propidium iodide pi  (Millipore)

 
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    Name:
    Propidium iodide
    Description:

    Catalog Number:
    P4170
    Price:
    None
    Applications:
    Fluorescent stain for nucleic acids. Cell membrane integrity excludes propidium iodide from staining viable and apoptotic cells. Propidium iodide may be used in flow cytometry to evaluate cell viability when used with other dyes that stain viable cells or cells that are early in the apoptosis process.
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    Structured Review

    Millipore propidium iodide pi
    Propidium iodide

    https://www.bioz.com/result/propidium iodide pi/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    propidium iodide pi - by Bioz Stars, 2021-04
    97/100 stars

    Images

    1) Product Images from "Nucleobase Derivatives as Building Blocks to Form Ru(II)-Based Complexes with High Cytotoxicity"

    Article Title: Nucleobase Derivatives as Building Blocks to Form Ru(II)-Based Complexes with High Cytotoxicity

    Journal: ACS Omega

    doi: 10.1021/acsomega.9b01921

    Effect of complexes 1 and 2 on apoptosis induction in HL-60 cells, as determined by flow cytometry using annexin V-FITC/propidium iodide staining after 24 h incubation. (A) Representative flow cytometric dot plots. (B) Quantification of viable (annexin V-FITC/PI double negative cells), early apoptosis (annexin V-FITC positive, but PI negative cells), late apoptosis (annexin V-FITC/PI double positive cells), and necrosis cells (PI positive, but annexin V-FITC negative cells). Negative control (CTL) was treated with vehicle (0.1% DMSO), and doxorubicin (DOX, 1 μM) was used as positive control. Data are presented as mean ± S.E.M. of three independent experiments performed in duplicate. Ten thousand events were evaluated per experiment, and cellular debris omitted from analysis. * P
    Figure Legend Snippet: Effect of complexes 1 and 2 on apoptosis induction in HL-60 cells, as determined by flow cytometry using annexin V-FITC/propidium iodide staining after 24 h incubation. (A) Representative flow cytometric dot plots. (B) Quantification of viable (annexin V-FITC/PI double negative cells), early apoptosis (annexin V-FITC positive, but PI negative cells), late apoptosis (annexin V-FITC/PI double positive cells), and necrosis cells (PI positive, but annexin V-FITC negative cells). Negative control (CTL) was treated with vehicle (0.1% DMSO), and doxorubicin (DOX, 1 μM) was used as positive control. Data are presented as mean ± S.E.M. of three independent experiments performed in duplicate. Ten thousand events were evaluated per experiment, and cellular debris omitted from analysis. * P

    Techniques Used: Flow Cytometry, Cytometry, Staining, Incubation, Negative Control, CTL Assay, Positive Control

    Effect of complexes 1 and 2 in the cell cycle distribution of HL-60 cells, as determined by flow cytometry using propidium iodide staining after 24 h incubation. (A) Representative flow cytometric histograms. (B) Quantification of sub-G 1 (internucleosomal DNA fragmentation), G 0 /G 1 , S, and G 2 /M percentage distribution. Negative control (CTL) was treated with vehicle (0.1% DMSO), and doxorubicin (DOX, 1 μM) was used as a positive control. Data are presented as mean ± S.E.M. of three independent experiments performed in duplicate. Ten thousand events were evaluated per experiment, and cellular debris omitted from analysis. * P
    Figure Legend Snippet: Effect of complexes 1 and 2 in the cell cycle distribution of HL-60 cells, as determined by flow cytometry using propidium iodide staining after 24 h incubation. (A) Representative flow cytometric histograms. (B) Quantification of sub-G 1 (internucleosomal DNA fragmentation), G 0 /G 1 , S, and G 2 /M percentage distribution. Negative control (CTL) was treated with vehicle (0.1% DMSO), and doxorubicin (DOX, 1 μM) was used as a positive control. Data are presented as mean ± S.E.M. of three independent experiments performed in duplicate. Ten thousand events were evaluated per experiment, and cellular debris omitted from analysis. * P

    Techniques Used: Flow Cytometry, Cytometry, Staining, Incubation, Negative Control, CTL Assay, Positive Control

    2) Product Images from "Antimicrobial Peptide AMP-17 Affects Candida albicans by Disrupting Its Cell Wall and Cell Membrane Integrity"

    Article Title: Antimicrobial Peptide AMP-17 Affects Candida albicans by Disrupting Its Cell Wall and Cell Membrane Integrity

    Journal: Infection and Drug Resistance

    doi: 10.2147/IDR.S250278

    Effect of AMP-17 on permeability of C. albicans cell membrane (600×). C. albicans cells were incubated with 0 (control), 20, 40, and 80 μg/mL AMP-17 at 37°C for 12 h. After staining with 20 μg/mL propidium iodide, the red fluorescence in samples was detected by confocal laser scanning microscopy at 488 nm and 525 nm of excitation and emission, respectively. The bars indicate 10 μm.
    Figure Legend Snippet: Effect of AMP-17 on permeability of C. albicans cell membrane (600×). C. albicans cells were incubated with 0 (control), 20, 40, and 80 μg/mL AMP-17 at 37°C for 12 h. After staining with 20 μg/mL propidium iodide, the red fluorescence in samples was detected by confocal laser scanning microscopy at 488 nm and 525 nm of excitation and emission, respectively. The bars indicate 10 μm.

    Techniques Used: Permeability, Incubation, Staining, Fluorescence, Confocal Laser Scanning Microscopy

    3) Product Images from "The in vitro and in vivo anti-melanoma effects of hydroxyapatite nanoparticles: influences of material factors"

    Article Title: The in vitro and in vivo anti-melanoma effects of hydroxyapatite nanoparticles: influences of material factors

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S184792

    ( A ) Density maps of FITC Annexin V apoptosis detection results and ( B ) the rates of apoptosis and necrosis obtained by double-staining with Annexin V-FITC/PI of A375 cells after culturing with various HANPs at the concentration of 200 μg/mL for 1 and 3 days (three duplicates for each experiment). Abbreviations: FITC, fluorescein isothiocyanate; PI, propidium iodide; HANP, hydroxyapatite nanoparticle.
    Figure Legend Snippet: ( A ) Density maps of FITC Annexin V apoptosis detection results and ( B ) the rates of apoptosis and necrosis obtained by double-staining with Annexin V-FITC/PI of A375 cells after culturing with various HANPs at the concentration of 200 μg/mL for 1 and 3 days (three duplicates for each experiment). Abbreviations: FITC, fluorescein isothiocyanate; PI, propidium iodide; HANP, hydroxyapatite nanoparticle.

