Assay, Titration

    AQP1 functions as a genetically encoded reporter for diffusion-weighted MRI. ( a ) Illustration of the impact of aquaporin expression on water diffusion across the cell membrane and the resulting decrease in diffusion-weighted signal intensity. ( b ) Diffusion-weighted images of CHO, U87 and Neuro 2a cell pellets expressing AQP1 or GFP, acquired using a b -value of ∼1,000 s mm −2 . Scale bars, 3 mm. ( c ) ADC of water in CHO, U87 and Neuro 2a cells expressing AQP1 relative to GFP controls, measured at Δ eff =398 ms. Transgene expression in CHO cells was induced with 1 μg ml −1 doxycycline, whereas U87 and Neuro 2a cells express AQP1 from a constitutive promoter. n =4 (U87, Neuro 2a) and 5 (CHO) biological replicates. ( d ) Longitudinal ( T 1 ) and ( e ) transverse ( T 2 ) relaxation rates in cells expressing AQP1 or GFP. n =3 (Neuro 2a, CHO) or 4 (U87) biological replicates. ( f ) Cell viability on AQP1 or GFP expression. n =12 (resazurin assay), 6 (ATP content), 4 (LDH release) and 3 (ethidium staining) biological replicates. Error bars±s.e.m. ( g ) Phase-contrast images of CHO, U87 and Neuro 2a cells expressing AQP1 or GFP. Scale bars, 10 μm.

    Journal: Nature Communications

    Article Title: Non-invasive imaging using reporter genes altering cellular water permeability

    doi: 10.1038/ncomms13891

    Figure Lengend Snippet: AQP1 functions as a genetically encoded reporter for diffusion-weighted MRI. ( a ) Illustration of the impact of aquaporin expression on water diffusion across the cell membrane and the resulting decrease in diffusion-weighted signal intensity. ( b ) Diffusion-weighted images of CHO, U87 and Neuro 2a cell pellets expressing AQP1 or GFP, acquired using a b -value of ∼1,000 s mm −2 . Scale bars, 3 mm. ( c ) ADC of water in CHO, U87 and Neuro 2a cells expressing AQP1 relative to GFP controls, measured at Δ eff =398 ms. Transgene expression in CHO cells was induced with 1 μg ml −1 doxycycline, whereas U87 and Neuro 2a cells express AQP1 from a constitutive promoter. n =4 (U87, Neuro 2a) and 5 (CHO) biological replicates. ( d ) Longitudinal ( T 1 ) and ( e ) transverse ( T 2 ) relaxation rates in cells expressing AQP1 or GFP. n =3 (Neuro 2a, CHO) or 4 (U87) biological replicates. ( f ) Cell viability on AQP1 or GFP expression. n =12 (resazurin assay), 6 (ATP content), 4 (LDH release) and 3 (ethidium staining) biological replicates. Error bars±s.e.m. ( g ) Phase-contrast images of CHO, U87 and Neuro 2a cells expressing AQP1 or GFP. Scale bars, 10 μm.

    Article Snippet: Determination of cell viability Cell viability was determined using four different approaches including staining with ethidium homodimer-1 (Thermo Fisher) and measurement of resazurin reduction (CellTiter-Blue assay, Promega), ATP content (CellTiter-Glo assay, Promega) and lactate dehydrogenase release (CytoOne, Promega).

    Techniques: Diffusion-based Assay, Magnetic Resonance Imaging, Expressing, Mass Spectrometry, Resazurin Assay, Staining