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  • 99
    Thermo Fisher triton x100
    Effect of Triton X-100 on the migration of single stranded DNA in polyacrylamide gels. The gel illustrates the smearing of single stranded DNA when loaded on polyacrylamide gels in the presence of Triton X-100. The DNA sample was the same in all lanes and contained a labeled single stranded circle of 235 nucleotides. Two minor bands that show no smearing most probably correspond to contaminating double stranded DNA, as smearing was always observed with many different single strands. Three different brands of <t>Triton-X100</t> denoted T1–T3 were tested at a final concentration of 0.05%: T1 and T2 were ordinary grade, T3 was highly purified, oxidant-free Triton-X100, sold in glass ampules sealed under nitrogen (Pierce). Lane 1: control, no Triton-X100 added; lane 2: deionized T1; lane 3: T1, untreated; lane 4: T1, heat-treated; lane 5: T1, SDS added at a concentration of 0.005%; lane 6: T3, untreated; lane 7: T1, washed with a 5M NaCl solution; lane 8: T2, untreated. Note that addition of SDS to the samples before loading the gel was a very efficient way to suppress smearing (lane 5).
    Triton X100, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2754 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 2754 article reviews
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    triton x100 - by Bioz Stars, 2020-07
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    99
    Millipore triton x100
    IFITM3 expression inhibits SFV capsid release . A) SFV (200 pfu/cell) was bound to cells for 1 h at 4°C prior to incubation at 37°C for the indicated times. At each time point the cells were fixed and permeabilized with 0.1% <t>Triton‐X100</t> and labeled with serum against the SFV capsid, which was detected with AF594. Confocal sections are displayed. Following internalization, virus particles were detected in cells, indicated by the typical punctate association of virus with endosomes. By 40 min after warm‐up diffuse cytosolic fluorescence was seen in A549 cells, indicating release of the viral capsid protein to the cytosol. In A549 cells treated with 10 µ m monensin, and in OS‐IFITM3‐HA expressing cells, the staining remains associated with puncta and the cytosolic staining was not observed even at 60 min after warm‐up. Nuclei were detected with Hoechst staining. Scale bar represents 15 µm. B) Quantification of cytosolic fluorescence and significance testing is described in Materials and Methods . The data are from three independent infections with 3–7 images taken at each condition, with a total of at least 60 cells analyzed per cell line, per condition. Data presented is the average fluorescence intensity, normalized to the background intensity at 0 min of warming. *p
    Triton X100, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 9142 article reviews
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    99
    Bio-Rad triton x100
    Membrane expression and functionality of hLDLR expressed by the CHO-hLDLR-EGFP cell line. (A) Representative confocal photomicrographs of immunostained CHO-hTfR-EGFP (negative control) and CHO-hLDLR-EGFP cells (green) with an anti-LDLR antibody diluted at 1/100 (red) following triton <t>X100</t> permeabilization of the cell membrane. Cell nuclei are labeled with Hoechst#33258 at 0.5 μg/mL (blue). Co-labeling appears in yellow in the merged pictures. Only the cells with stable expression of the hLDLR-EGFP construct express the membrane receptor. (B) Representative confocal photomicrographs of CHO-hTfR-EGFP and CHO-hLDLR-EGFP cells (green) after a 10 min incubation period with DiI-LDL 15 μg/mL (red). Cell nuclei are labeled with Hoechst#33258 (blue). Co-labeling appears in yellow in the merged pictures. DiI-LDL is essentially bound to the CHO-hLDLR-EGFP cells indicating that the hLDLR-EGFP chimera receptor binds its ligand. (C) Western blots performed on cell membrane preparations of CHO cells expressing hLDLR-EGFP, rLDLR-EGFP and mLDLR-EGFP compared to CHO WT, using anti-hLDLR (1/800) and anti-rat and mouse LDLR antibodies (1/1000). These bands are also labeled with an anti-EGFP antibody. Bands of 140 kDa and 190 kDa correspond respectively to the immature and mature LDLR-EGFP fusion proteins (arrows). Actin was used to check loading of equal amounts of protein.
    Triton X100, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Fisher Scientific triton x100
    Membrane expression and functionality of hLDLR expressed by the CHO-hLDLR-EGFP cell line. (A) Representative confocal photomicrographs of immunostained CHO-hTfR-EGFP (negative control) and CHO-hLDLR-EGFP cells (green) with an anti-LDLR antibody diluted at 1/100 (red) following triton <t>X100</t> permeabilization of the cell membrane. Cell nuclei are labeled with Hoechst#33258 at 0.5 μg/mL (blue). Co-labeling appears in yellow in the merged pictures. Only the cells with stable expression of the hLDLR-EGFP construct express the membrane receptor. (B) Representative confocal photomicrographs of CHO-hTfR-EGFP and CHO-hLDLR-EGFP cells (green) after a 10 min incubation period with DiI-LDL 15 μg/mL (red). Cell nuclei are labeled with Hoechst#33258 (blue). Co-labeling appears in yellow in the merged pictures. DiI-LDL is essentially bound to the CHO-hLDLR-EGFP cells indicating that the hLDLR-EGFP chimera receptor binds its ligand. (C) Western blots performed on cell membrane preparations of CHO cells expressing hLDLR-EGFP, rLDLR-EGFP and mLDLR-EGFP compared to CHO WT, using anti-hLDLR (1/800) and anti-rat and mouse LDLR antibodies (1/1000). These bands are also labeled with an anti-EGFP antibody. Bands of 140 kDa and 190 kDa correspond respectively to the immature and mature LDLR-EGFP fusion proteins (arrows). Actin was used to check loading of equal amounts of protein.
    Triton X100, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 163 article reviews
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    93
    Promega triton x100
    Inhibition of phospholipid synthesis affects the accessibility of the replication complexes. HeLa cells after choline deprivation were infected with poliovirus at an MOI of 10 PFU/cell and incubated in a choline-free or a choline-supplemented medium after infection. A. Confocal images of the cells permeabilized with either 0.2% Triton <t>X100</t> (strong permeabilization) or 0.02% saponin (mild permeabilization) and stained for a viral antigen 2B (red). Nuclear DNA is stained with Hoechst 33342 (blue). Arrows indicate areas protected upon mild permeabilization in cells incubated with choline-supplemented medium. B. Visualization of dsRNA in HeLa cells processed and infected as in A after Triton X100 permeabilization. Red arrows indicate dsRNA association with the nuclear envelope (PN) in choline-deprived cells; white arrows show big perinuclear blobs (CYT) of dsRNA signal reflecting normal development of replication membranes in choline-supplemented cells. C. HeLa cells were infected with poliovirus at an MOI of 10 PFU/cell after choline deprivation and incubated in a choline-free or a choline-supplemented medium for 4 h p.i. The cells were permeabilized with digitonin and treated (lines 3 and 4) or non-treated (lines 1 and 2) with proteinase K. * indicates protein fragments generated upon proteinase K treatment. Actin is shown as a loading control.
    Triton X100, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 202 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 202 article reviews
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    92
    EM Science Inc triton x100
    Inhibition of phospholipid synthesis affects the accessibility of the replication complexes. HeLa cells after choline deprivation were infected with poliovirus at an MOI of 10 PFU/cell and incubated in a choline-free or a choline-supplemented medium after infection. A. Confocal images of the cells permeabilized with either 0.2% Triton <t>X100</t> (strong permeabilization) or 0.02% saponin (mild permeabilization) and stained for a viral antigen 2B (red). Nuclear DNA is stained with Hoechst 33342 (blue). Arrows indicate areas protected upon mild permeabilization in cells incubated with choline-supplemented medium. B. Visualization of dsRNA in HeLa cells processed and infected as in A after Triton X100 permeabilization. Red arrows indicate dsRNA association with the nuclear envelope (PN) in choline-deprived cells; white arrows show big perinuclear blobs (CYT) of dsRNA signal reflecting normal development of replication membranes in choline-supplemented cells. C. HeLa cells were infected with poliovirus at an MOI of 10 PFU/cell after choline deprivation and incubated in a choline-free or a choline-supplemented medium for 4 h p.i. The cells were permeabilized with digitonin and treated (lines 3 and 4) or non-treated (lines 1 and 2) with proteinase K. * indicates protein fragments generated upon proteinase K treatment. Actin is shown as a loading control.
    Triton X100, supplied by EM Science Inc, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 8 article reviews
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    94
    Roche triton x100
    TcK2 is located in the endosomal compartment and is phosphorylated in non-proliferating parasite stages of T. cruzi . (A) Wild type epimastigotes (Epi), metacyclics (Meta), intracellular amastigotes (Ama) and cell-released trypomastigotes (Trypo) were fixed, permeabilized, and incubated with anti-BiP antibodies (Bip, green) and mAb 5D10 (TcK2, red). The images show individual immunofluorescence and DAPI labeling, DIC images and the merged immunofluorescence labeling from an enlarged field (Bars = 5 μm). (B) Wild type (WT) or HA-tagged K2 (TcK2-HA) epimastigotes were adsorbed to glass slides, fixed with paraformaldehyde and permeabilized with 0.1% Triton <t>X100.</t> Adhered cells were then stained with mAb 5D10 (TcK2, green) or anti-HA (HA, red), and DAPI (blue). The images show individual labeling and the fluorescence signals merged with differential interference contrast (DIC). (C) Western blots of total extracts of parasites of the indicated stages revealed by anti-TcK2 (anti-KD) antibody. Anti-β-tubulin was used as a loading control. The graph shows the mean and standard deviation of the TcK2 [Up] /TcK2 [Low] ratio (n = 3).