    Techniques Used: Double Staining, Concentration Assay

    4) Product Images from "Overexpression of tissue inhibitors of metalloproteinase 2 up-regulates NF-?B activity in melanoma cells"

    Article Title: Overexpression of tissue inhibitors of metalloproteinase 2 up-regulates NF-?B activity in melanoma cells

    Journal: Journal of Molecular Signaling

    doi: 10.1186/1750-2187-4-4

    Apoptosis in A2058, A2058T2-1, and A2058T2R-7 with or without TNF-α treatment . After 6 h of treatment with TNF, 1 × 10 6 adherent cells were trypsinized and incubated with FITC-conjugated annexin V and propidium iodide (PI) for 15 min in the dark. Cells were analyzed by flow cytometry. The percentages of cells in each groups within the gated areas are indicated. In the gate areas, the up-right panel is for the population of later apoptotic cells and the low-right panel is for the early apoptosis cells. Dot plots of early apoptotic cells have increased Annexin V FITC fluorescence only, whereas necrotic and late apoptotic cells have increased annexin V and PI fluorescence. Axes are labelled with arbitrary fluorescence units. Data are from a representative of three independent experiments that gave similar results.
    Figure Legend Snippet: Apoptosis in A2058, A2058T2-1, and A2058T2R-7 with or without TNF-α treatment . After 6 h of treatment with TNF, 1 × 10 6 adherent cells were trypsinized and incubated with FITC-conjugated annexin V and propidium iodide (PI) for 15 min in the dark. Cells were analyzed by flow cytometry. The percentages of cells in each groups within the gated areas are indicated. In the gate areas, the up-right panel is for the population of later apoptotic cells and the low-right panel is for the early apoptosis cells. Dot plots of early apoptotic cells have increased Annexin V FITC fluorescence only, whereas necrotic and late apoptotic cells have increased annexin V and PI fluorescence. Axes are labelled with arbitrary fluorescence units. Data are from a representative of three independent experiments that gave similar results.

    Techniques Used: Incubation, Flow Cytometry, Cytometry, Fluorescence

    5) Product Images from "Inhibition of proliferation of prostate cancer cell line, PC-3, in vitro and in vivo using (-)-gossypol"

    Article Title: Inhibition of proliferation of prostate cancer cell line, PC-3, in vitro and in vivo using (-)-gossypol

    Journal: Asian Journal of Andrology

    doi: 10.1038/aja.2009.87

    Dose-dependent induction of apoptosis by (−)-gossypol in PC-3 cells. PC-3 cells (10 5 ) were treated with different concentrations of (−)-gossypol for 48 h. Cells were then stained with annexin V-FITC and propidium iodide and analysed using
    Figure Legend Snippet: Dose-dependent induction of apoptosis by (−)-gossypol in PC-3 cells. PC-3 cells (10 5 ) were treated with different concentrations of (−)-gossypol for 48 h. Cells were then stained with annexin V-FITC and propidium iodide and analysed using

    Techniques Used: Staining

    6) Product Images from "Overcoming acquired resistance to HSP90 inhibition by targeting JAK-STAT signalling in triple-negative breast cancer"

    Article Title: Overcoming acquired resistance to HSP90 inhibition by targeting JAK-STAT signalling in triple-negative breast cancer

    Journal: BMC Cancer

    doi: 10.1186/s12885-019-5295-z

    Resistance to HSP90i in CR2 and CR3. a Cross-resistance to HSP90i. Hs578T, CR2 and CR3 cells were treated with increasing concentrations of either ganetespib, NVP-AUY922 or 17-AAG for 72 h and subjected to resazurin-based cell viability assay. Cell viability (%) for each treatment was expressed relative to the DMSO-treated control. Representative graph from at least three independent experiments each performed in triplicate and error bars indicate SEM. The table represents the IC 50 values of compounds in the cell lines. The IC 50 values in respective clones were compared with the values in the parental Hs578T cells. *, *** and **** indicate p -value ≤0.05, ≤0.001 and ≤ 0.0001 respectively; by Student’s t-test. b Absence of downregulated expression of HSP90 client protein and induction of apoptosis. Hs578T, CR2 and CR3 cells were treated with 30 nM ganetespib for 24 h. Lysates were subjected to western blotting analysis and blotted with indicated antibodies. Level of cleaved PARP was also determined to assess apoptosis. PPIB and β-actin were used as loading controls. Representative images from two experiments are shown. c G2/M cell cycle arrest observed in parental Hs578T cells only. Hs578T, CR2 and CR3 cells were treated with 30 nM ganetespib for 24 h and pulse labelled with BrdU for 20 min. Cells were stained with propidium iodide and anti-BrdU antibody before analysed by flow cytometry. The top images represent the cell population with BrdU staining and PI staining, which consist of G0/G1- (bottom left), G2/M- (bottom right) and S-phase. The graph represents the mean percentage of cells in each phase of cell cycle from two independent experiments. Error bars indicate SEM
    Figure Legend Snippet: Resistance to HSP90i in CR2 and CR3. a Cross-resistance to HSP90i. Hs578T, CR2 and CR3 cells were treated with increasing concentrations of either ganetespib, NVP-AUY922 or 17-AAG for 72 h and subjected to resazurin-based cell viability assay. Cell viability (%) for each treatment was expressed relative to the DMSO-treated control. Representative graph from at least three independent experiments each performed in triplicate and error bars indicate SEM. The table represents the IC 50 values of compounds in the cell lines. The IC 50 values in respective clones were compared with the values in the parental Hs578T cells. *, *** and **** indicate p -value ≤0.05, ≤0.001 and ≤ 0.0001 respectively; by Student’s t-test. b Absence of downregulated expression of HSP90 client protein and induction of apoptosis. Hs578T, CR2 and CR3 cells were treated with 30 nM ganetespib for 24 h. Lysates were subjected to western blotting analysis and blotted with indicated antibodies. Level of cleaved PARP was also determined to assess apoptosis. PPIB and β-actin were used as loading controls. Representative images from two experiments are shown. c G2/M cell cycle arrest observed in parental Hs578T cells only. Hs578T, CR2 and CR3 cells were treated with 30 nM ganetespib for 24 h and pulse labelled with BrdU for 20 min. Cells were stained with propidium iodide and anti-BrdU antibody before analysed by flow cytometry. The top images represent the cell population with BrdU staining and PI staining, which consist of G0/G1- (bottom left), G2/M- (bottom right) and S-phase. The graph represents the mean percentage of cells in each phase of cell cycle from two independent experiments. Error bars indicate SEM

    Techniques Used: Viability Assay, Clone Assay, Expressing, Western Blot, Staining, Flow Cytometry, Cytometry, BrdU Staining