    Triton X100, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 3420 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 3420 article reviews
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    92
    Avantor triton x100
    TcK2 is located in the endosomal compartment and is phosphorylated in non-proliferating parasite stages of T. cruzi . (A) Wild type epimastigotes (Epi), metacyclics (Meta), intracellular amastigotes (Ama) and cell-released trypomastigotes (Trypo) were fixed, permeabilized, and incubated with anti-BiP antibodies (Bip, green) and mAb 5D10 (TcK2, red). The images show individual immunofluorescence and DAPI labeling, DIC images and the merged immunofluorescence labeling from an enlarged field (Bars = 5 μm). (B) Wild type (WT) or HA-tagged K2 (TcK2-HA) epimastigotes were adsorbed to glass slides, fixed with paraformaldehyde and permeabilized with 0.1% Triton <t>X100.</t> Adhered cells were then stained with mAb 5D10 (TcK2, green) or anti-HA (HA, red), and DAPI (blue). The images show individual labeling and the fluorescence signals merged with differential interference contrast (DIC). (C) Western blots of total extracts of parasites of the indicated stages revealed by anti-TcK2 (anti-KD) antibody. Anti-β-tubulin was used as a loading control. The graph shows the mean and standard deviation of the TcK2 [Up] /TcK2 [Low] ratio (n = 3).
    Triton X100, supplied by Avantor, used in various techniques. Bioz Stars score: 92/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 47 article reviews
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    92
    Carl Roth GmbH triton x100
    The impact of crosslinking on the purification of TF – RNCs. (a,b) Polysome profiles in the absence of crosslinker or after DSP or EDC ex vivo crosslinking and sucrose gradient centrifugation. Depicted are data from experiments in which E. coli MC4100 cells were grown in LB medium and harvested as described in step 1 , option C. The lysate was either crosslinked ex vivo with DSP or EDC (step 7 , option B) or left untreated (step 7 , option A). Undigested (a) and digested (b) lysates were run on a sucrose gradient (step 18 , option B). The digestion was performed with a reduced MNase concentration of 15 U/A 260 to partially retain di- and trisomes for comparison. ‘30S’ and ‘50S’ depict the peaks of the small and large ribosomal subunits, respectively. The monosome peak is labeled with ‘70S’. To compare polysome profiles quantitatively, the curves were normalized to the same area underneath all ribosomal peaks. (c) Gel analysis of the DSP crosslinker titration. 200 ml of E. coli MC4100 ∆ tig::Kan + pTrc-tig-TEV-Avi cells were grown and harvested according to step 1 , option A. Cells were resuspended in 2 ml of buffer A (50 mM HEPES pH 7.5, 1 M potassium acetate, 10 mM MgAc 2 , 1 mM PMSF, 1 mM chloramphenicol, 0.4% Triton <t>X100,</t> 0.1% NP-40, 1 mg/ml lysozyme, 2.5 µg/ml RNase-free DNase I). Lysis and purification of TF – RNCs was performed as described including DSP ex vivo crosslinking (step 7 , option B) and sucrose cushion centrifugation (step 18 , option A) with the following exceptions: For crosslinking either 3 mg of DSP (‘1×’), 15 mg of DSP (‘5×’) or DSMO only (‘−‘) were used. Ultracentrifugation was done with 1 M potassium acetate instead of 1 M NaCl in the sucrose cushion buffer causing the high amount of non-crosslinked TF that copelleted without DSP addition (‘−‘). For AP only half of Strep-Tactin slurry and TEV protease were used. Either non-reducing (‘non-red.’) or reducing (‘red.’) sample buffer was used for SDS-PAGE. Gels were stained with coomassie or used for western blotting employing a polyclonal α-TF antibody. Crosslinks are abbreviated with ‘X’. (d) Gel analysis of the EDC crosslinker titration. E. coli MC4100 ∆ tig::Kan + pTrc-tig-TEV-Avi cells were grown in 1 l LB, harvested as described in step 1 , option A, and resuspended in 6 ml of lysis buffer. Ex vivo crosslinking (step 7 , option B) was performed with 2.5 mM (‘0.125×’), 10 mM (‘0.5×’), 20 mM (‘1×’) and 80 mM (‘4×’) EDC. TF–RNCs were purified as described, including sucrose cushion centrifugation (step 18 , option A), eluted from the affinity matrix by boiling in reducing sample buffer, and analyzed by SDS-PAGE or western blotting using polyclonal antibodies against TF and L23. Crosslinks are abbreviated with ‘X’.
    Triton X100, supplied by Carl Roth GmbH, used in various techniques. Bioz Stars score: 92/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/triton x100/product/Carl Roth GmbH
    Average 92 stars, based on 32 article reviews
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    triton x100 - by Bioz Stars, 2020-07
    92/100 stars
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    92
    Valiant triton x100
    The impact of crosslinking on the purification of TF – RNCs. (a,b) Polysome profiles in the absence of crosslinker or after DSP or EDC ex vivo crosslinking and sucrose gradient centrifugation. Depicted are data from experiments in which E. coli MC4100 cells were grown in LB medium and harvested as described in step 1 , option C. The lysate was either crosslinked ex vivo with DSP or EDC (step 7 , option B) or left untreated (step 7 , option A). Undigested (a) and digested (b) lysates were run on a sucrose gradient (step 18 , option B). The digestion was performed with a reduced MNase concentration of 15 U/A 260 to partially retain di- and trisomes for comparison. ‘30S’ and ‘50S’ depict the peaks of the small and large ribosomal subunits, respectively. The monosome peak is labeled with ‘70S’. To compare polysome profiles quantitatively, the curves were normalized to the same area underneath all ribosomal peaks. (c) Gel analysis of the DSP crosslinker titration. 200 ml of E. coli MC4100 ∆ tig::Kan + pTrc-tig-TEV-Avi cells were grown and harvested according to step 1 , option A. Cells were resuspended in 2 ml of buffer A (50 mM HEPES pH 7.5, 1 M potassium acetate, 10 mM MgAc 2 , 1 mM PMSF, 1 mM chloramphenicol, 0.4% Triton <t>X100,</t> 0.1% NP-40, 1 mg/ml lysozyme, 2.5 µg/ml RNase-free DNase I). Lysis and purification of TF – RNCs was performed as described including DSP ex vivo crosslinking (step 7 , option B) and sucrose cushion centrifugation (step 18 , option A) with the following exceptions: For crosslinking either 3 mg of DSP (‘1×’), 15 mg of DSP (‘5×’) or DSMO only (‘−‘) were used. Ultracentrifugation was done with 1 M potassium acetate instead of 1 M NaCl in the sucrose cushion buffer causing the high amount of non-crosslinked TF that copelleted without DSP addition (‘−‘). For AP only half of Strep-Tactin slurry and TEV protease were used. Either non-reducing (‘non-red.’) or reducing (‘red.’) sample buffer was used for SDS-PAGE. Gels were stained with coomassie or used for western blotting employing a polyclonal α-TF antibody. Crosslinks are abbreviated with ‘X’. (d) Gel analysis of the EDC crosslinker titration. E. coli MC4100 ∆ tig::Kan + pTrc-tig-TEV-Avi cells were grown in 1 l LB, harvested as described in step 1 , option A, and resuspended in 6 ml of lysis buffer. Ex vivo crosslinking (step 7 , option B) was performed with 2.5 mM (‘0.125×’), 10 mM (‘0.5×’), 20 mM (‘1×’) and 80 mM (‘4×’) EDC. TF–RNCs were purified as described, including sucrose cushion centrifugation (step 18 , option A), eluted from the affinity matrix by boiling in reducing sample buffer, and analyzed by SDS-PAGE or western blotting using polyclonal antibodies against TF and L23. Crosslinks are abbreviated with ‘X’.
    Triton X100, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/triton x100/product/Valiant
    Average 92 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    triton x100 - by Bioz Stars, 2020-07
    92/100 stars
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    93
    Merck & Co triton x100
    The impact of crosslinking on the purification of TF – RNCs. (a,b) Polysome profiles in the absence of crosslinker or after DSP or EDC ex vivo crosslinking and sucrose gradient centrifugation. Depicted are data from experiments in which E. coli MC4100 cells were grown in LB medium and harvested as described in step 1 , option C. The lysate was either crosslinked ex vivo with DSP or EDC (step 7 , option B) or left untreated (step 7 , option A). Undigested (a) and digested (b) lysates were run on a sucrose gradient (step 18 , option B). The digestion was performed with a reduced MNase concentration of 15 U/A 260 to partially retain di- and trisomes for comparison. ‘30S’ and ‘50S’ depict the peaks of the small and large ribosomal subunits, respectively. The monosome peak is labeled with ‘70S’. To compare polysome profiles quantitatively, the curves were normalized to the same area underneath all ribosomal peaks. (c) Gel analysis of the DSP crosslinker titration. 200 ml of E. coli MC4100 ∆ tig::Kan + pTrc-tig-TEV-Avi cells were grown and harvested according to step 1 , option A. Cells were resuspended in 2 ml of buffer A (50 mM HEPES pH 7.5, 1 M potassium acetate, 10 mM MgAc 2 , 1 mM PMSF, 1 mM chloramphenicol, 0.4% Triton <t>X100,</t> 0.1% NP-40, 1 mg/ml lysozyme, 2.5 µg/ml RNase-free DNase I). Lysis and purification of TF – RNCs was performed as described including DSP ex vivo crosslinking (step 7 , option B) and sucrose cushion centrifugation (step 18 , option A) with the following exceptions: For crosslinking either 3 mg of DSP (‘1×’), 15 mg of DSP (‘5×’) or DSMO only (‘−‘) were used. Ultracentrifugation was done with 1 M potassium acetate instead of 1 M NaCl in the sucrose cushion buffer causing the high amount of non-crosslinked TF that copelleted without DSP addition (‘−‘). For AP only half of Strep-Tactin slurry and TEV protease were used. Either non-reducing (‘non-red.’) or reducing (‘red.’) sample buffer was used for SDS-PAGE. Gels were stained with coomassie or used for western blotting employing a polyclonal α-TF antibody. Crosslinks are abbreviated with ‘X’. (d) Gel analysis of the EDC crosslinker titration. E. coli MC4100 ∆ tig::Kan + pTrc-tig-TEV-Avi cells were grown in 1 l LB, harvested as described in step 1 , option A, and resuspended in 6 ml of lysis buffer. Ex vivo crosslinking (step 7 , option B) was performed with 2.5 mM (‘0.125×’), 10 mM (‘0.5×’), 20 mM (‘1×’) and 80 mM (‘4×’) EDC. TF–RNCs were purified as described, including sucrose cushion centrifugation (step 18 , option A), eluted from the affinity matrix by boiling in reducing sample buffer, and analyzed by SDS-PAGE or western blotting using polyclonal antibodies against TF and L23. Crosslinks are abbreviated with ‘X’.
    Triton X100, supplied by Merck & Co, used in various techniques. Bioz Stars score: 93/100, based on 159 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/triton x100/product/Merck & Co
    Average 93 stars, based on 159 article reviews
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    triton x100 - by Bioz Stars, 2020-07
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    92
    Amresco triton x100
    The impact of crosslinking on the purification of TF – RNCs. (a,b) Polysome profiles in the absence of crosslinker or after DSP or EDC ex vivo crosslinking and sucrose gradient centrifugation. Depicted are data from experiments in which E. coli MC4100 cells were grown in LB medium and harvested as described in step 1 , option C. The lysate was either crosslinked ex vivo with DSP or EDC (step 7 , option B) or left untreated (step 7 , option A). Undigested (a) and digested (b) lysates were run on a sucrose gradient (step 18 , option B). The digestion was performed with a reduced MNase concentration of 15 U/A 260 to partially retain di- and trisomes for comparison. ‘30S’ and ‘50S’ depict the peaks of the small and large ribosomal subunits, respectively. The monosome peak is labeled with ‘70S’. To compare polysome profiles quantitatively, the curves were normalized to the same area underneath all ribosomal peaks. (c) Gel analysis of the DSP crosslinker titration. 200 ml of E. coli MC4100 ∆ tig::Kan + pTrc-tig-TEV-Avi cells were grown and harvested according to step 1 , option A. Cells were resuspended in 2 ml of buffer A (50 mM HEPES pH 7.5, 1 M potassium acetate, 10 mM MgAc 2 , 1 mM PMSF, 1 mM chloramphenicol, 0.4% Triton <t>X100,</t> 0.1% NP-40, 1 mg/ml lysozyme, 2.5 µg/ml RNase-free DNase I). Lysis and purification of TF – RNCs was performed as described including DSP ex vivo crosslinking (step 7 , option B) and sucrose cushion centrifugation (step 18 , option A) with the following exceptions: For crosslinking either 3 mg of DSP (‘1×’), 15 mg of DSP (‘5×’) or DSMO only (‘−‘) were used. Ultracentrifugation was done with 1 M potassium acetate instead of 1 M NaCl in the sucrose cushion buffer causing the high amount of non-crosslinked TF that copelleted without DSP addition (‘−‘). For AP only half of Strep-Tactin slurry and TEV protease were used. Either non-reducing (‘non-red.’) or reducing (‘red.’) sample buffer was used for SDS-PAGE. Gels were stained with coomassie or used for western blotting employing a polyclonal α-TF antibody. Crosslinks are abbreviated with ‘X’. (d) Gel analysis of the EDC crosslinker titration. E. coli MC4100 ∆ tig::Kan + pTrc-tig-TEV-Avi cells were grown in 1 l LB, harvested as described in step 1 , option A, and resuspended in 6 ml of lysis buffer. Ex vivo crosslinking (step 7 , option B) was performed with 2.5 mM (‘0.125×’), 10 mM (‘0.5×’), 20 mM (‘1×’) and 80 mM (‘4×’) EDC. TF–RNCs were purified as described, including sucrose cushion centrifugation (step 18 , option A), eluted from the affinity matrix by boiling in reducing sample buffer, and analyzed by SDS-PAGE or western blotting using polyclonal antibodies against TF and L23. Crosslinks are abbreviated with ‘X’.
    Triton X100, supplied by Amresco, used in various techniques. Bioz Stars score: 92/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/triton x100/product/Amresco
    Average 92 stars, based on 38 article reviews
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    triton x100 - by Bioz Stars, 2020-07
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    93
    Applichem triton x100
    Cell culture model for osteoblastic differentiation of pluripotent human bone marrow-derived MSC. ( A ) Representative FACS analysis out of 20 of an MSC defining surface marker panel confirming homogeneity of isolated cells. More than 98% of cells are positive for CD73, CD90 and CD105 and negative for CD11b, CD14, CD19, CD34, CD45, and HLA-DR compared to isotype control (grey). ( B ) Chondroblastic differentiation of MSC after incubation for 32 days with chondroblast induction medium demonstrated by western blot analysis for chondrocyte-specific type II collagen. Two representative cell preparations out of 20 are shown. α-Tubulin served as loading control. ( C ) Adipocytic differentiation of MSC after incubation for 21 days with adipocyte induction medium demonstrated by staining of intracellular lipid droplets with Oil red O. Representative experiment out of 20, phase contrast microscopy, original magnification × 200, scale bar = 50 µm. ( D ) Osteoblastic differentiation of MSC after incubation for 21 days with osteoblast induction medium demonstrated by staining of extracellular hydroxyapatite deposits with Alizarin red S. Representative experiment out of 20, phase contrast microscopy, original magnification <t>x100,</t> scale bar = 50 µm. ( E ) Baseline expression of vascular smooth muscle marker proteins in MSC: SM22α, smooth muscle calponin (sm-Calponin), myosin light chain kinase (MLCK), smooth muscle-α-actin (SMA). Western blot analyses of three representative cell preparations out of 20 are shown. α-Tubulin served as loading control. ( F ) Calcium transients without (control) and after pretreatment with nimodipine (Nimodipine) in MSC at base line showing expression of functional voltage-dependent dihydropyridine-sensitive (Ca v 1.2) L-type Ca 2+ channels. Representative experiment out of three; 20–30 individual cells were studied for each tracing.
    Triton X100, supplied by Applichem, used in various techniques. Bioz Stars score: 93/100, based on 122 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 122 article reviews
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    triton x100 - by Bioz Stars, 2020-07
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    92
    Merck KGaA triton x100
    LDH- and hCG-levels in culture supernatants of placental explants, BeWo cells, and primary cytotrophoblast cells, exposed to neutral (Neu), positively (Pos), and negatively charged (Neg) dPG-NPs for 24 h, at concentrations of 1 µM and 10 nM and to 1% Triton ® <t>X100.</t> LDH data are shown in relative units as ratio to control values, while hCG data are presented in mU mL −1 . *( p
    Triton X100, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 155 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/triton x100/product/Merck KGaA
    Average 92 stars, based on 155 article reviews
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    triton x100 - by Bioz Stars, 2020-07
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    92
    Nacalai triton x100
    LDH- and hCG-levels in culture supernatants of placental explants, BeWo cells, and primary cytotrophoblast cells, exposed to neutral (Neu), positively (Pos), and negatively charged (Neg) dPG-NPs for 24 h, at concentrations of 1 µM and 10 nM and to 1% Triton ® <t>X100.</t> LDH data are shown in relative units as ratio to control values, while hCG data are presented in mU mL −1 . *( p
    Triton X100, supplied by Nacalai, used in various techniques. Bioz Stars score: 92/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Ricca Chemical Company triton x100
    LDH- and hCG-levels in culture supernatants of placental explants, BeWo cells, and primary cytotrophoblast cells, exposed to neutral (Neu), positively (Pos), and negatively charged (Neg) dPG-NPs for 24 h, at concentrations of 1 µM and 10 nM and to 1% Triton ® <t>X100.</t> LDH data are shown in relative units as ratio to control values, while hCG data are presented in mU mL −1 . *( p
    Triton X100, supplied by Ricca Chemical Company, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FUJIFILM triton x100
    LDH- and hCG-levels in culture supernatants of placental explants, BeWo cells, and primary cytotrophoblast cells, exposed to neutral (Neu), positively (Pos), and negatively charged (Neg) dPG-NPs for 24 h, at concentrations of 1 µM and 10 nM and to 1% Triton ® <t>X100.</t> LDH data are shown in relative units as ratio to control values, while hCG data are presented in mU mL −1 . *( p
    Triton X100, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    scharlau triton x100
    LDH- and hCG-levels in culture supernatants of placental explants, BeWo cells, and primary cytotrophoblast cells, exposed to neutral (Neu), positively (Pos), and negatively charged (Neg) dPG-NPs for 24 h, at concentrations of 1 µM and 10 nM and to 1% Triton ® <t>X100.</t> LDH data are shown in relative units as ratio to control values, while hCG data are presented in mU mL −1 . *( p
    Triton X100, supplied by scharlau, used in various techniques. Bioz Stars score: 90/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Kodak Corp triton x100
    LDH- and hCG-levels in culture supernatants of placental explants, BeWo cells, and primary cytotrophoblast cells, exposed to neutral (Neu), positively (Pos), and negatively charged (Neg) dPG-NPs for 24 h, at concentrations of 1 µM and 10 nM and to 1% Triton ® <t>X100.</t> LDH data are shown in relative units as ratio to control values, while hCG data are presented in mU mL −1 . *( p
    Triton X100, supplied by Kodak Corp, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effect of Triton X-100 on the migration of single stranded DNA in polyacrylamide gels. The gel illustrates the smearing of single stranded DNA when loaded on polyacrylamide gels in the presence of Triton X-100. The DNA sample was the same in all lanes and contained a labeled single stranded circle of 235 nucleotides. Two minor bands that show no smearing most probably correspond to contaminating double stranded DNA, as smearing was always observed with many different single strands. Three different brands of Triton-X100 denoted T1–T3 were tested at a final concentration of 0.05%: T1 and T2 were ordinary grade, T3 was highly purified, oxidant-free Triton-X100, sold in glass ampules sealed under nitrogen (Pierce). Lane 1: control, no Triton-X100 added; lane 2: deionized T1; lane 3: T1, untreated; lane 4: T1, heat-treated; lane 5: T1, SDS added at a concentration of 0.005%; lane 6: T3, untreated; lane 7: T1, washed with a 5M NaCl solution; lane 8: T2, untreated. Note that addition of SDS to the samples before loading the gel was a very efficient way to suppress smearing (lane 5).