    7) Product Images from "Involvement of cyclin B1 in progesterone-mediated cell growth inhibition, G2/M cell cycle arrest, and apoptosis in human endometrial cell"

    Article Title: Involvement of cyclin B1 in progesterone-mediated cell growth inhibition, G2/M cell cycle arrest, and apoptosis in human endometrial cell

    Journal: Reproductive Biology and Endocrinology : RB & E

    doi: 10.1186/1477-7827-7-144

    Arrest of cell cycle in G2/M phase induced by progesterone and Alp . Human endometrial cells were treated with progesterone alone or in combination with Alp. Cell cycles were analyzed by flow cytometry. A: Cell cycle analyzed with propidium iodide (PI) staining followed by flow cytometry. B: Comparison of the proportions of cells in G2/M phase. The proportion of cells in G2/M phase was significantly increased after hECs were treated with progesterone or progesterone and Alp (*P
    Figure Legend Snippet: Arrest of cell cycle in G2/M phase induced by progesterone and Alp . Human endometrial cells were treated with progesterone alone or in combination with Alp. Cell cycles were analyzed by flow cytometry. A: Cell cycle analyzed with propidium iodide (PI) staining followed by flow cytometry. B: Comparison of the proportions of cells in G2/M phase. The proportion of cells in G2/M phase was significantly increased after hECs were treated with progesterone or progesterone and Alp (*P

    Techniques Used: ALP Assay, Flow Cytometry, Cytometry, Staining

    8) Product Images from "Excess biglycan causes eyelid malformation by perturbing muscle development and TGF-? signaling"

    Article Title: Excess biglycan causes eyelid malformation by perturbing muscle development and TGF-? signaling

    Journal: Developmental biology

    doi: 10.1016/j.ydbio.2004.09.022

    Expression of TGF-α, EGF, and HB-EGF, and activation of EGFR and cJun during eyelid morphogenesis of Kera-Bgn mice. (A) Western blot analysis of TGF-α, EGF, HB-EGF, EGFR, pEGFR, cJun, and pcJun of eyelid extracts from E13.5 embryos shows downregulated expression of HB-EGF and cJun, and phosphorylation of EGFR and cJun in Kera-Bgn (Tg) mice. (B) In situ hybridization detects TGF-α mRNA in epithelium and migrating mesenchymal cells of wild-type (Wt) and Kera-Bgn (Tg) eyelid. Immunohistochemistry shows TGF-α distribution is limited to the upper one-third of eyelid stroma adjacent to the epithelium of Kera-Bgn (Tg) mice, but it is more evenly distributed along the stroma of wild-type (Wt) mice. The expression of HB-EGF (green) is also downregulated in the eyelids of Kera-Bgn mice (counter stained with propidium iodide, red). (C) EGFR (red) and pEGFR (green) are detected in migrating mesenchymal cells and epidermal epithelium of E13.5 wild-type (Wt) eyelid, whereas lower pEGFR positive signals are observed in Kera-Bgn (Tg) littermates. (D) cJun and pcJun expression (green) in migrating mesenchymal cells. The tissue sections are counterstained with propidium iodide (red). cJun is expressed at E13.5 by both wild-type (Wt) and Kera-Bgn (Tg) eyelid mesenchymal cells. Fewer cells express pcJun in Kera-Bgn (Tg) eyelid stroma compared to wild-type littermates. (E) Diagrams illustrating the EGFR signaling mediated by TGF-α during eyelid morphogenesis in wild-type (Wt) and Kera-Bgn (Tg) mice. The presence of excess biglycan in transgenic mice sequesters TGF-α and consequently perturbs the autocrine or paracrine loop of EGFR signaling pathways via HB-EGF and impairs mesenchymal cell migration.
    Figure Legend Snippet: Expression of TGF-α, EGF, and HB-EGF, and activation of EGFR and cJun during eyelid morphogenesis of Kera-Bgn mice. (A) Western blot analysis of TGF-α, EGF, HB-EGF, EGFR, pEGFR, cJun, and pcJun of eyelid extracts from E13.5 embryos shows downregulated expression of HB-EGF and cJun, and phosphorylation of EGFR and cJun in Kera-Bgn (Tg) mice. (B) In situ hybridization detects TGF-α mRNA in epithelium and migrating mesenchymal cells of wild-type (Wt) and Kera-Bgn (Tg) eyelid. Immunohistochemistry shows TGF-α distribution is limited to the upper one-third of eyelid stroma adjacent to the epithelium of Kera-Bgn (Tg) mice, but it is more evenly distributed along the stroma of wild-type (Wt) mice. The expression of HB-EGF (green) is also downregulated in the eyelids of Kera-Bgn mice (counter stained with propidium iodide, red). (C) EGFR (red) and pEGFR (green) are detected in migrating mesenchymal cells and epidermal epithelium of E13.5 wild-type (Wt) eyelid, whereas lower pEGFR positive signals are observed in Kera-Bgn (Tg) littermates. (D) cJun and pcJun expression (green) in migrating mesenchymal cells. The tissue sections are counterstained with propidium iodide (red). cJun is expressed at E13.5 by both wild-type (Wt) and Kera-Bgn (Tg) eyelid mesenchymal cells. Fewer cells express pcJun in Kera-Bgn (Tg) eyelid stroma compared to wild-type littermates. (E) Diagrams illustrating the EGFR signaling mediated by TGF-α during eyelid morphogenesis in wild-type (Wt) and Kera-Bgn (Tg) mice. The presence of excess biglycan in transgenic mice sequesters TGF-α and consequently perturbs the autocrine or paracrine loop of EGFR signaling pathways via HB-EGF and impairs mesenchymal cell migration.

    Techniques Used: Expressing, Activation Assay, Mouse Assay, Western Blot, In Situ Hybridization, Immunohistochemistry, Staining, Transgenic Assay, Migration

    9) Product Images from "Small molecule inhibitor screening identifified HSP90 inhibitor 17-AAG as potential therapeutic agent for gallbladder cancer"

    Article Title: Small molecule inhibitor screening identifified HSP90 inhibitor 17-AAG as potential therapeutic agent for gallbladder cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.15410

    Effect of 17-AAG and GA on apoptosis in human GBC cells ( A and B ) G-415 and ( C and D ) GB-d1 cells were treated with 17-AAG or GA and harvested after 24, 48, and 72 hours for annexin V /propidium iodide (PI) staining and analized by flow cytometry. Data are shown as mean ± SD (* P
    Figure Legend Snippet: Effect of 17-AAG and GA on apoptosis in human GBC cells ( A and B ) G-415 and ( C and D ) GB-d1 cells were treated with 17-AAG or GA and harvested after 24, 48, and 72 hours for annexin V /propidium iodide (PI) staining and analized by flow cytometry. Data are shown as mean ± SD (* P