    Journal: PLoS ONE

    Article Title: Construction of DNA Hemicatenanes from Two Small Circular DNA Molecules

    doi: 10.1371/journal.pone.0119368

    Figure Lengend Snippet: Effect of Triton X-100 on the migration of single stranded DNA in polyacrylamide gels. The gel illustrates the smearing of single stranded DNA when loaded on polyacrylamide gels in the presence of Triton X-100. The DNA sample was the same in all lanes and contained a labeled single stranded circle of 235 nucleotides. Two minor bands that show no smearing most probably correspond to contaminating double stranded DNA, as smearing was always observed with many different single strands. Three different brands of Triton-X100 denoted T1–T3 were tested at a final concentration of 0.05%: T1 and T2 were ordinary grade, T3 was highly purified, oxidant-free Triton-X100, sold in glass ampules sealed under nitrogen (Pierce). Lane 1: control, no Triton-X100 added; lane 2: deionized T1; lane 3: T1, untreated; lane 4: T1, heat-treated; lane 5: T1, SDS added at a concentration of 0.005%; lane 6: T3, untreated; lane 7: T1, washed with a 5M NaCl solution; lane 8: T2, untreated. Note that addition of SDS to the samples before loading the gel was a very efficient way to suppress smearing (lane 5).

    Article Snippet: Deionization of Triton-X100 with mixed-bed ion exchange resin, or use of highly-purified Triton-X100 sold in glass ampules sealed under nitrogen (Pierce), did not significantly change the result ( ).

    Techniques: Migration, Labeling, Concentration Assay, Purification

    Erk1 is associated with focal adhesions. BxPC-3 cells were stimulated with 10 nM of mature TGFβ 1 for ten minutes followed by preparation of the Triton-X100 nonsoluble fraction and precipitation with α V β 6 integrin antibodies. The precipitate was then re-precipitated with anti-FAK antibodies (sc-1688) and analyzed with an Erk1 antibody (sc-93). In part, the cells were preincubated with a TGFβ 1 antibody [ 10 ].

    Journal: Molecular Cancer

    Article Title: TGF?1 activates c-Jun and Erk1 via ?V?6 integrin

    doi: 10.1186/1476-4598-2-33

    Figure Lengend Snippet: Erk1 is associated with focal adhesions. BxPC-3 cells were stimulated with 10 nM of mature TGFβ 1 for ten minutes followed by preparation of the Triton-X100 nonsoluble fraction and precipitation with α V β 6 integrin antibodies. The precipitate was then re-precipitated with anti-FAK antibodies (sc-1688) and analyzed with an Erk1 antibody (sc-93). In part, the cells were preincubated with a TGFβ 1 antibody [ 10 ].

    Article Snippet: After preparation of the cytoskeletal fraction by Triton-X100 extraction [ , , ], slides were stained using Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for Actin, Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes) for αV β6 integrin, and Alexa Fluor 350 donkey anti-rabbit IgG (Molecular Probes) for TGFβ1 labeling [ ].

    Techniques:

    Paxillin and c-Jun are associated with focal adhesions. BxPC-3 cells were stimulated with 10 nM of mature TGFβ 1 for ten minutes followed by preparation of the Triton-X100 nonsoluble fraction and precipitation with α V β 6 integrin antibodies. The precipitate was then re-precipitated with anti-FAK antibodies (sc-1688) and analyzed with antibodies against p-Paxillin Y118 (2541, Cell Signaling) and p-c-Jun S63 (9621, Cell Signaling). In part, the cells were preincubated with a TGFβ-RII antibody (Santa Cruz), BAPTA-AM and Cytochalasin D, respectively [ 10 ].

    Journal: Molecular Cancer

    Article Title: TGF?1 activates c-Jun and Erk1 via ?V?6 integrin

    doi: 10.1186/1476-4598-2-33

    Figure Lengend Snippet: Paxillin and c-Jun are associated with focal adhesions. BxPC-3 cells were stimulated with 10 nM of mature TGFβ 1 for ten minutes followed by preparation of the Triton-X100 nonsoluble fraction and precipitation with α V β 6 integrin antibodies. The precipitate was then re-precipitated with anti-FAK antibodies (sc-1688) and analyzed with antibodies against p-Paxillin Y118 (2541, Cell Signaling) and p-c-Jun S63 (9621, Cell Signaling). In part, the cells were preincubated with a TGFβ-RII antibody (Santa Cruz), BAPTA-AM and Cytochalasin D, respectively [ 10 ].

    Article Snippet: After preparation of the cytoskeletal fraction by Triton-X100 extraction [ , , ], slides were stained using Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for Actin, Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes) for αV β6 integrin, and Alexa Fluor 350 donkey anti-rabbit IgG (Molecular Probes) for TGFβ1 labeling [ ].

    Techniques:

    MEKK1 is associated with focal adhesions. BxPC-3 cells were stimulated with 10 nM of mature TGFβ 1 for ten minutes followed by preparation of the Triton-X100 nonsoluble fraction and precipitation with α V β 6 integrin antibodies. The precipitate was then re-precipitated with anti-FAK antibodies (sc-1688) and analyzed with a MEKK1 antibody (sc-448). In part, the cells were preincubated with a TGFβ 1 antibody [ 10 ].

    Journal: Molecular Cancer

    Article Title: TGF?1 activates c-Jun and Erk1 via ?V?6 integrin

    doi: 10.1186/1476-4598-2-33

    Figure Lengend Snippet: MEKK1 is associated with focal adhesions. BxPC-3 cells were stimulated with 10 nM of mature TGFβ 1 for ten minutes followed by preparation of the Triton-X100 nonsoluble fraction and precipitation with α V β 6 integrin antibodies. The precipitate was then re-precipitated with anti-FAK antibodies (sc-1688) and analyzed with a MEKK1 antibody (sc-448). In part, the cells were preincubated with a TGFβ 1 antibody [ 10 ].

    Article Snippet: After preparation of the cytoskeletal fraction by Triton-X100 extraction [ , , ], slides were stained using Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for Actin, Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes) for αV β6 integrin, and Alexa Fluor 350 donkey anti-rabbit IgG (Molecular Probes) for TGFβ1 labeling [ ].

    Techniques:

    Pak1 is associated with focal adhesions. BxPC-3 cells were stimulated with 10 nM of mature TGFβ 1 for ten minutes followed by preparation of the Triton-X100 nonsoluble fraction and precipitation with α V β 6 integrin antibodies. The precipitate was then reprecipitated with anti-FAK antibodies (sc-1688), followed by re-precipitation with anti-phospho S (ab9334, Abcam), anti-phospho Thr (ab2286), and analyzed with a Pak1 antibody (71-9300, Zymed). In part, the cells were preincubated with 50 μM PP2 (Calbiochem), and 50 μM PD98059 (Calbiochem), respectively [ 10 ].

    Journal: Molecular Cancer

    Article Title: TGF?1 activates c-Jun and Erk1 via ?V?6 integrin

    doi: 10.1186/1476-4598-2-33

    Figure Lengend Snippet: Pak1 is associated with focal adhesions. BxPC-3 cells were stimulated with 10 nM of mature TGFβ 1 for ten minutes followed by preparation of the Triton-X100 nonsoluble fraction and precipitation with α V β 6 integrin antibodies. The precipitate was then reprecipitated with anti-FAK antibodies (sc-1688), followed by re-precipitation with anti-phospho S (ab9334, Abcam), anti-phospho Thr (ab2286), and analyzed with a Pak1 antibody (71-9300, Zymed). In part, the cells were preincubated with 50 μM PP2 (Calbiochem), and 50 μM PD98059 (Calbiochem), respectively [ 10 ].

    Article Snippet: After preparation of the cytoskeletal fraction by Triton-X100 extraction [ , , ], slides were stained using Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for Actin, Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes) for αV β6 integrin, and Alexa Fluor 350 donkey anti-rabbit IgG (Molecular Probes) for TGFβ1 labeling [ ].

    Techniques:

    Colocalization of TGFβ 1 , α V β 6 integrin and the cytoskeleton. 10,000 cells(SW48, DLD1, HeLa, keratinocytes) were cultured on glass coverslips in DMEM supplemented with 17% of heat inactivated fetal bovine serum and stimulated with 10 nM of mature TGFβ 1 (from R D Systems) for ten minutes. After preparation of the cytoskeletal fraction by Triton-X100 extraction [ 10 ], slides were stained using Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for actin (sc-8432, Santa Cruz), Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes) for α V β 6 integrin (sc-6617 and sc-6632), and Alexa Fluor 350 donkey anti-rabbit IgG (Molecular Probes) for TGFβ 1 (sc-146) labeling and viewed using a Zeiss LSM-510 confocal microscope [ 10 ]. Magnification 1000 ×.

    Journal: Molecular Cancer

    Article Title: TGF?1 activates c-Jun and Erk1 via ?V?6 integrin

    doi: 10.1186/1476-4598-2-33

    Figure Lengend Snippet: Colocalization of TGFβ 1 , α V β 6 integrin and the cytoskeleton. 10,000 cells(SW48, DLD1, HeLa, keratinocytes) were cultured on glass coverslips in DMEM supplemented with 17% of heat inactivated fetal bovine serum and stimulated with 10 nM of mature TGFβ 1 (from R D Systems) for ten minutes. After preparation of the cytoskeletal fraction by Triton-X100 extraction [ 10 ], slides were stained using Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for actin (sc-8432, Santa Cruz), Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes) for α V β 6 integrin (sc-6617 and sc-6632), and Alexa Fluor 350 donkey anti-rabbit IgG (Molecular Probes) for TGFβ 1 (sc-146) labeling and viewed using a Zeiss LSM-510 confocal microscope [ 10 ]. Magnification 1000 ×.

    Article Snippet: After preparation of the cytoskeletal fraction by Triton-X100 extraction [ , , ], slides were stained using Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for Actin, Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes) for αV β6 integrin, and Alexa Fluor 350 donkey anti-rabbit IgG (Molecular Probes) for TGFβ1 labeling [ ].

    Techniques: Cell Culture, Staining, Labeling, Microscopy

    IFITM3 expression inhibits SFV capsid release . A) SFV (200 pfu/cell) was bound to cells for 1 h at 4°C prior to incubation at 37°C for the indicated times. At each time point the cells were fixed and permeabilized with 0.1% Triton‐X100 and labeled with serum against the SFV capsid, which was detected with AF594. Confocal sections are displayed. Following internalization, virus particles were detected in cells, indicated by the typical punctate association of virus with endosomes. By 40 min after warm‐up diffuse cytosolic fluorescence was seen in A549 cells, indicating release of the viral capsid protein to the cytosol. In A549 cells treated with 10 µ m monensin, and in OS‐IFITM3‐HA expressing cells, the staining remains associated with puncta and the cytosolic staining was not observed even at 60 min after warm‐up. Nuclei were detected with Hoechst staining. Scale bar represents 15 µm. B) Quantification of cytosolic fluorescence and significance testing is described in Materials and Methods . The data are from three independent infections with 3–7 images taken at each condition, with a total of at least 60 cells analyzed per cell line, per condition. Data presented is the average fluorescence intensity, normalized to the background intensity at 0 min of warming. *p

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Alphavirus Restriction by IFITM Proteins

    doi: 10.1111/tra.12416

    Figure Lengend Snippet: IFITM3 expression inhibits SFV capsid release . A) SFV (200 pfu/cell) was bound to cells for 1 h at 4°C prior to incubation at 37°C for the indicated times. At each time point the cells were fixed and permeabilized with 0.1% Triton‐X100 and labeled with serum against the SFV capsid, which was detected with AF594. Confocal sections are displayed. Following internalization, virus particles were detected in cells, indicated by the typical punctate association of virus with endosomes. By 40 min after warm‐up diffuse cytosolic fluorescence was seen in A549 cells, indicating release of the viral capsid protein to the cytosol. In A549 cells treated with 10 µ m monensin, and in OS‐IFITM3‐HA expressing cells, the staining remains associated with puncta and the cytosolic staining was not observed even at 60 min after warm‐up. Nuclei were detected with Hoechst staining. Scale bar represents 15 µm. B) Quantification of cytosolic fluorescence and significance testing is described in Materials and Methods . The data are from three independent infections with 3–7 images taken at each condition, with a total of at least 60 cells analyzed per cell line, per condition. Data presented is the average fluorescence intensity, normalized to the background intensity at 0 min of warming. *p

    Article Snippet: Immunofluorescence staining and microscopy and flow cytometry Immunofluorescence staining and microscopy was performed as previously described with the exception that 0.1% Triton‐X100 (Tx100, Sigma) was used for permeabilization in experiments to detect the SFV capsid protein.

    Techniques: Expressing, Incubation, Labeling, Fluorescence, Staining

    Membrane expression and functionality of hLDLR expressed by the CHO-hLDLR-EGFP cell line. (A) Representative confocal photomicrographs of immunostained CHO-hTfR-EGFP (negative control) and CHO-hLDLR-EGFP cells (green) with an anti-LDLR antibody diluted at 1/100 (red) following triton X100 permeabilization of the cell membrane. Cell nuclei are labeled with Hoechst#33258 at 0.5 μg/mL (blue). Co-labeling appears in yellow in the merged pictures. Only the cells with stable expression of the hLDLR-EGFP construct express the membrane receptor. (B) Representative confocal photomicrographs of CHO-hTfR-EGFP and CHO-hLDLR-EGFP cells (green) after a 10 min incubation period with DiI-LDL 15 μg/mL (red). Cell nuclei are labeled with Hoechst#33258 (blue). Co-labeling appears in yellow in the merged pictures. DiI-LDL is essentially bound to the CHO-hLDLR-EGFP cells indicating that the hLDLR-EGFP chimera receptor binds its ligand. (C) Western blots performed on cell membrane preparations of CHO cells expressing hLDLR-EGFP, rLDLR-EGFP and mLDLR-EGFP compared to CHO WT, using anti-hLDLR (1/800) and anti-rat and mouse LDLR antibodies (1/1000). These bands are also labeled with an anti-EGFP antibody. Bands of 140 kDa and 190 kDa correspond respectively to the immature and mature LDLR-EGFP fusion proteins (arrows). Actin was used to check loading of equal amounts of protein.