    Techniques Used: Staining, Flow Cytometry, Cytometry

    10) Product Images from "Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity"

    Article Title: Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36718-0

    Tc HMGB overexpression alters cell cycle progression. ( A ) Flow Cytometry analysis of the cell cycle progression. On the left, Flow Cytometry analysis of cultured T . cruzi Dm28c/p Tc INDEX-GW- Tc HMGB(HA) 2 epimastigotes at different times post-tetracycline induction (p.i.). Histograms are plotted as number of events vs. propidium iodide absorbance (PI-A). On the right, bar graph with the percentages of cells in the different phases of the cell cycle. **p
    Figure Legend Snippet: Tc HMGB overexpression alters cell cycle progression. ( A ) Flow Cytometry analysis of the cell cycle progression. On the left, Flow Cytometry analysis of cultured T . cruzi Dm28c/p Tc INDEX-GW- Tc HMGB(HA) 2 epimastigotes at different times post-tetracycline induction (p.i.). Histograms are plotted as number of events vs. propidium iodide absorbance (PI-A). On the right, bar graph with the percentages of cells in the different phases of the cell cycle. **p

    Techniques Used: Over Expression, Flow Cytometry, Cytometry, Cell Culture

    Related Articles

    Incubation:

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    Article Snippet: Glass slides with HEp-2 cells attached (ANA-screen, Immuno Concepts, Sacramento, CA or Bion Enterprises) were also used. .. Cells were incubated with propidium iodine (Sigma) or the following antibodies: purified Fab ANA15 (2–5 μg/ml), donor serum (1:640), preparation bacterial Fab supernatant (1:2), rabbit anti-human serum (1:100), mouse anti-human rough endoplasmic reticulum antibody (1:50; Dako), mouse anti-human nucleoli antibody (1:2; Biodesign International, Kennebunkport, ME), and mouse anti-human α-tubulin antibody (1:100). .. The cells were washed with PBS and incubated with FITC-labeled (Fab′)2 goat anti-human IgG (Fab′)2 antibody, Texas Red-labeled goat anti-mouse IgG antibody, or CY-5-labeled goat anti-rabbit IgG antibody (all from Jackson ImmunoResearch).

    Article Title: Release of Membrane-Bound Vesicles and Inhibition of Tumor Cell Adhesion by the Peptide Neopetrosiamide A
    Article Snippet: .. All cells were then pooled, collected through centrifugation, re-suspended in 100 µl binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl2 ) and incubated with 5 µl FITC Annexin V (BD Biosciences) and 10 µl of propidium iodide (PI; 50 µg/ml; Sigma) for 15 min at room temperature. .. Staining for PI (relative fluorescence intensity measured in FL2 channel) and Annexin V (measured in FL1 channel) was assessed by flow cytometry on a FACSCalibur instrument (BD Biosciences) followed by data analysis using FlowJo software (Tree Star Inc).

    Article Title: Ontogenetic Changes in Auxin Biosynthesis and Distribution Determine the Organogenic Activity of the Shoot Apical Meristem in pin1 Mutants
    Article Snippet: .. To visualize the three-dimensional (3D) structure of the xylem strands, inflorescence stems of WT plants (n = 15) and pin1 mutants (n = 34) were fixed in FAA, hydrated in a graded ethanol series, incubated in 10% KOH for 2–4 days at 37 °C, rinsed in water, and stained with 0.05 mg/mL propidium iodide (PI; Sigma-Aldrich, Steinheim, Germany) for 1 h. .. Auxin Immunolocalization Apical parts of 1 cm long of the inflorescences of WT plants (n = 6) and pin1 mutants (n = 24) were incubated at 4 °C in 3% N -(3-Dimethylaminopropyl)-N ′-ethylcarbodiimide hydrochloride (EDAC; Sigma-Aldrich) dissolved in 0.1× phosphate buffered saline (PBS) (pH = 7.4; Sigma-Aldrich) with 0.1% Triton X-100 (POCH, Gliwice, Poland) for 4 h. Then, the material was fixed 24 h in FAA, rinsed in 50% ethanol, and hydrated in a graded ethanol series.

    Purification:

    Article Title: Cloning and expression of a novel human antibody-antigen pair associated with Felty's syndrome
    Article Snippet: Glass slides with HEp-2 cells attached (ANA-screen, Immuno Concepts, Sacramento, CA or Bion Enterprises) were also used. .. Cells were incubated with propidium iodine (Sigma) or the following antibodies: purified Fab ANA15 (2–5 μg/ml), donor serum (1:640), preparation bacterial Fab supernatant (1:2), rabbit anti-human serum (1:100), mouse anti-human rough endoplasmic reticulum antibody (1:50; Dako), mouse anti-human nucleoli antibody (1:2; Biodesign International, Kennebunkport, ME), and mouse anti-human α-tubulin antibody (1:100). .. The cells were washed with PBS and incubated with FITC-labeled (Fab′)2 goat anti-human IgG (Fab′)2 antibody, Texas Red-labeled goat anti-mouse IgG antibody, or CY-5-labeled goat anti-rabbit IgG antibody (all from Jackson ImmunoResearch).

    Staining:

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    Article Title: Nestin-positive mesenchymal stem cells favour the astroglial lineage in neural progenitors and stem cells by releasing active BMP4
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    Article Title: Vpr R77Q is associated with long-term nonprogressive HIV infection and impaired induction of apoptosis
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    Article Title: Ontogenetic Changes in Auxin Biosynthesis and Distribution Determine the Organogenic Activity of the Shoot Apical Meristem in pin1 Mutants
    Article Snippet: .. To visualize the three-dimensional (3D) structure of the xylem strands, inflorescence stems of WT plants (n = 15) and pin1 mutants (n = 34) were fixed in FAA, hydrated in a graded ethanol series, incubated in 10% KOH for 2–4 days at 37 °C, rinsed in water, and stained with 0.05 mg/mL propidium iodide (PI; Sigma-Aldrich, Steinheim, Germany) for 1 h. .. Auxin Immunolocalization Apical parts of 1 cm long of the inflorescences of WT plants (n = 6) and pin1 mutants (n = 24) were incubated at 4 °C in 3% N -(3-Dimethylaminopropyl)-N ′-ethylcarbodiimide hydrochloride (EDAC; Sigma-Aldrich) dissolved in 0.1× phosphate buffered saline (PBS) (pH = 7.4; Sigma-Aldrich) with 0.1% Triton X-100 (POCH, Gliwice, Poland) for 4 h. Then, the material was fixed 24 h in FAA, rinsed in 50% ethanol, and hydrated in a graded ethanol series.

    MTT Assay:

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    Centrifugation:

    Article Title: Release of Membrane-Bound Vesicles and Inhibition of Tumor Cell Adhesion by the Peptide Neopetrosiamide A
    Article Snippet: .. All cells were then pooled, collected through centrifugation, re-suspended in 100 µl binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl2 ) and incubated with 5 µl FITC Annexin V (BD Biosciences) and 10 µl of propidium iodide (PI; 50 µg/ml; Sigma) for 15 min at room temperature. .. Staining for PI (relative fluorescence intensity measured in FL2 channel) and Annexin V (measured in FL1 channel) was assessed by flow cytometry on a FACSCalibur instrument (BD Biosciences) followed by data analysis using FlowJo software (Tree Star Inc).

    Binding Assay:

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    Article Snippet: .. All cells were then pooled, collected through centrifugation, re-suspended in 100 µl binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl2 ) and incubated with 5 µl FITC Annexin V (BD Biosciences) and 10 µl of propidium iodide (PI; 50 µg/ml; Sigma) for 15 min at room temperature. .. Staining for PI (relative fluorescence intensity measured in FL2 channel) and Annexin V (measured in FL1 channel) was assessed by flow cytometry on a FACSCalibur instrument (BD Biosciences) followed by data analysis using FlowJo software (Tree Star Inc).

    Marker:

    Article Title: Nestin-positive mesenchymal stem cells favour the astroglial lineage in neural progenitors and stem cells by releasing active BMP4
    Article Snippet: .. BrdU and propidium iodide incorporation After 24 hours or 3 days of culture, BrdU (20 μM, Sigma) which is a S-phase marker, or propidium iodide (400 mg/ml, Sigma) was added to the differentiating neurosphere cultures for 18 hours before fixation and staining. ..

    End Labeling:

    Article Title: Vpr R77Q is associated with long-term nonprogressive HIV infection and impaired induction of apoptosis
    Article Snippet: Cells were plated at 106 cells per well and incubated with varying concentrations of Vpr wild-type or Vpr R77Q peptides in isotonic buffer for 30 minutes followed by culture in complete media. .. Cells were collected at 8–20 hours and stained either with annexin V FITC (BD Pharmingen, San Diego, California, USA) and 1 mg/ml propidium iodide (Sigma-Aldrich) or by terminal deoxyuridine nucleotide end labeling (TUNEL; Boehringer Mannheim, Indianapolis, Indiana, USA) according to the manufacturers’ instructions. .. Mitochondrial transmembrane potential (Δψm ) was quantified using 40 μM DiOC6 (Molecular Probes, Eugene, Oregon, USA) according to the manufacturer’s instructions, and 4 μM dihydroethidine (Molecular Probes) was used to determine superoxide anion generation as a measure of reactive oxygen species (ROS) production.

    TUNEL Assay:

    Article Title: Vpr R77Q is associated with long-term nonprogressive HIV infection and impaired induction of apoptosis
    Article Snippet: Cells were plated at 106 cells per well and incubated with varying concentrations of Vpr wild-type or Vpr R77Q peptides in isotonic buffer for 30 minutes followed by culture in complete media. .. Cells were collected at 8–20 hours and stained either with annexin V FITC (BD Pharmingen, San Diego, California, USA) and 1 mg/ml propidium iodide (Sigma-Aldrich) or by terminal deoxyuridine nucleotide end labeling (TUNEL; Boehringer Mannheim, Indianapolis, Indiana, USA) according to the manufacturers’ instructions. .. Mitochondrial transmembrane potential (Δψm ) was quantified using 40 μM DiOC6 (Molecular Probes, Eugene, Oregon, USA) according to the manufacturer’s instructions, and 4 μM dihydroethidine (Molecular Probes) was used to determine superoxide anion generation as a measure of reactive oxygen species (ROS) production.

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    Millipore propidium iodide pi
    Increased sensitivity to lysenin of U937 versus THP-1 cells. ( A ) Equal numbers of U937 and THP-1 cells were stimulated with 13.75 ng lysenin/10 6 cells and cell permeabilization was monitored using <t>propidium</t> iodide influx after 5 min (left panel) or 15 min (right panel) post toxin addition by FACS; ( B ) cells were stimulated with 13.75 ng lysenin/10 6 cells and cell lysis after 5 min (left panel) and 15 min (right panel) were analyzed by FACS; ( C ) cells were stimulated 32 ng lysenin/10 6 cells for 30 min. Subsequently, the unbound toxin was removed and the cells were recovered in full RPMI media for 24 h. Percentages of living cells at T = 24 h were assessed using the Alamar blue ® assay. Mean ± SEM is shown (n = 3, * p
    Propidium Iodide Pi, supplied by Millipore, 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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    99
    Millipore rnase a
    Cell cycle analysis. (A) MDA-MB-231 cells (1 × 10 5 ) were treated with 10 nM docetaxel and/or 1 µM ABT-737 for indicated time periods and stained with 50 µg/mL of propidium iodide including 100 µg/mL of <t>RNase</t> A for 30 minutes at 37℃. Top, middle, and bottom rows were treated with ABT-737, docetaxel, and both drugs, respectively. Graphs depict data of flow cytometry results after treatment for 72 hours according to cell cycle (B) and treatment agents (C). Values correspond to mean ± standard deviation of 3 independent experiments. Statistical significance was assessed by unpaired Student t-test. NS, not significant. * P
    Rnase A, supplied by Millipore, 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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    99
    Millipore propidium iodide
    Metformin activates apoptotic cell death; ( A ) MDA-MB-231 cells were plated in 96 well plate and then either left untreated or treated with millimolar (upper graphs) or micromolar (lower graphs) doses of metformin. ADP/ATP ratio was determined as indicated in Material and Methods; ( B ) MDA-MB-231 cells were seeded on glass coverslip and then left untreated or treated with 30 μM metformin. At the end of the treatment, cells were stained with 10 μg/mL JC-1 as indicated under Material and Methods. Mitochondria fluorescence was observed by confocal microscopy. First row: Red fluorescence representing JC1 aggregates. Second row: Green fluorescence representing JC1 monomers. Third row: Merging of the first two rows. Alternatively, cells were treated with 30 μM metformin and stained with JC-1 as above. Red (FL2H) and green (FL1H) fluorescence intensity was quantified by Flow Cytometry; ( C )upper panel: MDA-MB-231 cells were kept in the presence or absence of metformin for the time indicated and then processed to obtain mitochondrial fractions. BAX and CYCS expression levels were determined by Western blot as indicated under Material and Methods. Densitometric analysis of the gels was performed as indicated under Material and Methods. PHB and CDK4 were used as loading and purity control, respectively. Lower panel: MDA-MB-231 cells were kept in the presence or absence of metformin for the time indicated and then processed to obtain whole cellular extracts. BAX and CYCS expression levels were determined by Western blot as indicated under Material and Methods. Densitometric analysis of the gels was performed as indicated under Material and Methods. CDK4 and PHB were used as loading control. Right panel: MCF-7 cells were kept in the presence or absence of metformin for 20 days and then processed to obtain mitochondrial or whole cellular extracts. BAX and CYCS expression levels were determined by Western blot as indicated under Material and Methods. Densitometric analysis of the gels was performed as indicated under Material and Methods. PHB and CDK4 were used as purity and loading controls; ( D ) MDA-MB-231 cells were kept in the presence or absence of metformin for 20 days. At the end of the treatment cells were harvested and percentage of sub-G 1 (M1) cells was determined by <t>propidium</t> iodide staining as described in the Material and Methods section. All experiments in this figure were repeated three times. * Significantly different from control untreated cells.
    Propidium Iodide, supplied by Millipore, 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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    Increased sensitivity to lysenin of U937 versus THP-1 cells. ( A ) Equal numbers of U937 and THP-1 cells were stimulated with 13.75 ng lysenin/10 6 cells and cell permeabilization was monitored using propidium iodide influx after 5 min (left panel) or 15 min (right panel) post toxin addition by FACS; ( B ) cells were stimulated with 13.75 ng lysenin/10 6 cells and cell lysis after 5 min (left panel) and 15 min (right panel) were analyzed by FACS; ( C ) cells were stimulated 32 ng lysenin/10 6 cells for 30 min. Subsequently, the unbound toxin was removed and the cells were recovered in full RPMI media for 24 h. Percentages of living cells at T = 24 h were assessed using the Alamar blue ® assay. Mean ± SEM is shown (n = 3, * p

    Journal: Toxins

    Article Title: Small Pore-Forming Toxins Different Membrane Area Binding and Ca2+ Permeability of Pores Determine Cellular Resistance of Monocytic Cells †

    doi: 10.3390/toxins13020126

    Figure Lengend Snippet: Increased sensitivity to lysenin of U937 versus THP-1 cells. ( A ) Equal numbers of U937 and THP-1 cells were stimulated with 13.75 ng lysenin/10 6 cells and cell permeabilization was monitored using propidium iodide influx after 5 min (left panel) or 15 min (right panel) post toxin addition by FACS; ( B ) cells were stimulated with 13.75 ng lysenin/10 6 cells and cell lysis after 5 min (left panel) and 15 min (right panel) were analyzed by FACS; ( C ) cells were stimulated 32 ng lysenin/10 6 cells for 30 min. Subsequently, the unbound toxin was removed and the cells were recovered in full RPMI media for 24 h. Percentages of living cells at T = 24 h were assessed using the Alamar blue ® assay. Mean ± SEM is shown (n = 3, * p

    Article Snippet: In brief, 2 × 106 cells in Ca2+ Tyrode’s buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 10 mM glucose, 10 mM HEPES (pH 7.4), 2.5 mM Ca2+ ) were incubated with 20 μg/mL propidium iodide (PI) (Sigma-Aldrich, Munich, Germany).

    Techniques: FACS, Lysis, Alamar Blue Assay

    Enhanced permeabilization to aerolysin treatment of U937 versus THP-1 cells. ( A ) Equal numbers of U937 and THP-1. cells were stimulated with increasing concentrations of aerolysin [ng/10 6 cells] and cell permeabilization was monitored using propidium iodide influx after 5 min (left panel) or 15 min (right panel) by FACS; ( B ) cells were stimulated with 20 ng aerolysin/10 6 cells and cell lysis after 5 min (left panel) and 15 min (right panel) were analyzed by FACS; ( C ) cells were stimulated with 2 ng aerolysin/10 6 cells for 30 min. Subsequently, the unbound toxin was removed, and the cells were recovered in full RPMI media for 24 h. Percentages of living cells at T = 24 h were assessed using the Alamar blue ® assay. Mean ± SEM is shown (n = 3, * p

    Journal: Toxins

    Article Title: Small Pore-Forming Toxins Different Membrane Area Binding and Ca2+ Permeability of Pores Determine Cellular Resistance of Monocytic Cells †

    doi: 10.3390/toxins13020126

    Figure Lengend Snippet: Enhanced permeabilization to aerolysin treatment of U937 versus THP-1 cells. ( A ) Equal numbers of U937 and THP-1. cells were stimulated with increasing concentrations of aerolysin [ng/10 6 cells] and cell permeabilization was monitored using propidium iodide influx after 5 min (left panel) or 15 min (right panel) by FACS; ( B ) cells were stimulated with 20 ng aerolysin/10 6 cells and cell lysis after 5 min (left panel) and 15 min (right panel) were analyzed by FACS; ( C ) cells were stimulated with 2 ng aerolysin/10 6 cells for 30 min. Subsequently, the unbound toxin was removed, and the cells were recovered in full RPMI media for 24 h. Percentages of living cells at T = 24 h were assessed using the Alamar blue ® assay. Mean ± SEM is shown (n = 3, * p

    Article Snippet: In brief, 2 × 106 cells in Ca2+ Tyrode’s buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 10 mM glucose, 10 mM HEPES (pH 7.4), 2.5 mM Ca2+ ) were incubated with 20 μg/mL propidium iodide (PI) (Sigma-Aldrich, Munich, Germany).

    Techniques: FACS, Lysis, Alamar Blue Assay

    Icariin induces apoptosis in U266 cells. (A) U266 cells (1 × 10 6 cells/well) were treated with various indicated concentrations for 24 h. Then 10 μg proteins of those whole cell extracts was loaded on 15% SDS–PAGE gel followed by western blot analysis against Bcl-2, Bcl-xl, Survivin, IAP-1, IAP-2, COX-2, VEGF, and MMP-9. (B) U266 cells (1 × 10 6 cells/well) were treated with various indicated concentrations for 8 h. Total RNA was extracted and equal amounts were prepared to probe for Bcl-2, Bcl-xl, and Survivin by RT-PCR. (C) U266 cells (1 × 10 6 cells/well) were treated with various indicated concentrations for 24 h. Equal amounts of whole cell lysates were prepared and performed to detect caspase-3 and PARP by western blotting. (D) After Icariin treatment (0, 50, 100 μM for 24 h), cells were washed with PBS and digested with RNase A for 1 h, then stained with propidium iodide and analyzed cell for cycle division using flow cytometry. (E) After U266 cells (1 × 10 6 cells/well) were incubated with 100 μM icariin for 24 h, stained with Annexin V-FITC for 15 min and add PI then were analyzed by flow cytometry. (F) U266 cells (1 × 10 4 cells/well) were incubated with icariin (0, 50, 100 μM) for different indicated time periods. After incubation, difference in degree of cell proliferation was analyzed by MTT assay. (G) U266 cells (1 × 10 4 cells/well) and PBMC cells (1 × 10 4 cells/well) were incubated with 100 μM icariin for 24 h. The results shown are representative of three independent experiments.

    Journal: Frontiers in Pharmacology

    Article Title: Anti-myeloma Effects of Icariin Are Mediated Through the Attenuation of JAK/STAT3-Dependent Signaling Cascade

    doi: 10.3389/fphar.2018.00531

    Figure Lengend Snippet: Icariin induces apoptosis in U266 cells. (A) U266 cells (1 × 10 6 cells/well) were treated with various indicated concentrations for 24 h. Then 10 μg proteins of those whole cell extracts was loaded on 15% SDS–PAGE gel followed by western blot analysis against Bcl-2, Bcl-xl, Survivin, IAP-1, IAP-2, COX-2, VEGF, and MMP-9. (B) U266 cells (1 × 10 6 cells/well) were treated with various indicated concentrations for 8 h. Total RNA was extracted and equal amounts were prepared to probe for Bcl-2, Bcl-xl, and Survivin by RT-PCR. (C) U266 cells (1 × 10 6 cells/well) were treated with various indicated concentrations for 24 h. Equal amounts of whole cell lysates were prepared and performed to detect caspase-3 and PARP by western blotting. (D) After Icariin treatment (0, 50, 100 μM for 24 h), cells were washed with PBS and digested with RNase A for 1 h, then stained with propidium iodide and analyzed cell for cycle division using flow cytometry. (E) After U266 cells (1 × 10 6 cells/well) were incubated with 100 μM icariin for 24 h, stained with Annexin V-FITC for 15 min and add PI then were analyzed by flow cytometry. (F) U266 cells (1 × 10 4 cells/well) were incubated with icariin (0, 50, 100 μM) for different indicated time periods. After incubation, difference in degree of cell proliferation was analyzed by MTT assay. (G) U266 cells (1 × 10 4 cells/well) and PBMC cells (1 × 10 4 cells/well) were incubated with 100 μM icariin for 24 h. The results shown are representative of three independent experiments.

    Article Snippet: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), Tris base, glycine, NaCl, sodium dodecylsulfate (SDS), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: SDS Page, Western Blot, Reverse Transcription Polymerase Chain Reaction, Staining, Flow Cytometry, Cytometry, Incubation, MTT Assay

    Icariin and Bortezomib induce apoptosis by caspase-3 and PARP in U266 cells. (A) To confirm the synergistic effect between icariin and bortezomib on cell cycle, U266 cells (1 × 10 6 cells/well) were incubated with icariin (10 μM) and bortezomib (1 nM) for 24h then treated with RNase A for 1h. After staining with propidium iodide, cells were analyzed by flow cytometry. (B) U266 cells were treated with icariin and bortezomib for 24 h. Cells were fixed and stained with TUNEL assay reagent, then analyzed with a flow cytometer. (C,D) We confirm the synergistic effect for induced apoptosis by western blot analysis. U266 cells (1 × 10 6 cells/well) were treated with 10 μM icariin and 1 nM bortezomib for 24 h. Same amounts of whole cell lysates were prepared and probed using Bcl-2, Bcl-xl, Survivin, IAP-1, IAP-2, COX-2, VEGF, MMP-9, caspase-3, and PARP antibodies then analyzed by Western blotting. (E) To confirm the anti-cancer effect of icariin and bortezomib, U266 cells (1 × 10 6 cells/well) were incubated with icariin (10 μM) and bortezomib (1 nM) for 24h then proteins were resolved on SDS–PAGE and probed against p21 antibody. (F) U266 cells were transfected with STAT3 siRNA or scramble siRNA for 48 h, then 100 μM of icariin were treated for 24 h. The Whole cell lysates were prepared and 15 μg proteins were resolved on SDS–PAGE and probed against PARP antibody. b-actin was used as internal controls. (G) U266 cells were transfected with STAT3 siRNA or scramble siRNA for 48 h, then 100 μM of icariin were treated for 24 h. Then cell viability was analyzed by MTT assay. The results shown are representative of three independent experiments.

    Journal: Frontiers in Pharmacology

    Article Title: Anti-myeloma Effects of Icariin Are Mediated Through the Attenuation of JAK/STAT3-Dependent Signaling Cascade

    doi: 10.3389/fphar.2018.00531

    Figure Lengend Snippet: Icariin and Bortezomib induce apoptosis by caspase-3 and PARP in U266 cells. (A) To confirm the synergistic effect between icariin and bortezomib on cell cycle, U266 cells (1 × 10 6 cells/well) were incubated with icariin (10 μM) and bortezomib (1 nM) for 24h then treated with RNase A for 1h. After staining with propidium iodide, cells were analyzed by flow cytometry. (B) U266 cells were treated with icariin and bortezomib for 24 h. Cells were fixed and stained with TUNEL assay reagent, then analyzed with a flow cytometer. (C,D) We confirm the synergistic effect for induced apoptosis by western blot analysis. U266 cells (1 × 10 6 cells/well) were treated with 10 μM icariin and 1 nM bortezomib for 24 h. Same amounts of whole cell lysates were prepared and probed using Bcl-2, Bcl-xl, Survivin, IAP-1, IAP-2, COX-2, VEGF, MMP-9, caspase-3, and PARP antibodies then analyzed by Western blotting. (E) To confirm the anti-cancer effect of icariin and bortezomib, U266 cells (1 × 10 6 cells/well) were incubated with icariin (10 μM) and bortezomib (1 nM) for 24h then proteins were resolved on SDS–PAGE and probed against p21 antibody. (F) U266 cells were transfected with STAT3 siRNA or scramble siRNA for 48 h, then 100 μM of icariin were treated for 24 h. The Whole cell lysates were prepared and 15 μg proteins were resolved on SDS–PAGE and probed against PARP antibody. b-actin was used as internal controls. (G) U266 cells were transfected with STAT3 siRNA or scramble siRNA for 48 h, then 100 μM of icariin were treated for 24 h. Then cell viability was analyzed by MTT assay. The results shown are representative of three independent experiments.

    Article Snippet: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), Tris base, glycine, NaCl, sodium dodecylsulfate (SDS), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: Incubation, Staining, Flow Cytometry, Cytometry, TUNEL Assay, Western Blot, SDS Page, Transfection, MTT Assay

    Cell cycle analysis. (A) MDA-MB-231 cells (1 × 10 5 ) were treated with 10 nM docetaxel and/or 1 µM ABT-737 for indicated time periods and stained with 50 µg/mL of propidium iodide including 100 µg/mL of RNase A for 30 minutes at 37℃. Top, middle, and bottom rows were treated with ABT-737, docetaxel, and both drugs, respectively. Graphs depict data of flow cytometry results after treatment for 72 hours according to cell cycle (B) and treatment agents (C). Values correspond to mean ± standard deviation of 3 independent experiments. Statistical significance was assessed by unpaired Student t-test. NS, not significant. * P

    Journal: Annals of Surgical Treatment and Research

    Article Title: ABT-737 ameliorates docetaxel resistance in triple negative breast cancer cell line

    doi: 10.4174/astr.2018.95.5.240

    Figure Lengend Snippet: Cell cycle analysis. (A) MDA-MB-231 cells (1 × 10 5 ) were treated with 10 nM docetaxel and/or 1 µM ABT-737 for indicated time periods and stained with 50 µg/mL of propidium iodide including 100 µg/mL of RNase A for 30 minutes at 37℃. Top, middle, and bottom rows were treated with ABT-737, docetaxel, and both drugs, respectively. Graphs depict data of flow cytometry results after treatment for 72 hours according to cell cycle (B) and treatment agents (C). Values correspond to mean ± standard deviation of 3 independent experiments. Statistical significance was assessed by unpaired Student t-test. NS, not significant. * P

    Article Snippet: After centrifugation at 850 g for 5 minutes at 4℃, cell pellet was incubated with 100-µg/mL RNase A (Sigma-Aldrich) and 50-µg/mL propidium iodide (Thermo Fisher Scientific) at 37℃ for 30 minutes.

    Techniques: Cell Cycle Assay, Multiple Displacement Amplification, Staining, Flow Cytometry, Cytometry, Standard Deviation

    Metformin activates apoptotic cell death; ( A ) MDA-MB-231 cells were plated in 96 well plate and then either left untreated or treated with millimolar (upper graphs) or micromolar (lower graphs) doses of metformin. ADP/ATP ratio was determined as indicated in Material and Methods; ( B ) MDA-MB-231 cells were seeded on glass coverslip and then left untreated or treated with 30 μM metformin. At the end of the treatment, cells were stained with 10 μg/mL JC-1 as indicated under Material and Methods. Mitochondria fluorescence was observed by confocal microscopy. First row: Red fluorescence representing JC1 aggregates. Second row: Green fluorescence representing JC1 monomers. Third row: Merging of the first two rows. Alternatively, cells were treated with 30 μM metformin and stained with JC-1 as above. Red (FL2H) and green (FL1H) fluorescence intensity was quantified by Flow Cytometry; ( C )upper panel: MDA-MB-231 cells were kept in the presence or absence of metformin for the time indicated and then processed to obtain mitochondrial fractions. BAX and CYCS expression levels were determined by Western blot as indicated under Material and Methods. Densitometric analysis of the gels was performed as indicated under Material and Methods. PHB and CDK4 were used as loading and purity control, respectively. Lower panel: MDA-MB-231 cells were kept in the presence or absence of metformin for the time indicated and then processed to obtain whole cellular extracts. BAX and CYCS expression levels were determined by Western blot as indicated under Material and Methods. Densitometric analysis of the gels was performed as indicated under Material and Methods. CDK4 and PHB were used as loading control. Right panel: MCF-7 cells were kept in the presence or absence of metformin for 20 days and then processed to obtain mitochondrial or whole cellular extracts. BAX and CYCS expression levels were determined by Western blot as indicated under Material and Methods. Densitometric analysis of the gels was performed as indicated under Material and Methods. PHB and CDK4 were used as purity and loading controls; ( D ) MDA-MB-231 cells were kept in the presence or absence of metformin for 20 days. At the end of the treatment cells were harvested and percentage of sub-G 1 (M1) cells was determined by propidium iodide staining as described in the Material and Methods section. All experiments in this figure were repeated three times. * Significantly different from control untreated cells.

    Journal: Cells

    Article Title: Metformin Impairs Glutamine Metabolism and Autophagy in Tumour Cells

    doi: 10.3390/cells8010049

    Figure Lengend Snippet: Metformin activates apoptotic cell death; ( A ) MDA-MB-231 cells were plated in 96 well plate and then either left untreated or treated with millimolar (upper graphs) or micromolar (lower graphs) doses of metformin. ADP/ATP ratio was determined as indicated in Material and Methods; ( B ) MDA-MB-231 cells were seeded on glass coverslip and then left untreated or treated with 30 μM metformin. At the end of the treatment, cells were stained with 10 μg/mL JC-1 as indicated under Material and Methods. Mitochondria fluorescence was observed by confocal microscopy. First row: Red fluorescence representing JC1 aggregates. Second row: Green fluorescence representing JC1 monomers. Third row: Merging of the first two rows. Alternatively, cells were treated with 30 μM metformin and stained with JC-1 as above. Red (FL2H) and green (FL1H) fluorescence intensity was quantified by Flow Cytometry; ( C )upper panel: MDA-MB-231 cells were kept in the presence or absence of metformin for the time indicated and then processed to obtain mitochondrial fractions. BAX and CYCS expression levels were determined by Western blot as indicated under Material and Methods. Densitometric analysis of the gels was performed as indicated under Material and Methods. PHB and CDK4 were used as loading and purity control, respectively. Lower panel: MDA-MB-231 cells were kept in the presence or absence of metformin for the time indicated and then processed to obtain whole cellular extracts. BAX and CYCS expression levels were determined by Western blot as indicated under Material and Methods. Densitometric analysis of the gels was performed as indicated under Material and Methods. CDK4 and PHB were used as loading control. Right panel: MCF-7 cells were kept in the presence or absence of metformin for 20 days and then processed to obtain mitochondrial or whole cellular extracts. BAX and CYCS expression levels were determined by Western blot as indicated under Material and Methods. Densitometric analysis of the gels was performed as indicated under Material and Methods. PHB and CDK4 were used as purity and loading controls; ( D ) MDA-MB-231 cells were kept in the presence or absence of metformin for 20 days. At the end of the treatment cells were harvested and percentage of sub-G 1 (M1) cells was determined by propidium iodide staining as described in the Material and Methods section. All experiments in this figure were repeated three times. * Significantly different from control untreated cells.

    Article Snippet: Samples were stained with 50 μg/mL Propidium Iodide (PI, P4864; Sigma-Aldrich) in PBS for 2 h at 4 °C cover light.

    Techniques: Multiple Displacement Amplification, Staining, Fluorescence, Confocal Microscopy, Flow Cytometry, Cytometry, Expressing, Western Blot