    Journal: PLoS ONE

    Article Title: Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos

    doi: 10.1371/journal.pone.0191052

    Figure Lengend Snippet: Membrane expression and functionality of hLDLR expressed by the CHO-hLDLR-EGFP cell line. (A) Representative confocal photomicrographs of immunostained CHO-hTfR-EGFP (negative control) and CHO-hLDLR-EGFP cells (green) with an anti-LDLR antibody diluted at 1/100 (red) following triton X100 permeabilization of the cell membrane. Cell nuclei are labeled with Hoechst#33258 at 0.5 μg/mL (blue). Co-labeling appears in yellow in the merged pictures. Only the cells with stable expression of the hLDLR-EGFP construct express the membrane receptor. (B) Representative confocal photomicrographs of CHO-hTfR-EGFP and CHO-hLDLR-EGFP cells (green) after a 10 min incubation period with DiI-LDL 15 μg/mL (red). Cell nuclei are labeled with Hoechst#33258 (blue). Co-labeling appears in yellow in the merged pictures. DiI-LDL is essentially bound to the CHO-hLDLR-EGFP cells indicating that the hLDLR-EGFP chimera receptor binds its ligand. (C) Western blots performed on cell membrane preparations of CHO cells expressing hLDLR-EGFP, rLDLR-EGFP and mLDLR-EGFP compared to CHO WT, using anti-hLDLR (1/800) and anti-rat and mouse LDLR antibodies (1/1000). These bands are also labeled with an anti-EGFP antibody. Bands of 140 kDa and 190 kDa correspond respectively to the immature and mature LDLR-EGFP fusion proteins (arrows). Actin was used to check loading of equal amounts of protein.

    Article Snippet: In order to detect the Fc conjugates in tissues of injected mice, organs (liver and adrenals), were mechanically dissociated in PBS 0.1% Triton X100, snap frozen, thawed and sonicated 10 x 10 s. Protein content in membrane and tissue extracts were quantified using the BioRad DC Protein Assay (Bio-Rad, Hercules, CA, USA) following manufacturer’s instructions.

    Techniques: Expressing, Negative Control, Labeling, Construct, Incubation, Western Blot

    Inhibition of phospholipid synthesis affects the accessibility of the replication complexes. HeLa cells after choline deprivation were infected with poliovirus at an MOI of 10 PFU/cell and incubated in a choline-free or a choline-supplemented medium after infection. A. Confocal images of the cells permeabilized with either 0.2% Triton X100 (strong permeabilization) or 0.02% saponin (mild permeabilization) and stained for a viral antigen 2B (red). Nuclear DNA is stained with Hoechst 33342 (blue). Arrows indicate areas protected upon mild permeabilization in cells incubated with choline-supplemented medium. B. Visualization of dsRNA in HeLa cells processed and infected as in A after Triton X100 permeabilization. Red arrows indicate dsRNA association with the nuclear envelope (PN) in choline-deprived cells; white arrows show big perinuclear blobs (CYT) of dsRNA signal reflecting normal development of replication membranes in choline-supplemented cells. C. HeLa cells were infected with poliovirus at an MOI of 10 PFU/cell after choline deprivation and incubated in a choline-free or a choline-supplemented medium for 4 h p.i. The cells were permeabilized with digitonin and treated (lines 3 and 4) or non-treated (lines 1 and 2) with proteinase K. * indicates protein fragments generated upon proteinase K treatment. Actin is shown as a loading control.

    Journal: PLoS Pathogens

    Article Title: Phospholipid synthesis fueled by lipid droplets drives the structural development of poliovirus replication organelles

    doi: 10.1371/journal.ppat.1007280

    Figure Lengend Snippet: Inhibition of phospholipid synthesis affects the accessibility of the replication complexes. HeLa cells after choline deprivation were infected with poliovirus at an MOI of 10 PFU/cell and incubated in a choline-free or a choline-supplemented medium after infection. A. Confocal images of the cells permeabilized with either 0.2% Triton X100 (strong permeabilization) or 0.02% saponin (mild permeabilization) and stained for a viral antigen 2B (red). Nuclear DNA is stained with Hoechst 33342 (blue). Arrows indicate areas protected upon mild permeabilization in cells incubated with choline-supplemented medium. B. Visualization of dsRNA in HeLa cells processed and infected as in A after Triton X100 permeabilization. Red arrows indicate dsRNA association with the nuclear envelope (PN) in choline-deprived cells; white arrows show big perinuclear blobs (CYT) of dsRNA signal reflecting normal development of replication membranes in choline-supplemented cells. C. HeLa cells were infected with poliovirus at an MOI of 10 PFU/cell after choline deprivation and incubated in a choline-free or a choline-supplemented medium for 4 h p.i. The cells were permeabilized with digitonin and treated (lines 3 and 4) or non-treated (lines 1 and 2) with proteinase K. * indicates protein fragments generated upon proteinase K treatment. Actin is shown as a loading control.

    Article Snippet: Triton-X100 was from Promega.

    Techniques: Inhibition, Infection, Incubation, Staining, Generated

    TcK2 is located in the endosomal compartment and is phosphorylated in non-proliferating parasite stages of T. cruzi . (A) Wild type epimastigotes (Epi), metacyclics (Meta), intracellular amastigotes (Ama) and cell-released trypomastigotes (Trypo) were fixed, permeabilized, and incubated with anti-BiP antibodies (Bip, green) and mAb 5D10 (TcK2, red). The images show individual immunofluorescence and DAPI labeling, DIC images and the merged immunofluorescence labeling from an enlarged field (Bars = 5 μm). (B) Wild type (WT) or HA-tagged K2 (TcK2-HA) epimastigotes were adsorbed to glass slides, fixed with paraformaldehyde and permeabilized with 0.1% Triton X100. Adhered cells were then stained with mAb 5D10 (TcK2, green) or anti-HA (HA, red), and DAPI (blue). The images show individual labeling and the fluorescence signals merged with differential interference contrast (DIC). (C) Western blots of total extracts of parasites of the indicated stages revealed by anti-TcK2 (anti-KD) antibody. Anti-β-tubulin was used as a loading control. The graph shows the mean and standard deviation of the TcK2 [Up] /TcK2 [Low] ratio (n = 3).

    Journal: PLoS Pathogens

    Article Title: A Membrane-bound eIF2 Alpha Kinase Located in Endosomes Is Regulated by Heme and Controls Differentiation and ROS Levels in Trypanosoma cruzi

    doi: 10.1371/journal.ppat.1004618

    Figure Lengend Snippet: TcK2 is located in the endosomal compartment and is phosphorylated in non-proliferating parasite stages of T. cruzi . (A) Wild type epimastigotes (Epi), metacyclics (Meta), intracellular amastigotes (Ama) and cell-released trypomastigotes (Trypo) were fixed, permeabilized, and incubated with anti-BiP antibodies (Bip, green) and mAb 5D10 (TcK2, red). The images show individual immunofluorescence and DAPI labeling, DIC images and the merged immunofluorescence labeling from an enlarged field (Bars = 5 μm). (B) Wild type (WT) or HA-tagged K2 (TcK2-HA) epimastigotes were adsorbed to glass slides, fixed with paraformaldehyde and permeabilized with 0.1% Triton X100. Adhered cells were then stained with mAb 5D10 (TcK2, green) or anti-HA (HA, red), and DAPI (blue). The images show individual labeling and the fluorescence signals merged with differential interference contrast (DIC). (C) Western blots of total extracts of parasites of the indicated stages revealed by anti-TcK2 (anti-KD) antibody. Anti-β-tubulin was used as a loading control. The graph shows the mean and standard deviation of the TcK2 [Up] /TcK2 [Low] ratio (n = 3).

    Article Snippet: Alternatively, extracts were prepared by lysis of 1 × 107 cells in 50 μl of ice-cold 50 mM NaCl, 20 mM Tris-HCl (pH 7.4) containing 1% Triton X100, 1 mM phenylmethanesulfonyl fluoride (PMSF), and the cOmplete protease inhibitor cocktail, EDTA-free (Roche).

    Techniques: Incubation, Immunofluorescence, Labeling, Staining, Fluorescence, Western Blot, Standard Deviation

    T. cruzi K2 is a membrane bound protein kinase that undergoes autophosphorylation and phosphorylates the parasite eIF2α. (A) Comparative topology of T. cruzi (TcK2) and Mus musculus (Mm) PERK indicating the signal sequence (SS), the transmembrane (TMD) and the kinase (KD) domains. (B) Western blot of total extracts of T. cruzi epimastigotes incubated with an antiserum from a rabbit immunized with a recombinant protein corresponding to T. brucei K2 KD or with a mAb 5D10 prepared against a recombinant K2 fragment located as indicated in panel (A). (C) Western blot of the 100,000 g supernatant (Sol) (1 h centrifugation) from parasites extracts; of the supernatant from the previous pellet after extraction with 1% Triton X100 in the same buffer extraction (Memb) and of the insoluble fraction after detergent extraction (Pellet). T. cruzi epimastigotes were lysed in 0.1 M NaCl, 50 mM Tris-HCl (pH 7.4), 2 mM MgCl 2 , 10 mM ethylene-glycol tetra-acetic acid, containing the cOmplete cocktail by three freezing and thawing cycles. The blots were probed with anti-TcK2-KD and anti-Hsp70 antibodies. (D) Western blot of lysates of epimastigotes (Tot), or the supernatants of epimastigotes lysed in the presence of indicted amounts of digitonin as described [ 21 ] and probed with and anti-TcK2 and anti-BiP and anti-HsP70 antibodies. (E) SDS-PAGE of 6XHis-tagged TceIF2α incubated in the presence of indicated proteins for 15 min with [ 32 P]-γ-ATP. The upper panel shows the autoradiogram and the bottom panel the same gel stained with Coomassie Blue R250. (F) and (G) are blots of, respectively, total extracts of T. cruzi epimastigotes treated with CIAP or of cells pre-incubated for 2 h without (−) or with (+) 10 nM of calyculin A. Anti-β-tubulin was used as a loading control. The histograms below the gel represent the relative amounts of the upper phosphorylated band of TcK2 (TcK2 [Up] ) and the fast migrating band of TcK2 (TcK2 [Low] ). The values are mean and standard deviations of triplicates. Asterisks indicate statistically significant differences (p

    Journal: PLoS Pathogens

    Article Title: A Membrane-bound eIF2 Alpha Kinase Located in Endosomes Is Regulated by Heme and Controls Differentiation and ROS Levels in Trypanosoma cruzi

    doi: 10.1371/journal.ppat.1004618

    Figure Lengend Snippet: T. cruzi K2 is a membrane bound protein kinase that undergoes autophosphorylation and phosphorylates the parasite eIF2α. (A) Comparative topology of T. cruzi (TcK2) and Mus musculus (Mm) PERK indicating the signal sequence (SS), the transmembrane (TMD) and the kinase (KD) domains. (B) Western blot of total extracts of T. cruzi epimastigotes incubated with an antiserum from a rabbit immunized with a recombinant protein corresponding to T. brucei K2 KD or with a mAb 5D10 prepared against a recombinant K2 fragment located as indicated in panel (A). (C) Western blot of the 100,000 g supernatant (Sol) (1 h centrifugation) from parasites extracts; of the supernatant from the previous pellet after extraction with 1% Triton X100 in the same buffer extraction (Memb) and of the insoluble fraction after detergent extraction (Pellet). T. cruzi epimastigotes were lysed in 0.1 M NaCl, 50 mM Tris-HCl (pH 7.4), 2 mM MgCl 2 , 10 mM ethylene-glycol tetra-acetic acid, containing the cOmplete cocktail by three freezing and thawing cycles. The blots were probed with anti-TcK2-KD and anti-Hsp70 antibodies. (D) Western blot of lysates of epimastigotes (Tot), or the supernatants of epimastigotes lysed in the presence of indicted amounts of digitonin as described [ 21 ] and probed with and anti-TcK2 and anti-BiP and anti-HsP70 antibodies. (E) SDS-PAGE of 6XHis-tagged TceIF2α incubated in the presence of indicated proteins for 15 min with [ 32 P]-γ-ATP. The upper panel shows the autoradiogram and the bottom panel the same gel stained with Coomassie Blue R250. (F) and (G) are blots of, respectively, total extracts of T. cruzi epimastigotes treated with CIAP or of cells pre-incubated for 2 h without (−) or with (+) 10 nM of calyculin A. Anti-β-tubulin was used as a loading control. The histograms below the gel represent the relative amounts of the upper phosphorylated band of TcK2 (TcK2 [Up] ) and the fast migrating band of TcK2 (TcK2 [Low] ). The values are mean and standard deviations of triplicates. Asterisks indicate statistically significant differences (p

    Article Snippet: Alternatively, extracts were prepared by lysis of 1 × 107 cells in 50 μl of ice-cold 50 mM NaCl, 20 mM Tris-HCl (pH 7.4) containing 1% Triton X100, 1 mM phenylmethanesulfonyl fluoride (PMSF), and the cOmplete protease inhibitor cocktail, EDTA-free (Roche).

    Techniques: Sequencing, Western Blot, Incubation, Recombinant, Centrifugation, SDS Page, Staining

    The impact of crosslinking on the purification of TF – RNCs. (a,b) Polysome profiles in the absence of crosslinker or after DSP or EDC ex vivo crosslinking and sucrose gradient centrifugation. Depicted are data from experiments in which E. coli MC4100 cells were grown in LB medium and harvested as described in step 1 , option C. The lysate was either crosslinked ex vivo with DSP or EDC (step 7 , option B) or left untreated (step 7 , option A). Undigested (a) and digested (b) lysates were run on a sucrose gradient (step 18 , option B). The digestion was performed with a reduced MNase concentration of 15 U/A 260 to partially retain di- and trisomes for comparison. ‘30S’ and ‘50S’ depict the peaks of the small and large ribosomal subunits, respectively. The monosome peak is labeled with ‘70S’. To compare polysome profiles quantitatively, the curves were normalized to the same area underneath all ribosomal peaks. (c) Gel analysis of the DSP crosslinker titration. 200 ml of E. coli MC4100 ∆ tig::Kan + pTrc-tig-TEV-Avi cells were grown and harvested according to step 1 , option A. Cells were resuspended in 2 ml of buffer A (50 mM HEPES pH 7.5, 1 M potassium acetate, 10 mM MgAc 2 , 1 mM PMSF, 1 mM chloramphenicol, 0.4% Triton X100, 0.1% NP-40, 1 mg/ml lysozyme, 2.5 µg/ml RNase-free DNase I). Lysis and purification of TF – RNCs was performed as described including DSP ex vivo crosslinking (step 7 , option B) and sucrose cushion centrifugation (step 18 , option A) with the following exceptions: For crosslinking either 3 mg of DSP (‘1×’), 15 mg of DSP (‘5×’) or DSMO only (‘−‘) were used. Ultracentrifugation was done with 1 M potassium acetate instead of 1 M NaCl in the sucrose cushion buffer causing the high amount of non-crosslinked TF that copelleted without DSP addition (‘−‘). For AP only half of Strep-Tactin slurry and TEV protease were used. Either non-reducing (‘non-red.’) or reducing (‘red.’) sample buffer was used for SDS-PAGE. Gels were stained with coomassie or used for western blotting employing a polyclonal α-TF antibody. Crosslinks are abbreviated with ‘X’. (d) Gel analysis of the EDC crosslinker titration. E. coli MC4100 ∆ tig::Kan + pTrc-tig-TEV-Avi cells were grown in 1 l LB, harvested as described in step 1 , option A, and resuspended in 6 ml of lysis buffer. Ex vivo crosslinking (step 7 , option B) was performed with 2.5 mM (‘0.125×’), 10 mM (‘0.5×’), 20 mM (‘1×’) and 80 mM (‘4×’) EDC. TF–RNCs were purified as described, including sucrose cushion centrifugation (step 18 , option A), eluted from the affinity matrix by boiling in reducing sample buffer, and analyzed by SDS-PAGE or western blotting using polyclonal antibodies against TF and L23. Crosslinks are abbreviated with ‘X’.

    Journal: Nature protocols

    Article Title: Selective ribosome profiling as a tool to study the interaction of chaperones and targeting factors with nascent polypeptide chains and ribosomes

    doi: 10.1038/nprot.2013.133

    Figure Lengend Snippet: The impact of crosslinking on the purification of TF – RNCs. (a,b) Polysome profiles in the absence of crosslinker or after DSP or EDC ex vivo crosslinking and sucrose gradient centrifugation. Depicted are data from experiments in which E. coli MC4100 cells were grown in LB medium and harvested as described in step 1 , option C. The lysate was either crosslinked ex vivo with DSP or EDC (step 7 , option B) or left untreated (step 7 , option A). Undigested (a) and digested (b) lysates were run on a sucrose gradient (step 18 , option B). The digestion was performed with a reduced MNase concentration of 15 U/A 260 to partially retain di- and trisomes for comparison. ‘30S’ and ‘50S’ depict the peaks of the small and large ribosomal subunits, respectively. The monosome peak is labeled with ‘70S’. To compare polysome profiles quantitatively, the curves were normalized to the same area underneath all ribosomal peaks. (c) Gel analysis of the DSP crosslinker titration. 200 ml of E. coli MC4100 ∆ tig::Kan + pTrc-tig-TEV-Avi cells were grown and harvested according to step 1 , option A. Cells were resuspended in 2 ml of buffer A (50 mM HEPES pH 7.5, 1 M potassium acetate, 10 mM MgAc 2 , 1 mM PMSF, 1 mM chloramphenicol, 0.4% Triton X100, 0.1% NP-40, 1 mg/ml lysozyme, 2.5 µg/ml RNase-free DNase I). Lysis and purification of TF – RNCs was performed as described including DSP ex vivo crosslinking (step 7 , option B) and sucrose cushion centrifugation (step 18 , option A) with the following exceptions: For crosslinking either 3 mg of DSP (‘1×’), 15 mg of DSP (‘5×’) or DSMO only (‘−‘) were used. Ultracentrifugation was done with 1 M potassium acetate instead of 1 M NaCl in the sucrose cushion buffer causing the high amount of non-crosslinked TF that copelleted without DSP addition (‘−‘). For AP only half of Strep-Tactin slurry and TEV protease were used. Either non-reducing (‘non-red.’) or reducing (‘red.’) sample buffer was used for SDS-PAGE. Gels were stained with coomassie or used for western blotting employing a polyclonal α-TF antibody. Crosslinks are abbreviated with ‘X’. (d) Gel analysis of the EDC crosslinker titration. E. coli MC4100 ∆ tig::Kan + pTrc-tig-TEV-Avi cells were grown in 1 l LB, harvested as described in step 1 , option A, and resuspended in 6 ml of lysis buffer. Ex vivo crosslinking (step 7 , option B) was performed with 2.5 mM (‘0.125×’), 10 mM (‘0.5×’), 20 mM (‘1×’) and 80 mM (‘4×’) EDC. TF–RNCs were purified as described, including sucrose cushion centrifugation (step 18 , option A), eluted from the affinity matrix by boiling in reducing sample buffer, and analyzed by SDS-PAGE or western blotting using polyclonal antibodies against TF and L23. Crosslinks are abbreviated with ‘X’.

    Article Snippet: Phenylmethylsulfonylfluoride (PMSF, Roth, 6367) Triton X100 (Roth, 3051) Nonidet P 40 Substitute (NP-40, Sigma, 74385) RNase-free DNase I (Roche, 04716728001) HCl 37% (wt/wt) (VWR, 20252.335) CAUTION Hydrochloric acid is corrosive.

    Techniques: Purification, Ex Vivo, Gradient Centrifugation, Concentration Assay, Labeling, Titration, Lysis, Centrifugation, SDS Page, Staining, Western Blot

    Cell culture model for osteoblastic differentiation of pluripotent human bone marrow-derived MSC. ( A ) Representative FACS analysis out of 20 of an MSC defining surface marker panel confirming homogeneity of isolated cells. More than 98% of cells are positive for CD73, CD90 and CD105 and negative for CD11b, CD14, CD19, CD34, CD45, and HLA-DR compared to isotype control (grey). ( B ) Chondroblastic differentiation of MSC after incubation for 32 days with chondroblast induction medium demonstrated by western blot analysis for chondrocyte-specific type II collagen. Two representative cell preparations out of 20 are shown. α-Tubulin served as loading control. ( C ) Adipocytic differentiation of MSC after incubation for 21 days with adipocyte induction medium demonstrated by staining of intracellular lipid droplets with Oil red O. Representative experiment out of 20, phase contrast microscopy, original magnification × 200, scale bar = 50 µm. ( D ) Osteoblastic differentiation of MSC after incubation for 21 days with osteoblast induction medium demonstrated by staining of extracellular hydroxyapatite deposits with Alizarin red S. Representative experiment out of 20, phase contrast microscopy, original magnification x100, scale bar = 50 µm. ( E ) Baseline expression of vascular smooth muscle marker proteins in MSC: SM22α, smooth muscle calponin (sm-Calponin), myosin light chain kinase (MLCK), smooth muscle-α-actin (SMA). Western blot analyses of three representative cell preparations out of 20 are shown. α-Tubulin served as loading control. ( F ) Calcium transients without (control) and after pretreatment with nimodipine (Nimodipine) in MSC at base line showing expression of functional voltage-dependent dihydropyridine-sensitive (Ca v 1.2) L-type Ca 2+ channels. Representative experiment out of three; 20–30 individual cells were studied for each tracing.

    Journal: Scientific Reports

    Article Title: mTORC1 and mTORC2 Differentially Regulate Cell Fate Programs to Coordinate Osteoblastic Differentiation in Mesenchymal Stromal Cells

    doi: 10.1038/s41598-019-56237-w

    Figure Lengend Snippet: Cell culture model for osteoblastic differentiation of pluripotent human bone marrow-derived MSC. ( A ) Representative FACS analysis out of 20 of an MSC defining surface marker panel confirming homogeneity of isolated cells. More than 98% of cells are positive for CD73, CD90 and CD105 and negative for CD11b, CD14, CD19, CD34, CD45, and HLA-DR compared to isotype control (grey). ( B ) Chondroblastic differentiation of MSC after incubation for 32 days with chondroblast induction medium demonstrated by western blot analysis for chondrocyte-specific type II collagen. Two representative cell preparations out of 20 are shown. α-Tubulin served as loading control. ( C ) Adipocytic differentiation of MSC after incubation for 21 days with adipocyte induction medium demonstrated by staining of intracellular lipid droplets with Oil red O. Representative experiment out of 20, phase contrast microscopy, original magnification × 200, scale bar = 50 µm. ( D ) Osteoblastic differentiation of MSC after incubation for 21 days with osteoblast induction medium demonstrated by staining of extracellular hydroxyapatite deposits with Alizarin red S. Representative experiment out of 20, phase contrast microscopy, original magnification x100, scale bar = 50 µm. ( E ) Baseline expression of vascular smooth muscle marker proteins in MSC: SM22α, smooth muscle calponin (sm-Calponin), myosin light chain kinase (MLCK), smooth muscle-α-actin (SMA). Western blot analyses of three representative cell preparations out of 20 are shown. α-Tubulin served as loading control. ( F ) Calcium transients without (control) and after pretreatment with nimodipine (Nimodipine) in MSC at base line showing expression of functional voltage-dependent dihydropyridine-sensitive (Ca v 1.2) L-type Ca 2+ channels. Representative experiment out of three; 20–30 individual cells were studied for each tracing.

    Article Snippet: Cells were lysed with 250 µl ALP lysis buffer (150 mM Tris pH 10.0 (Roth), 0.1 mM ZnCl2 , 0.1 mM MgCl2 , 1% Triton-X100 (all from Applichem)) at room temperature under constant agitation for 30 minutes.

    Techniques: Cell Culture, Derivative Assay, FACS, Marker, Isolation, Incubation, Western Blot, Staining, Microscopy, Expressing, Functional Assay

    LDH- and hCG-levels in culture supernatants of placental explants, BeWo cells, and primary cytotrophoblast cells, exposed to neutral (Neu), positively (Pos), and negatively charged (Neg) dPG-NPs for 24 h, at concentrations of 1 µM and 10 nM and to 1% Triton ® X100. LDH data are shown in relative units as ratio to control values, while hCG data are presented in mU mL −1 . *( p

    Journal: Nanotoxicology

    Article Title: Dendritic polyglycerol nanoparticles show charge dependent bio-distribution in early human placental explants and reduce hCG secretion

    doi: 10.1080/17435390.2018.1425496

    Figure Lengend Snippet: LDH- and hCG-levels in culture supernatants of placental explants, BeWo cells, and primary cytotrophoblast cells, exposed to neutral (Neu), positively (Pos), and negatively charged (Neg) dPG-NPs for 24 h, at concentrations of 1 µM and 10 nM and to 1% Triton ® X100. LDH data are shown in relative units as ratio to control values, while hCG data are presented in mU mL −1 . *( p

    Article Snippet: Unexposed cells and tissues were used as negative controls, Triton® X100 (Merck KGaA; 1% vol. diluted in medium) exposure was used to generate positive controls.

    Techniques: