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  • pbs  (Lonza)
    99
    Lonza pbs
    House dust mite <t>(HDM)-induced</t> airway resistance diagram. The airway resistance was abrogated upon oral administration of 20 mg/kg TP (prebiotic), or 10 5 or 10 7 cfu/mouse LGG (probiotic) or TP in combination with 10 5 or 10 7 cfu/mouse of LGG (synbiotic). Lung resistance (RL) measured in response to increasing doses of methacholine. <t>PBS-PBS</t> (control negative group): PBS sensitized, challenged, and treated mice, PBS-TP: PBS sensitized and challenged, and TP (20 mg/kg) treated mice, PBS-TP- LGG E7: PBS sensitized and challenged, and synbiotic (with 10 7 cfu/mouse LGG) treated mice, HDM-PBS (control positive group): HDM sensitized and challenged, and PBS treated mice, HDM-CS: HDM sensitized and challenged, and corticosteroid treated mice, HDM- LGG E5: HDM sensitized and challenged, and 10 5 cfu/mouse probiotic treated mice, HDM-LGG E7: HDM sensitized and challenged, and 10 7 cfu/mouse probiotic treated mice. HDM-TP: HDM sensitized and challenged, and prebiotic treated mice, HDM-TP-LGG E5: HDM sensitized and challenged, and synbiotic (with 10 5 cfu/mouse LGG) treated mice, HDM-TP-LGG E7: HDM sensitized and challenged, and synbiotic (with 10 7 cfu/mouse LGG) treated mice. Results are shown as mean ± SEM . * P
    Pbs, supplied by Lonza, used in various techniques. Bioz Stars score: 99/100, based on 1233 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    LI-COR odyssey blocking buffer
    T-cell anergy in chronic infection. ( A ) We assessed CTL priming and recall responses as described in the diagram. The value in each panel represents the percentage of OVA-specific (tetramer-positive) CD8 + T cells in the total CD8 + T-cell population. ( B , C ) We assessed cell proliferation as described in the diagrams. Cell divisions were monitored by analyzing CFSE dilution. Histograms show the FACS profiles of ( B ) CD8 + /CFSE + T cells from the in vitro proliferation assay or ( C ) PE-tetramer + /CFSE + T cells from the in vivo proliferation assay. The numbers of cell divisions were indicated on top of each panel, and the percentages of dividing cells were indicated on top of the number of divisions. ( D ) Peripheral blood samples from naive mice or chronically AdVova-infected mice were stained with PE-CD45RA, FITC-CD8 and PE-Cy5-labeled Abs and then analyzed by flow cytometry. Naive CD8 + T cells with positive PE-CD45RA and FITC-CD8 staining were gated (rectangle) for further measurement of the expression of co-stimulatory or inhibitory molecules (solid lines on the right). Mean fluorescence intensity (MFI) numbers are indicated in each panel. Dotted lines (on the left) represent isotype-matched controls. The control MFI numbers for the upper panel’s PD-L1/BTLA and CD40 were 1.28 and 1.80, respectively, while the control MFI numbers for the lower panel’s PD-L1/BTLA and CD40 were 2.51 and 1.80, respectively. ( E ) The expression of anergy-associated genes was assessed by RT-PCR using mRNA purified from naive CD8 + T cells derived from naive or chronically infected mouse splenocytes. The numbers of mRNA fold changes (the ratio of mRNA expression of each gene in naive CD8 + T cells derived from AdVova-infected mice vs WT B6 mice) are indicated. ( F ) Western blotting analysis. The protein extracts were analyzed by western blotting. Each protein band intensity was quantitated using a computational densitometer in the <t>ODYSSEY</t> software. The expression ratio (ER) represents the protein expression of each gene normalized to the matching β-actin control. The relative protein expression (RPE) represents the ratio of the ER of each gene in AdVova-infected B6 mice vs that in naive B6 mice. * P
    Odyssey Blocking Buffer, supplied by LI-COR, used in various techniques. Bioz Stars score: 99/100, based on 23218 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    odyssey blocking buffer - by Bioz Stars, 2020-09
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    94
    Thermo Fisher pbs
    Pharmacological inhibition of CXCR4 does not impact the tissue exit of wildtype or Ccr7 −/− T cells. ( A–B ) Chemotaxis of mouse splenocytes was tested toward mouse CXCL12 ( A ) and CCL21 ( B ) in a Transwell chemotaxis assay in the presence or absence of CXCR4 inhibitor <t>AMD3100</t> at indicated concentrations. Data represent mean ±SD of triplicate wells. One of two similar experiments is shown. ( C–E ) Mice carrying 3-week-old skin granulomas in the footpads were systemically treated with either <t>PBS</t> or 1000 µg/kg/h AMD3100 through subcutaneously implanted mini osmotic pumps. 12 h after implantation, a mixture of fluorescently labeled Ccr7 −/− and wildtype splenocytes were transferred into the inflamed footpads. The numbers and phenotypes of cells that egressed from the skin and entered the draining lymph node were determined by flow cytometry 12 h after transfer. The numbers of wildtype ( C ) and Ccr7 −/− ( D ) lymphocyte subsets that migrated to the draining lymph nodes are shown. Data points represent individually analyzed mice of groups of 7–10 recipient mice per group; horizontal lines indicate the mean of each group. One of two experiments with similar results ( C and D ) or both experiments combined ( E ) are shown. WT, wildtype.
    Pbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 45979 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore pbs
    Influence of culture and measurement conditions on cell mechanical properties of DLD-1 colon carcinoma cells: ( a ) Elastic moduli of cells from four independent measurements, arbitrarily split into two groups (control 1 and control 2). ( b ) Cell elastic moduli measured for cell populations with different culture confluency (20, 60, and 100%) before harvesting. ( c ) Cell elastic moduli in response to different detachment times before measurement (20, 60, and 100 min). ( d ) Cell elastic moduli measured for three different measurement media: <t>PBS,</t> HEPES-buffer, and DMEM cell culture medium. ( e ) Cell elastic moduli measured after coating the devices with 1 wt % <t>pluronic</t> for 0, 20, and 40 min. ( f ) Cell elastic moduli measured with devices coated for 30 min with pluronic concentrations of 0.25, 1, and 4 wt %. n > 1200 for each condition. ε max and Δ p histogram matching was performed separately for each ( a )–( f ). Numbers show mean ± SE. Significant ( p
    Pbs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 34240 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pbs - by Bioz Stars, 2020-09
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    99
    Millipore phosphate buffered saline pbs
    Confirmation of E2 and Cap expression. A. Confirmation of E2 and Cap expression by immunofluorescence assay (IFA). The E2 and Cap proteins expression was verified by IFA in PK-15 cells infected with rPRVTJ-delgE/gI/TK-E2-Cap. Briefly, PK-15 cells were infected with classical swine fever virus (CSFV), porcine circovirus type 2 (PCV2) rPRVTJ-delgE/gI/TK-E2-Cap and PRV TJ at a multiplication of infectivity (MOI) of 1 or left un-infected (mock). At 48 hpi, the cells were fixed and analyzed by IFA using the anti-E2 MAb HQ06 and anti-Cap MAb 36A9 primary antibodies and fluorescein <t>isothiocyanate</t> (FITC)-labeled anti-mouse secondary antibody. Bars, 400 nm. The results were visualized under a fluorescent microscope (TE2000-U Nikon, Japan). B. Expression of E2 and Cap by Western blotting. To confirm E2 and Cap expression, PK-15 cells were either infected with the CSFV, rPRVTJ-delgE/gI/TK-E2-Cap, rPRVTJdelgE/gI/TK-Cap, rPRVTJ-delgE/gI/TK or mock infected. At 48 hpi, the cells were lysed and assayed by Western blotting using the anti-E2 MAb HQ06 and anti-Cap MAb 36A9 (1:300) in <t>PBS,</t> subsequently incubated with IRDye 800CW-labeled goat anti-mouse secondary antibody (Li-Cor). GAPDH was used as a loading control. The arrows indicate the representative bands of the CSFV E2 protein and PCV2 Cap fused with the PRV gG.
    Phosphate Buffered Saline Pbs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 41313 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    phosphate buffered saline pbs - by Bioz Stars, 2020-09
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    93
    Thermo Fisher dulbecco s phosphate buffered saline
    Effect of pro-inflammatory cytokines on the expression of human leukocyte antigens (HLAs) and co-stimulatory molecules on the surface of ADSCs. ADSCs were isolated from human adipose tissue and cultured in complete <t>Dulbecco’s</t> modified Eagle’s medium (DMEM). ADSCs that had been passaged fewer than eight times were used. ADSCs were stained with monoclonal antibodies (mAbs) against CD80 and CD86. ADSCs were additionally stained with mAbs against HLA-DR and HLA-ABC and for markers of NKG2DL with mAbs against MIC-A, MIC-B, ULBP1 and ULBP2/5/6. The data are representative of at least three experiments. IFN+IL-17: combination of IFN-γ and IL-17A/F; IFN+IL-17+IL-23: combination of IFN-γ, IL-17A/F and IL-23.
    Dulbecco S Phosphate Buffered Saline, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 6477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson pbs
    Safranin O/fast green (A, B) and hematoxylin/eosin (C, D) micrographs of unseeded (A, C) and human adipose-derived stem cell-seeded (B, D) <t>TDM-,</t> FN-, and <t>PBS-coated</t> multilayered, electrospun scaffolds cultured for 28 days. Scale bar=100 μm.
    Pbs, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 4681 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pbs - by Bioz Stars, 2020-09
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    92
    Trinity Biotech fitzgerald pb
    Differences in resting state functional connectivity between more effective versus less effective DLPFC stimulation sites. Coordinates are taken from Herbsman et al. 2009 (A–C) and <t>Fitzgerald</t> et al. 2009 (D–F). The top row (A, D) shows the DLPFC regions of interest compared in each study. The middle row (B, E) shows significant differences in resting state functional connectivity between the two sites (more effective – less effective). The border of our a-priori defined subgenual region of interest is show for reference in red. The bottom row (C, F) shows bar graphs of the correlation of each DLPFC site with the subgenual cingulate. In both cases the more effective DLPFC site is significantly more anticorrelated with the subgenual cingulate than the less effective site.
    Fitzgerald Pb, supplied by Trinity Biotech, used in various techniques. Bioz Stars score: 92/100, based on 2000 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    fitzgerald pb - by Bioz Stars, 2020-09
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    94
    Agilent technologies pbs
    Treatment of 005 GSC-derived tumors with systemic immune checkpoint inhibitors, intratumoral G47 Δ-mIL12, or the combination prolongs survival (A–B) Mice implanted with 2 × 10 4 005 GSCs on day 0, treated with G47Δ-mIL12 (5 × 10 5 pfu) or <t>PBS</t> injected IT on day 12 (upward arrow) and isotype control IgG (10 mg/kg), anti-(α)PD-L1 antibody (A) , or anti-(α)PD-1 antibody (B) injected IP on days 15, 18 and 21 (downward arrows). Values from a single experiment, with Mock (treated with PBS and IgG) and G47Δ-mIL12 groups the same in A and B. Median survival of Mock (33.5 days; n=6) was compared to anti-PD-1 (39 days; n=8, p=0.02), anti-PD-L1 (42 days; n=7, p=0.003), or G47Δ-mIL12 (39 days; n=8, p=0.01) by Log-rank analysis. Similarly, G47Δ-mIL12 was compared to the combination of G47Δ-mIL12 with anti-PD-1 (49 days; n=7, p=0.02) or -PD-L1 (50 days; n=7, p=0.03), and antibodies were compared to the combination of G47Δ-mIL12 with anti-PD-1 (p=0.053) or anti-PD-L1 (p=0.08). Experiment was conducted once (A) or twice (B). C. Mice implanted with 2 × 10 4 005 GSCs on day 0 and treated with G47Δ-mIL12 or PBS injected IT on day 8 and anti-(α)CTLA-4 antibody or isotype control IgG (5 mg/kg) injected IP on days 8, 11 and 14 (n=8/group, except for G47Δ-mIL12 n=7). Median survival of Mock (37.5 days) was compared to anti-CTLA-4 (45 days; p=0.002) or G47Δ-mIL12 (40 days; p=0.03) alone by Log-rank analysis. Similarly, combination of G47Δ-mIL12 with anti-CTLA-4 (58 days) was compared to anti-CTLA-4 (p=0.05) or G47Δ-mIL12 (p=0.008). Experiment was conducted 2 times. D. Mice implanted with 2 × 10 4 005 GSCs on day 0, treated with PBS (right; n=2), rat anti-(α)PD-1 antibody (middle; 200 μg/mouse; n=2), or rat anti-(α)PD-L1 antibody (left; 200 μg/mouse; n=2) injected IP on day 25, and sacrificed 3 hr later. Antibodies were detected with <t>HRP-conjugated</t> anti-(α)rat Ig (brown; right) or control HRP-conjugated anti-(α)rabbit Ig (left). * normal brain adjacent to tumor. Scale bar=100 μm. E. 005 GSCs (1.5 × 10 5 ) implanted on day 0, treated with PBS or G47Δ-mIL12 (5 × 10 5 .
    Pbs, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 5525 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 5525 article reviews
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    99
    GE Healthcare pbs
    Antimicrobial activities. (A) Outer membrane permeability of 4 kinds of microbe (2 Gram-negative and 2 Gram-positive bacteria) was measured using <t>PMAP36-P22</t> lysozyme fusion protein by EtBr influx assay. The activities were compared with <t>PBS</t> buffer as a control. The error bars show the standard deviation of the mean from three independent trials. Asterisks indicate statistically significant differences between groups. (****, Unpaired t -test, P
    Pbs, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 2461 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore d pbs
    Localization and expression level of MAD2 in fresh mouse oocytes with blocked microtubule polymerization. (A) Immunofluorescent staining of MAD2 (red) and <t>α-tubulin</t> (green). DNA (blue) was stained with DAPI. <t>D-PBS,</t> instead of the primary antibody, was used as the negative control. The inserted panel in the middle panel of the nocodazole group indicates only MAD2 signals. Scale bar = 10 μm. (B) Western blotting and expression levels of MAD2 in oocytes treated with nocodazole. β-actin was used as a loading control.
    D Pbs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1631 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology pbs
    In vivo biotinylated and trimyc-tagged sdAbs in bacterial lysate allow a sensitive antigenic detection on slide or beads A) Immobilization of sdAbs using trimyc tag allows the direct use of bacterial lysates. Epoxy bead were coated with <t>9E10</t> (1 μg/ml) and incubated with bacterial lysate (50 nl/wells) containing sdAbs with three (●), one (■) or no (▲) c-myc followed by serial dilutions of biotinylated Nef. The captured antigen was detected with HRP-conjugated streptavidin. B) Immobilization of sdAbs using trimyc allows direct spotting of bacterial lysate. Nitrocellulose slides were coated with 9E10 (9E10) or <t>PBS</t> (Ø). Bacterial lysates containing sdAbs fused to three, one or no c-myc were spotted. Serial dilutions of biotinylated Nef were incubated and the captured antigen was detected with Alexa705-conjugated streptavidin. C): Streptavidin beads were coated with bacterial lysate (50 nl/wells) containing anti-Nef sdAbs biotinylated in vivo (●, ▲, ◆) or not (■, ▼, ○) and incubated with serial dilutions of Nef or Alexa488-conjugated Nef (◆, ○). The captured antigen was detected with a mouse anti-Nef antibody followed by a goat anti-mouse HRP (●,■) or Alexa488-conjugated mAb (▲, ▼). D) Nitrocellulose slides were incubated with streptavidin (S) or PBS (Ø). Bacterial lysates containing sdAbs biotinylated in vivo or unbiotinylated were spotted. Serial dilutions of Nef were incubated and the captured antigen was detected with a mouse anti-Nef antibody followed by a goat anti-mouse Alexa705-conjugated mAb. Standard deviation represents two experiments performed in triplicates.
    Pbs, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 5161 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pbs - by Bioz Stars, 2020-09
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    99
    Thermo Fisher bsa pbs
    Confocal images of TRPV1 (A–D) and SP immunolabeling (E–H) expression in (A, E) BA, (B, F) AICA, (C, G) SMA, and (D, H) RA. Arrows indicate labeled: longitudinal (a), circumferential (b) fibers. Insets in A–D, show negligible labeling in BA, AICA, SMA, and RA incubated with antigen-adsorbed TRPV1 antibodies and secondary antibodies. Insets in E–H show negligible labeling in the BA, AICA, SMA, and radiating arterioles incubated with 1% <t>BSA-PBS</t> and secondary antibodies. Scale bars in microns.
    Bsa Pbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6044 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Fisher Scientific pbs
    In vitro release kinetics of entrapped <t>FITC-dextran</t> from PSN microparticles in <t>PBS</t> (pH 7.4) at 37 °C. Data is shown as mean ± S.D. (n = 3).
    Pbs, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 93/100, based on 690 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pbs  (Abcam)
    94
    Abcam pbs
    Interleukin 11 (IL-11)/IL-11R signaling pathway induced the chemo-resistance through <t>JAK/STAT3</t> pathway. (A) Immunofluoresence of p-JAK2 in BGC823 and SGC7901 cells pre-treated with or without cultured medium of cancer-associated-fibroblasts (CAFs-CM)/rhIL-11 in the presence or absence of IL-11 neutralizing antibody. (B) Immunofluoresence of p-STAT3 in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (C) Western blotting of p-JAK2, total JAK2 and β-actin in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (D) Western blotting of p-STAT3, total STAT3, and β-actin in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (E) The cell viability of BGC823 cells treated with 6 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of ruxolitinib (5 μM). <t>PBS,</t> phosphate buffered saline; Dox, doxorubicin; Eto, etoposide. (F) The cell viability of SGC7901 cells treated with 4 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of ruxolitinib (5 μM). (G) The cell viability of BGC823 cells treated with 6 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the wild type or shSTAT3 cells. (H) The cell viability of SGC7901 cells treated with 4 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the wild type or shSTAT3 cells. The data was presented as the mean±standard error of mean from three independent experiments. ** p
    Pbs, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 5349 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher pbs phosphate buffered saline 10x ph 7 4
    Interleukin 11 (IL-11)/IL-11R signaling pathway induced the chemo-resistance through <t>JAK/STAT3</t> pathway. (A) Immunofluoresence of p-JAK2 in BGC823 and SGC7901 cells pre-treated with or without cultured medium of cancer-associated-fibroblasts (CAFs-CM)/rhIL-11 in the presence or absence of IL-11 neutralizing antibody. (B) Immunofluoresence of p-STAT3 in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (C) Western blotting of p-JAK2, total JAK2 and β-actin in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (D) Western blotting of p-STAT3, total STAT3, and β-actin in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (E) The cell viability of BGC823 cells treated with 6 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of ruxolitinib (5 μM). <t>PBS,</t> phosphate buffered saline; Dox, doxorubicin; Eto, etoposide. (F) The cell viability of SGC7901 cells treated with 4 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of ruxolitinib (5 μM). (G) The cell viability of BGC823 cells treated with 6 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the wild type or shSTAT3 cells. (H) The cell viability of SGC7901 cells treated with 4 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the wild type or shSTAT3 cells. The data was presented as the mean±standard error of mean from three independent experiments. ** p
    Pbs Phosphate Buffered Saline 10x Ph 7 4, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 291 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pbs  (Bio-Rad)
    99
    Bio-Rad pbs
    Cell electrotransfection and viability rates using Bio-Rad buffer, OptiMEM-GlutaMAX, and <t>PBS.</t> Electrotransfection efficiency of mouse <t>iPS</t> ( a and b ) and MEF cells ( c and d ). In each electroporation reaction, 20 µg (1.5–2.5 µg/µl) of the reporter plasmid (encoding mCherry) was pre-mixed with cells and underwent electroporation using the square-wave protocol consisted of 250 V for iPS and 300 V for MEF cells, 2 pulses, each 10 ms length, 10 s interval, and 4 mm cuvette. The reporter expression was assessed 36 h after electroporation under a fluorescence microscope. White and black bars are electrotransfection efficiency and cell viability, respectively. Bars with different A, B, C or a, b, c letters are significantly different ( p value
    Pbs, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1280 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pbs - by Bioz Stars, 2020-09
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    94
    Thermo Fisher sterile pbs
    High-sensitivity flow-cytometry analysis of fractionated <t>IDG</t> revealed the peculiar enrichment of EVs in the 12,000 g pellet. (a) A pool of all the IDG fractions was labelled with deep red CellTracker fluorescent dye and the gates set for fluorescent EV (green) detection, compared with unlabelled EVs (Red) and CellTracker in <t>PBS-loaded</t> (empty) IDG fractions (PBS, Blue). (b–c) Each IDG fraction of the 12,000 g (b) and 35,000 g (c) was labelled and analysed for the presence of esterase-positive EVs. Results are expressed as a count of events (mean ± SD; n = 3). (d) After setting the gates for size, using beads of known diameter, we analysed the CellTracker positive population in each fraction to determine the approximate size of the observed events. (e–f) Representative results obtained for IDG fractions F7 and F8 from 12,000 g (e) and 35,000 g (f) EVs are displayed. A green colour represents CellTracker positive events. The associated quantification results are available as Supplementary File 5.
    Sterile Pbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 3430 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Quantitative analysis of neurofilament protein accumulation proximal and distal to the constriction site after constriction for 2 h in the presence or absence of metabolic substrates. Each column represents the mean accumulation ratio for the number of cells indicated and the error bars represent the standard deviation about the mean as described in the legend to Fig. 5 . For these experiments, the L-15-based culture me dium was replaced with a simpler <t>Dulbecco's</t> <t>PBS-based</t> medium with or without 0.6% glucose and 0.055% sodium pyruvate (fed and starved, respectively). The mean proximal accumulation ratio in the presence of glucose and pyruvate was lower than for axons constricted for the same period of time in L-15-based medium (Fig. 5 A) but it was, nevertheless, reduced significantly when these two substrates were omitted ( P = 0.01, t test).
    Dulbecco S Pbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 3358 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Tagging of CDCA8 in HeLa cells with GFP-3xFLAG. (A) HeLa cells were arrested in mitosis by overnight treatment with <t>STLC</t> and collected via mitotic shake-off, followed by washing with <t>PBS.</t> The indicated drugs were added to the final wash of the cells, after which the cells were lysed with freeze–thaw cycles. Cell lysates were then immunoblotted for pT232-AURK (recognizing pAURKA, pAURKB, and pAURKC) and α-tubulin. The p value between the barasertib- and okadaic acid–treated cells is 0.069; n = 2. (B) Lysates of the parent and tagged cell line were prepared as in A. Magnetic beads preloaded with an antibody against FLAG were used to immunoprecipitate (IP) tagged proteins from the lysates, and the resulting proteins were blotted for FLAG after separation via SDS–PAGE gel electrophoresis; n = 2. (C) Lysates of the tagged cell line were prepared as in A, and IPs were done with beads loaded with antibodies against random IgG or FLAG, followed by blotting for FLAG as in A; n = 2. (D) HeLa cells expressing GFP-tagged CPC were fixed with 4% paraformaldehyde, stained with an antibody against tubulin, and imaged with a spinning-disk confocal microscope at 60× magnification. Scale bars, 10 μm. Cells were imaged on three separate occasions.
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    Localisation of the hBD2 peptide in epithelial bronchial 16HBE cells . 16HBE cells were seeded at 5 × 10 5 cells per well in 1 ml of DMEM/F12 on 18-mm-diameter cover slips in 12 well plates in triplicate and grown for 16 h at 37°C. After washing the cover slips with <t>PBS-BSA,</t> the cells were exposed to either latex beads, ethanol fixed conidia or ethanol fixed HF for 18 hours. Il-1β was used as a positive control. Following washing with PBS, the cells were fixed with a paraformaldehyde solution for 30 min at 37°C. The slides were then incubated in 1% BSA/PBS-Triton 0.05%, followed by a solution of 10% normal goat serum. After washing, polyclonal rabbit anti-human hBD2 at a dilution of 1:250 was applied as primary antibody overnight at 4°C, followed by incubation with FITC-labelled goat anti-rabbit secondary antibody at a dilution of 1:300 for 4 hours at room temperature. After washing, the cover slips were mounted on slides with ProLong antifade Vectashield. Samples were viewed with a Zeiss fluorescence microscope using ×400 magnification. The arrows indicate the cells stained with anti-hBD2 antibody. The percentage of stained cells was computed from triplicates of four experiments. Means followed by the same letter are not significantly different. +, presence; -, absence of Il-1β, A. fumigatus fixed organisms and latex beads. The punctuated localisation of the signal, which is concentrated adjacent to the nucleus (arrow), was observed. The data shown are representative of four independent experiments.
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    Image Search Results


    House dust mite (HDM)-induced airway resistance diagram. The airway resistance was abrogated upon oral administration of 20 mg/kg TP (prebiotic), or 10 5 or 10 7 cfu/mouse LGG (probiotic) or TP in combination with 10 5 or 10 7 cfu/mouse of LGG (synbiotic). Lung resistance (RL) measured in response to increasing doses of methacholine. PBS-PBS (control negative group): PBS sensitized, challenged, and treated mice, PBS-TP: PBS sensitized and challenged, and TP (20 mg/kg) treated mice, PBS-TP- LGG E7: PBS sensitized and challenged, and synbiotic (with 10 7 cfu/mouse LGG) treated mice, HDM-PBS (control positive group): HDM sensitized and challenged, and PBS treated mice, HDM-CS: HDM sensitized and challenged, and corticosteroid treated mice, HDM- LGG E5: HDM sensitized and challenged, and 10 5 cfu/mouse probiotic treated mice, HDM-LGG E7: HDM sensitized and challenged, and 10 7 cfu/mouse probiotic treated mice. HDM-TP: HDM sensitized and challenged, and prebiotic treated mice, HDM-TP-LGG E5: HDM sensitized and challenged, and synbiotic (with 10 5 cfu/mouse LGG) treated mice, HDM-TP-LGG E7: HDM sensitized and challenged, and synbiotic (with 10 7 cfu/mouse LGG) treated mice. Results are shown as mean ± SEM . * P

    Journal: Frontiers in Immunology

    Article Title: Crude Turmeric Extract Improves the Suppressive Effects of Lactobacillus rhamnosus GG on Allergic Inflammation in a Murine Model of House Dust Mite-Induced Asthma

    doi: 10.3389/fimmu.2020.01092

    Figure Lengend Snippet: House dust mite (HDM)-induced airway resistance diagram. The airway resistance was abrogated upon oral administration of 20 mg/kg TP (prebiotic), or 10 5 or 10 7 cfu/mouse LGG (probiotic) or TP in combination with 10 5 or 10 7 cfu/mouse of LGG (synbiotic). Lung resistance (RL) measured in response to increasing doses of methacholine. PBS-PBS (control negative group): PBS sensitized, challenged, and treated mice, PBS-TP: PBS sensitized and challenged, and TP (20 mg/kg) treated mice, PBS-TP- LGG E7: PBS sensitized and challenged, and synbiotic (with 10 7 cfu/mouse LGG) treated mice, HDM-PBS (control positive group): HDM sensitized and challenged, and PBS treated mice, HDM-CS: HDM sensitized and challenged, and corticosteroid treated mice, HDM- LGG E5: HDM sensitized and challenged, and 10 5 cfu/mouse probiotic treated mice, HDM-LGG E7: HDM sensitized and challenged, and 10 7 cfu/mouse probiotic treated mice. HDM-TP: HDM sensitized and challenged, and prebiotic treated mice, HDM-TP-LGG E5: HDM sensitized and challenged, and synbiotic (with 10 5 cfu/mouse LGG) treated mice, HDM-TP-LGG E7: HDM sensitized and challenged, and synbiotic (with 10 7 cfu/mouse LGG) treated mice. Results are shown as mean ± SEM . * P

    Article Snippet: Murine HDM-Induced Asthma Model BALB/c mice were intranasally sensitized on day 0 with 1 μg HDM (Greer Laboratories, Lenoir, USA)/40 μL PBS (Lonza, Walkersville, USA) or PBS alone under the mild anesthetic circumstance induced by isoflurane inhalation.

    Techniques: Mouse Assay

    Schematic overview of the experimental protocol. BALB/c mice ( n = 6/group) were intranasally sensitized (i.n.) with house dust mite (HDM) or PBS on day 0 and were challenged i.n. for five consecutive days ( 7 – 11 ) with HDM or PBS. The oral gavage treatment started 2 weeks prior to sensitization. By oral gavage, the mice were received 20 mg/kg TP (prebiotic), or 10 5 or 10 7 cfu/mouse LGG (probiotic) or turmeric in combination with 10 5 or 10 7 cfu/mouse of LGG (synbiotic) once a day, throughout the study. The mice were sacrificed on day 14.

    Journal: Frontiers in Immunology

    Article Title: Crude Turmeric Extract Improves the Suppressive Effects of Lactobacillus rhamnosus GG on Allergic Inflammation in a Murine Model of House Dust Mite-Induced Asthma

    doi: 10.3389/fimmu.2020.01092

    Figure Lengend Snippet: Schematic overview of the experimental protocol. BALB/c mice ( n = 6/group) were intranasally sensitized (i.n.) with house dust mite (HDM) or PBS on day 0 and were challenged i.n. for five consecutive days ( 7 – 11 ) with HDM or PBS. The oral gavage treatment started 2 weeks prior to sensitization. By oral gavage, the mice were received 20 mg/kg TP (prebiotic), or 10 5 or 10 7 cfu/mouse LGG (probiotic) or turmeric in combination with 10 5 or 10 7 cfu/mouse of LGG (synbiotic) once a day, throughout the study. The mice were sacrificed on day 14.

    Article Snippet: Murine HDM-Induced Asthma Model BALB/c mice were intranasally sensitized on day 0 with 1 μg HDM (Greer Laboratories, Lenoir, USA)/40 μL PBS (Lonza, Walkersville, USA) or PBS alone under the mild anesthetic circumstance induced by isoflurane inhalation.

    Techniques: Mouse Assay

    T-cell anergy in chronic infection. ( A ) We assessed CTL priming and recall responses as described in the diagram. The value in each panel represents the percentage of OVA-specific (tetramer-positive) CD8 + T cells in the total CD8 + T-cell population. ( B , C ) We assessed cell proliferation as described in the diagrams. Cell divisions were monitored by analyzing CFSE dilution. Histograms show the FACS profiles of ( B ) CD8 + /CFSE + T cells from the in vitro proliferation assay or ( C ) PE-tetramer + /CFSE + T cells from the in vivo proliferation assay. The numbers of cell divisions were indicated on top of each panel, and the percentages of dividing cells were indicated on top of the number of divisions. ( D ) Peripheral blood samples from naive mice or chronically AdVova-infected mice were stained with PE-CD45RA, FITC-CD8 and PE-Cy5-labeled Abs and then analyzed by flow cytometry. Naive CD8 + T cells with positive PE-CD45RA and FITC-CD8 staining were gated (rectangle) for further measurement of the expression of co-stimulatory or inhibitory molecules (solid lines on the right). Mean fluorescence intensity (MFI) numbers are indicated in each panel. Dotted lines (on the left) represent isotype-matched controls. The control MFI numbers for the upper panel’s PD-L1/BTLA and CD40 were 1.28 and 1.80, respectively, while the control MFI numbers for the lower panel’s PD-L1/BTLA and CD40 were 2.51 and 1.80, respectively. ( E ) The expression of anergy-associated genes was assessed by RT-PCR using mRNA purified from naive CD8 + T cells derived from naive or chronically infected mouse splenocytes. The numbers of mRNA fold changes (the ratio of mRNA expression of each gene in naive CD8 + T cells derived from AdVova-infected mice vs WT B6 mice) are indicated. ( F ) Western blotting analysis. The protein extracts were analyzed by western blotting. Each protein band intensity was quantitated using a computational densitometer in the ODYSSEY software. The expression ratio (ER) represents the protein expression of each gene normalized to the matching β-actin control. The relative protein expression (RPE) represents the ratio of the ER of each gene in AdVova-infected B6 mice vs that in naive B6 mice. * P

    Journal: Cellular and Molecular Immunology

    Article Title: Novel exosome-targeted T-cell-based vaccine counteracts T-cell anergy and converts CTL exhaustion in chronic infection via CD40L signaling through the mTORC1 pathway

    doi: 10.1038/cmi.2016.23

    Figure Lengend Snippet: T-cell anergy in chronic infection. ( A ) We assessed CTL priming and recall responses as described in the diagram. The value in each panel represents the percentage of OVA-specific (tetramer-positive) CD8 + T cells in the total CD8 + T-cell population. ( B , C ) We assessed cell proliferation as described in the diagrams. Cell divisions were monitored by analyzing CFSE dilution. Histograms show the FACS profiles of ( B ) CD8 + /CFSE + T cells from the in vitro proliferation assay or ( C ) PE-tetramer + /CFSE + T cells from the in vivo proliferation assay. The numbers of cell divisions were indicated on top of each panel, and the percentages of dividing cells were indicated on top of the number of divisions. ( D ) Peripheral blood samples from naive mice or chronically AdVova-infected mice were stained with PE-CD45RA, FITC-CD8 and PE-Cy5-labeled Abs and then analyzed by flow cytometry. Naive CD8 + T cells with positive PE-CD45RA and FITC-CD8 staining were gated (rectangle) for further measurement of the expression of co-stimulatory or inhibitory molecules (solid lines on the right). Mean fluorescence intensity (MFI) numbers are indicated in each panel. Dotted lines (on the left) represent isotype-matched controls. The control MFI numbers for the upper panel’s PD-L1/BTLA and CD40 were 1.28 and 1.80, respectively, while the control MFI numbers for the lower panel’s PD-L1/BTLA and CD40 were 2.51 and 1.80, respectively. ( E ) The expression of anergy-associated genes was assessed by RT-PCR using mRNA purified from naive CD8 + T cells derived from naive or chronically infected mouse splenocytes. The numbers of mRNA fold changes (the ratio of mRNA expression of each gene in naive CD8 + T cells derived from AdVova-infected mice vs WT B6 mice) are indicated. ( F ) Western blotting analysis. The protein extracts were analyzed by western blotting. Each protein band intensity was quantitated using a computational densitometer in the ODYSSEY software. The expression ratio (ER) represents the protein expression of each gene normalized to the matching β-actin control. The relative protein expression (RPE) represents the ratio of the ER of each gene in AdVova-infected B6 mice vs that in naive B6 mice. * P

    Article Snippet: The membrane was blocked by incubation for 2 h at room temperature with ODYSSEY blocking buffer (LI-COR Bioscience, Lincoln, NE, USA) and immunoblotted with Abs specific for GRAIL and ITCH at 4 °C overnight.

    Techniques: Infection, CTL Assay, FACS, In Vitro, Proliferation Assay, In Vivo, Mouse Assay, Staining, Labeling, Flow Cytometry, Cytometry, Expressing, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Purification, Derivative Assay, Western Blot, Software

    Pharmacological inhibition of CXCR4 does not impact the tissue exit of wildtype or Ccr7 −/− T cells. ( A–B ) Chemotaxis of mouse splenocytes was tested toward mouse CXCL12 ( A ) and CCL21 ( B ) in a Transwell chemotaxis assay in the presence or absence of CXCR4 inhibitor AMD3100 at indicated concentrations. Data represent mean ±SD of triplicate wells. One of two similar experiments is shown. ( C–E ) Mice carrying 3-week-old skin granulomas in the footpads were systemically treated with either PBS or 1000 µg/kg/h AMD3100 through subcutaneously implanted mini osmotic pumps. 12 h after implantation, a mixture of fluorescently labeled Ccr7 −/− and wildtype splenocytes were transferred into the inflamed footpads. The numbers and phenotypes of cells that egressed from the skin and entered the draining lymph node were determined by flow cytometry 12 h after transfer. The numbers of wildtype ( C ) and Ccr7 −/− ( D ) lymphocyte subsets that migrated to the draining lymph nodes are shown. Data points represent individually analyzed mice of groups of 7–10 recipient mice per group; horizontal lines indicate the mean of each group. One of two experiments with similar results ( C and D ) or both experiments combined ( E ) are shown. WT, wildtype.

    Journal: PLoS ONE

    Article Title: CXCR4 Is Dispensable for T Cell Egress from Chronically Inflamed Skin via the Afferent Lymph

    doi: 10.1371/journal.pone.0095626

    Figure Lengend Snippet: Pharmacological inhibition of CXCR4 does not impact the tissue exit of wildtype or Ccr7 −/− T cells. ( A–B ) Chemotaxis of mouse splenocytes was tested toward mouse CXCL12 ( A ) and CCL21 ( B ) in a Transwell chemotaxis assay in the presence or absence of CXCR4 inhibitor AMD3100 at indicated concentrations. Data represent mean ±SD of triplicate wells. One of two similar experiments is shown. ( C–E ) Mice carrying 3-week-old skin granulomas in the footpads were systemically treated with either PBS or 1000 µg/kg/h AMD3100 through subcutaneously implanted mini osmotic pumps. 12 h after implantation, a mixture of fluorescently labeled Ccr7 −/− and wildtype splenocytes were transferred into the inflamed footpads. The numbers and phenotypes of cells that egressed from the skin and entered the draining lymph node were determined by flow cytometry 12 h after transfer. The numbers of wildtype ( C ) and Ccr7 −/− ( D ) lymphocyte subsets that migrated to the draining lymph nodes are shown. Data points represent individually analyzed mice of groups of 7–10 recipient mice per group; horizontal lines indicate the mean of each group. One of two experiments with similar results ( C and D ) or both experiments combined ( E ) are shown. WT, wildtype.

    Article Snippet: Sterile-filtered AMD3100 in PBS (Sigma-Aldrich) or PBS alone (Invitrogen) was loaded into the osmotic pumps before equilibration in sterile PBS at 37°C for 12 h according to the manufacturer’s instructions.

    Techniques: Inhibition, Chemotaxis Assay, Mouse Assay, Labeling, Flow Cytometry, Cytometry

    Antheraea assama  (Saturniidae) larva, cocoon and silk gland. ( A ) Pre-spinning V instar feeding larva (about 8 cm long). ( B ) Golden silk cocoon (chrysalis, about 5 cm long). ( C ) One in a pair of silk glands (about 30 cm long) dissected from V instar pre-spinning larva of  A. assama , placed in a Petri plate with 1X PBS. It shows features of a typical lepidopteron silk gland with three distinct regions: ASG (Anterior Silk Gland), MSG (Middle Silk Gland), and PSG (Posterior Silk Gland). ( D ) Fluorescent micrographs of portions of ASG, MSG and PSG of II instar larval silk gland showing stacks of paired glandular epithelial cells (DAPI stained) that makeup the cross-sectional circumference of silk gland.

    Journal: Scientific Reports

    Article Title: Molecular architecture of silk fibroin of Indian golden silkmoth, Antheraea assama

    doi: 10.1038/srep12706

    Figure Lengend Snippet: Antheraea assama (Saturniidae) larva, cocoon and silk gland. ( A ) Pre-spinning V instar feeding larva (about 8 cm long). ( B ) Golden silk cocoon (chrysalis, about 5 cm long). ( C ) One in a pair of silk glands (about 30 cm long) dissected from V instar pre-spinning larva of A. assama , placed in a Petri plate with 1X PBS. It shows features of a typical lepidopteron silk gland with three distinct regions: ASG (Anterior Silk Gland), MSG (Middle Silk Gland), and PSG (Posterior Silk Gland). ( D ) Fluorescent micrographs of portions of ASG, MSG and PSG of II instar larval silk gland showing stacks of paired glandular epithelial cells (DAPI stained) that makeup the cross-sectional circumference of silk gland.

    Article Snippet: The silkglands were gently rinsed in 1X PBS (Phosphate-Buffered Saline, 0.1 M Na2 HPO4 , pH=7.0, 0.15 M NaCl) prepared with DEPC-treated water (Ambion) and were carefully dissected for their posterior silk gland regions, discarding the tissue junctions.

    Techniques: Staining

    Influence of culture and measurement conditions on cell mechanical properties of DLD-1 colon carcinoma cells: ( a ) Elastic moduli of cells from four independent measurements, arbitrarily split into two groups (control 1 and control 2). ( b ) Cell elastic moduli measured for cell populations with different culture confluency (20, 60, and 100%) before harvesting. ( c ) Cell elastic moduli in response to different detachment times before measurement (20, 60, and 100 min). ( d ) Cell elastic moduli measured for three different measurement media: PBS, HEPES-buffer, and DMEM cell culture medium. ( e ) Cell elastic moduli measured after coating the devices with 1 wt % pluronic for 0, 20, and 40 min. ( f ) Cell elastic moduli measured with devices coated for 30 min with pluronic concentrations of 0.25, 1, and 4 wt %. n > 1200 for each condition. ε max and Δ p histogram matching was performed separately for each ( a )–( f ). Numbers show mean ± SE. Significant ( p

    Journal: Biophysical Journal

    Article Title: Unbiased High-Precision Cell Mechanical Measurements with Microconstrictions

    doi: 10.1016/j.bpj.2017.02.018

    Figure Lengend Snippet: Influence of culture and measurement conditions on cell mechanical properties of DLD-1 colon carcinoma cells: ( a ) Elastic moduli of cells from four independent measurements, arbitrarily split into two groups (control 1 and control 2). ( b ) Cell elastic moduli measured for cell populations with different culture confluency (20, 60, and 100%) before harvesting. ( c ) Cell elastic moduli in response to different detachment times before measurement (20, 60, and 100 min). ( d ) Cell elastic moduli measured for three different measurement media: PBS, HEPES-buffer, and DMEM cell culture medium. ( e ) Cell elastic moduli measured after coating the devices with 1 wt % pluronic for 0, 20, and 40 min. ( f ) Cell elastic moduli measured with devices coated for 30 min with pluronic concentrations of 0.25, 1, and 4 wt %. n > 1200 for each condition. ε max and Δ p histogram matching was performed separately for each ( a )–( f ). Numbers show mean ± SE. Significant ( p

    Article Snippet: Before measurements, the device is coated with 1 wt % pluronic (BASF, Ludwigshafen, Germany; Cat. No. P2443, Sigma-Aldrich) in PBS for 30 min to decrease nonspecific cell binding to the channel walls ( , ).

    Techniques: Cell Culture

    Confirmation of E2 and Cap expression. A. Confirmation of E2 and Cap expression by immunofluorescence assay (IFA). The E2 and Cap proteins expression was verified by IFA in PK-15 cells infected with rPRVTJ-delgE/gI/TK-E2-Cap. Briefly, PK-15 cells were infected with classical swine fever virus (CSFV), porcine circovirus type 2 (PCV2) rPRVTJ-delgE/gI/TK-E2-Cap and PRV TJ at a multiplication of infectivity (MOI) of 1 or left un-infected (mock). At 48 hpi, the cells were fixed and analyzed by IFA using the anti-E2 MAb HQ06 and anti-Cap MAb 36A9 primary antibodies and fluorescein isothiocyanate (FITC)-labeled anti-mouse secondary antibody. Bars, 400 nm. The results were visualized under a fluorescent microscope (TE2000-U Nikon, Japan). B. Expression of E2 and Cap by Western blotting. To confirm E2 and Cap expression, PK-15 cells were either infected with the CSFV, rPRVTJ-delgE/gI/TK-E2-Cap, rPRVTJdelgE/gI/TK-Cap, rPRVTJ-delgE/gI/TK or mock infected. At 48 hpi, the cells were lysed and assayed by Western blotting using the anti-E2 MAb HQ06 and anti-Cap MAb 36A9 (1:300) in PBS, subsequently incubated with IRDye 800CW-labeled goat anti-mouse secondary antibody (Li-Cor). GAPDH was used as a loading control. The arrows indicate the representative bands of the CSFV E2 protein and PCV2 Cap fused with the PRV gG.

    Journal: Pathogens

    Article Title: Generation and Immunogenicity of a Recombinant Pseudorabies Virus Co-Expressing Classical Swine Fever Virus E2 Protein and Porcine Circovirus Type 2 Capsid Protein Based on Fosmid Library Platform

    doi: 10.3390/pathogens8040279

    Figure Lengend Snippet: Confirmation of E2 and Cap expression. A. Confirmation of E2 and Cap expression by immunofluorescence assay (IFA). The E2 and Cap proteins expression was verified by IFA in PK-15 cells infected with rPRVTJ-delgE/gI/TK-E2-Cap. Briefly, PK-15 cells were infected with classical swine fever virus (CSFV), porcine circovirus type 2 (PCV2) rPRVTJ-delgE/gI/TK-E2-Cap and PRV TJ at a multiplication of infectivity (MOI) of 1 or left un-infected (mock). At 48 hpi, the cells were fixed and analyzed by IFA using the anti-E2 MAb HQ06 and anti-Cap MAb 36A9 primary antibodies and fluorescein isothiocyanate (FITC)-labeled anti-mouse secondary antibody. Bars, 400 nm. The results were visualized under a fluorescent microscope (TE2000-U Nikon, Japan). B. Expression of E2 and Cap by Western blotting. To confirm E2 and Cap expression, PK-15 cells were either infected with the CSFV, rPRVTJ-delgE/gI/TK-E2-Cap, rPRVTJdelgE/gI/TK-Cap, rPRVTJ-delgE/gI/TK or mock infected. At 48 hpi, the cells were lysed and assayed by Western blotting using the anti-E2 MAb HQ06 and anti-Cap MAb 36A9 (1:300) in PBS, subsequently incubated with IRDye 800CW-labeled goat anti-mouse secondary antibody (Li-Cor). GAPDH was used as a loading control. The arrows indicate the representative bands of the CSFV E2 protein and PCV2 Cap fused with the PRV gG.

    Article Snippet: The cells were then air-dried and incubated with 100 µL (1:300 in PBS) of anti-E2 monoclonal antibody (MAb) HQ06 [ ] and anti-Cap MAb 36A9 (Ingenasa, Madrid, Spain) [ ] for 2 h at 37°C, subsequently washed thrice with phosphate-buffered saline (PBS), and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (Sigma-Aldrich, USA) for 1 h at 37°C.

    Techniques: Expressing, Immunofluorescence, Infection, Labeling, Microscopy, Western Blot, Incubation

    Effect of pro-inflammatory cytokines on the expression of human leukocyte antigens (HLAs) and co-stimulatory molecules on the surface of ADSCs. ADSCs were isolated from human adipose tissue and cultured in complete Dulbecco’s modified Eagle’s medium (DMEM). ADSCs that had been passaged fewer than eight times were used. ADSCs were stained with monoclonal antibodies (mAbs) against CD80 and CD86. ADSCs were additionally stained with mAbs against HLA-DR and HLA-ABC and for markers of NKG2DL with mAbs against MIC-A, MIC-B, ULBP1 and ULBP2/5/6. The data are representative of at least three experiments. IFN+IL-17: combination of IFN-γ and IL-17A/F; IFN+IL-17+IL-23: combination of IFN-γ, IL-17A/F and IL-23.

    Journal: Cells

    Article Title: Allogeneic ADSCs Induce the Production of Alloreactive Memory-CD8 T Cells through HLA-ABC Antigens

    doi: 10.3390/cells9051246

    Figure Lengend Snippet: Effect of pro-inflammatory cytokines on the expression of human leukocyte antigens (HLAs) and co-stimulatory molecules on the surface of ADSCs. ADSCs were isolated from human adipose tissue and cultured in complete Dulbecco’s modified Eagle’s medium (DMEM). ADSCs that had been passaged fewer than eight times were used. ADSCs were stained with monoclonal antibodies (mAbs) against CD80 and CD86. ADSCs were additionally stained with mAbs against HLA-DR and HLA-ABC and for markers of NKG2DL with mAbs against MIC-A, MIC-B, ULBP1 and ULBP2/5/6. The data are representative of at least three experiments. IFN+IL-17: combination of IFN-γ and IL-17A/F; IFN+IL-17+IL-23: combination of IFN-γ, IL-17A/F and IL-23.

    Article Snippet: Briefly, adipose tissue was finely cut with scissors and separated into adipocytes through digestion for 1 h in a water bath of 37 °C in Dulbecco’s phosphate-buffered saline (DPBS, Life Technologies, Grand Island, NY, USA) containing 0.1% collagenase type I (Life Technologies, Grand Island, NY, USA) [ ].

    Techniques: Expressing, Isolation, Cell Culture, Modification, Staining

    Safranin O/fast green (A, B) and hematoxylin/eosin (C, D) micrographs of unseeded (A, C) and human adipose-derived stem cell-seeded (B, D) TDM-, FN-, and PBS-coated multilayered, electrospun scaffolds cultured for 28 days. Scale bar=100 μm.

    Journal: Tissue Engineering. Part A

    Article Title: Multilayered Electrospun Scaffolds for Tendon Tissue Engineering

    doi: 10.1089/ten.tea.2013.0165

    Figure Lengend Snippet: Safranin O/fast green (A, B) and hematoxylin/eosin (C, D) micrographs of unseeded (A, C) and human adipose-derived stem cell-seeded (B, D) TDM-, FN-, and PBS-coated multilayered, electrospun scaffolds cultured for 28 days. Scale bar=100 μm.

    Article Snippet: Scaffolds were coated on each side by direct pipetting with human FN in PBS (BD Biosciences) at 4 μg/cm2 , TDM in PBS at 2 mg/cm2 , or an equal volume of PBS and allowed to dry.

    Techniques: Derivative Assay, Cell Culture

    Immunofluorescence of human type III collagen of (−) unseeded and (+) human adipose-derived stem cell-seeded TDM-, FN-, and PBS-coated multilayered, electrospun scaffolds cultured for 28 days. Scale bar=50 μm. Color images available

    Journal: Tissue Engineering. Part A

    Article Title: Multilayered Electrospun Scaffolds for Tendon Tissue Engineering

    doi: 10.1089/ten.tea.2013.0165

    Figure Lengend Snippet: Immunofluorescence of human type III collagen of (−) unseeded and (+) human adipose-derived stem cell-seeded TDM-, FN-, and PBS-coated multilayered, electrospun scaffolds cultured for 28 days. Scale bar=50 μm. Color images available

    Article Snippet: Scaffolds were coated on each side by direct pipetting with human FN in PBS (BD Biosciences) at 4 μg/cm2 , TDM in PBS at 2 mg/cm2 , or an equal volume of PBS and allowed to dry.

    Techniques: Immunofluorescence, Derivative Assay, Cell Culture

    Expression of type 1 collagen ( COL1A1 ), type III collagen ( COL3A1 ), decorin ( DCN ), and tenascin C ( TNC ) by human adipose-derived stem cells after seeding on TDM-, FN-, and PBS-coated multilayered electrospun scaffolds at day 4, 7, and 14 of culture, normalized

    Journal: Tissue Engineering. Part A

    Article Title: Multilayered Electrospun Scaffolds for Tendon Tissue Engineering

    doi: 10.1089/ten.tea.2013.0165

    Figure Lengend Snippet: Expression of type 1 collagen ( COL1A1 ), type III collagen ( COL3A1 ), decorin ( DCN ), and tenascin C ( TNC ) by human adipose-derived stem cells after seeding on TDM-, FN-, and PBS-coated multilayered electrospun scaffolds at day 4, 7, and 14 of culture, normalized

    Article Snippet: Scaffolds were coated on each side by direct pipetting with human FN in PBS (BD Biosciences) at 4 μg/cm2 , TDM in PBS at 2 mg/cm2 , or an equal volume of PBS and allowed to dry.

    Techniques: Expressing, Derivative Assay

    Immunofluorescence of human type I collagen of (−) unseeded and (+) human adipose-derived stem cell-seeded TDM-, FN-, and PBS-coated multilayered, electrospun scaffolds cultured for 28 days. Scale bar=50 μm. Color images available

    Journal: Tissue Engineering. Part A

    Article Title: Multilayered Electrospun Scaffolds for Tendon Tissue Engineering

    doi: 10.1089/ten.tea.2013.0165

    Figure Lengend Snippet: Immunofluorescence of human type I collagen of (−) unseeded and (+) human adipose-derived stem cell-seeded TDM-, FN-, and PBS-coated multilayered, electrospun scaffolds cultured for 28 days. Scale bar=50 μm. Color images available

    Article Snippet: Scaffolds were coated on each side by direct pipetting with human FN in PBS (BD Biosciences) at 4 μg/cm2 , TDM in PBS at 2 mg/cm2 , or an equal volume of PBS and allowed to dry.

    Techniques: Immunofluorescence, Derivative Assay, Cell Culture

    The Young's modulus of linear region (A) , yield strain (B) , and yield stress (C) of human adipose-derived stem-cell seeded TDM-, FN-, and PBS-coated multilayered electrospun scaffolds at day 0 and after 28 days of culture. Groups having different letters

    Journal: Tissue Engineering. Part A

    Article Title: Multilayered Electrospun Scaffolds for Tendon Tissue Engineering

    doi: 10.1089/ten.tea.2013.0165

    Figure Lengend Snippet: The Young's modulus of linear region (A) , yield strain (B) , and yield stress (C) of human adipose-derived stem-cell seeded TDM-, FN-, and PBS-coated multilayered electrospun scaffolds at day 0 and after 28 days of culture. Groups having different letters

    Article Snippet: Scaffolds were coated on each side by direct pipetting with human FN in PBS (BD Biosciences) at 4 μg/cm2 , TDM in PBS at 2 mg/cm2 , or an equal volume of PBS and allowed to dry.

    Techniques: Derivative Assay

    Evaluation of cellular toxicity and viability in cisplatin-treated HK-2 cells. HK-2 cells were treated with increasing concentrations of cisplatin (6.25–100 μM). (A) HK-2 cell proliferation was assessed by MTS assay after 6, 12, and 24 h of cisplatin treatment. (B) Cells were treated with the indicated concentrations of cisplatin for 24 h and cell death was measured. (C and D) HK-2 cells were washed twice with cold phosphate-buffered saline (PBS), stained with Annexin V/propidium iodide (PI), and cell death was determined by flow cytometry. (E and F) Cisplatin-induced cell viability and cytotoxicity assay using calcein AM and ethidium homodimer (EthD-1) fluorescence staining with confocal microscopy (E) and flow cytometry (F). Magnification of the images was ×40 and scale bars represent 75 μm. The mean ± S.D. (standard deviation) of three independent experiments are presented; * p

    Journal: PLoS ONE

    Article Title: Mechanism of Cisplatin-Induced Cytotoxicity Is Correlated to Impaired Metabolism Due to Mitochondrial ROS Generation

    doi: 10.1371/journal.pone.0135083

    Figure Lengend Snippet: Evaluation of cellular toxicity and viability in cisplatin-treated HK-2 cells. HK-2 cells were treated with increasing concentrations of cisplatin (6.25–100 μM). (A) HK-2 cell proliferation was assessed by MTS assay after 6, 12, and 24 h of cisplatin treatment. (B) Cells were treated with the indicated concentrations of cisplatin for 24 h and cell death was measured. (C and D) HK-2 cells were washed twice with cold phosphate-buffered saline (PBS), stained with Annexin V/propidium iodide (PI), and cell death was determined by flow cytometry. (E and F) Cisplatin-induced cell viability and cytotoxicity assay using calcein AM and ethidium homodimer (EthD-1) fluorescence staining with confocal microscopy (E) and flow cytometry (F). Magnification of the images was ×40 and scale bars represent 75 μm. The mean ± S.D. (standard deviation) of three independent experiments are presented; * p

    Article Snippet: Finally, the cells were diluted in 200 μL of phosphate-buffered saline (PBS) for analysis by flow cytometry (FACSAria III, BD Biosciences).

    Techniques: MTS Assay, Staining, Flow Cytometry, Cytometry, Cytotoxicity Assay, Ethidium Homodimer Assay, Fluorescence, Confocal Microscopy, Standard Deviation

    Differences in resting state functional connectivity between more effective versus less effective DLPFC stimulation sites. Coordinates are taken from Herbsman et al. 2009 (A–C) and Fitzgerald et al. 2009 (D–F). The top row (A, D) shows the DLPFC regions of interest compared in each study. The middle row (B, E) shows significant differences in resting state functional connectivity between the two sites (more effective – less effective). The border of our a-priori defined subgenual region of interest is show for reference in red. The bottom row (C, F) shows bar graphs of the correlation of each DLPFC site with the subgenual cingulate. In both cases the more effective DLPFC site is significantly more anticorrelated with the subgenual cingulate than the less effective site.

    Journal: Biological psychiatry

    Article Title: Efficacy of TMS targets for depression is related to intrinsic functional connectivity with the subgenual cingulate

    doi: 10.1016/j.biopsych.2012.04.028

    Figure Lengend Snippet: Differences in resting state functional connectivity between more effective versus less effective DLPFC stimulation sites. Coordinates are taken from Herbsman et al. 2009 (A–C) and Fitzgerald et al. 2009 (D–F). The top row (A, D) shows the DLPFC regions of interest compared in each study. The middle row (B, E) shows significant differences in resting state functional connectivity between the two sites (more effective – less effective). The border of our a-priori defined subgenual region of interest is show for reference in red. The bottom row (C, F) shows bar graphs of the correlation of each DLPFC site with the subgenual cingulate. In both cases the more effective DLPFC site is significantly more anticorrelated with the subgenual cingulate than the less effective site.

    Article Snippet: Fitzgerald PB, Hoy K, McQueen S, Maller JJ, Herring S, Segrave R, et al. A randomized trial of rTMS targeted with MRI based neuro-navigation in treatment-resistant depression.

    Techniques: Functional Assay

    Treatment of 005 GSC-derived tumors with systemic immune checkpoint inhibitors, intratumoral G47 Δ-mIL12, or the combination prolongs survival (A–B) Mice implanted with 2 × 10 4 005 GSCs on day 0, treated with G47Δ-mIL12 (5 × 10 5 pfu) or PBS injected IT on day 12 (upward arrow) and isotype control IgG (10 mg/kg), anti-(α)PD-L1 antibody (A) , or anti-(α)PD-1 antibody (B) injected IP on days 15, 18 and 21 (downward arrows). Values from a single experiment, with Mock (treated with PBS and IgG) and G47Δ-mIL12 groups the same in A and B. Median survival of Mock (33.5 days; n=6) was compared to anti-PD-1 (39 days; n=8, p=0.02), anti-PD-L1 (42 days; n=7, p=0.003), or G47Δ-mIL12 (39 days; n=8, p=0.01) by Log-rank analysis. Similarly, G47Δ-mIL12 was compared to the combination of G47Δ-mIL12 with anti-PD-1 (49 days; n=7, p=0.02) or -PD-L1 (50 days; n=7, p=0.03), and antibodies were compared to the combination of G47Δ-mIL12 with anti-PD-1 (p=0.053) or anti-PD-L1 (p=0.08). Experiment was conducted once (A) or twice (B). C. Mice implanted with 2 × 10 4 005 GSCs on day 0 and treated with G47Δ-mIL12 or PBS injected IT on day 8 and anti-(α)CTLA-4 antibody or isotype control IgG (5 mg/kg) injected IP on days 8, 11 and 14 (n=8/group, except for G47Δ-mIL12 n=7). Median survival of Mock (37.5 days) was compared to anti-CTLA-4 (45 days; p=0.002) or G47Δ-mIL12 (40 days; p=0.03) alone by Log-rank analysis. Similarly, combination of G47Δ-mIL12 with anti-CTLA-4 (58 days) was compared to anti-CTLA-4 (p=0.05) or G47Δ-mIL12 (p=0.008). Experiment was conducted 2 times. D. Mice implanted with 2 × 10 4 005 GSCs on day 0, treated with PBS (right; n=2), rat anti-(α)PD-1 antibody (middle; 200 μg/mouse; n=2), or rat anti-(α)PD-L1 antibody (left; 200 μg/mouse; n=2) injected IP on day 25, and sacrificed 3 hr later. Antibodies were detected with HRP-conjugated anti-(α)rat Ig (brown; right) or control HRP-conjugated anti-(α)rabbit Ig (left). * normal brain adjacent to tumor. Scale bar=100 μm. E. 005 GSCs (1.5 × 10 5 ) implanted on day 0, treated with PBS or G47Δ-mIL12 (5 × 10 5 .

    Journal: Cancer cell

    Article Title: MACROPHAGE POLARIZATION CONTRIBUTES TO GLIOBLASTOMA ERADICATION BY COMBINATION IMMUNOVIROTHERAPY AND IMMUNE CHECKPOINT BLOCKADE

    doi: 10.1016/j.ccell.2017.07.006

    Figure Lengend Snippet: Treatment of 005 GSC-derived tumors with systemic immune checkpoint inhibitors, intratumoral G47 Δ-mIL12, or the combination prolongs survival (A–B) Mice implanted with 2 × 10 4 005 GSCs on day 0, treated with G47Δ-mIL12 (5 × 10 5 pfu) or PBS injected IT on day 12 (upward arrow) and isotype control IgG (10 mg/kg), anti-(α)PD-L1 antibody (A) , or anti-(α)PD-1 antibody (B) injected IP on days 15, 18 and 21 (downward arrows). Values from a single experiment, with Mock (treated with PBS and IgG) and G47Δ-mIL12 groups the same in A and B. Median survival of Mock (33.5 days; n=6) was compared to anti-PD-1 (39 days; n=8, p=0.02), anti-PD-L1 (42 days; n=7, p=0.003), or G47Δ-mIL12 (39 days; n=8, p=0.01) by Log-rank analysis. Similarly, G47Δ-mIL12 was compared to the combination of G47Δ-mIL12 with anti-PD-1 (49 days; n=7, p=0.02) or -PD-L1 (50 days; n=7, p=0.03), and antibodies were compared to the combination of G47Δ-mIL12 with anti-PD-1 (p=0.053) or anti-PD-L1 (p=0.08). Experiment was conducted once (A) or twice (B). C. Mice implanted with 2 × 10 4 005 GSCs on day 0 and treated with G47Δ-mIL12 or PBS injected IT on day 8 and anti-(α)CTLA-4 antibody or isotype control IgG (5 mg/kg) injected IP on days 8, 11 and 14 (n=8/group, except for G47Δ-mIL12 n=7). Median survival of Mock (37.5 days) was compared to anti-CTLA-4 (45 days; p=0.002) or G47Δ-mIL12 (40 days; p=0.03) alone by Log-rank analysis. Similarly, combination of G47Δ-mIL12 with anti-CTLA-4 (58 days) was compared to anti-CTLA-4 (p=0.05) or G47Δ-mIL12 (p=0.008). Experiment was conducted 2 times. D. Mice implanted with 2 × 10 4 005 GSCs on day 0, treated with PBS (right; n=2), rat anti-(α)PD-1 antibody (middle; 200 μg/mouse; n=2), or rat anti-(α)PD-L1 antibody (left; 200 μg/mouse; n=2) injected IP on day 25, and sacrificed 3 hr later. Antibodies were detected with HRP-conjugated anti-(α)rat Ig (brown; right) or control HRP-conjugated anti-(α)rabbit Ig (left). * normal brain adjacent to tumor. Scale bar=100 μm. E. 005 GSCs (1.5 × 10 5 ) implanted on day 0, treated with PBS or G47Δ-mIL12 (5 × 10 5 .

    Article Snippet: Ten μm sections were air-dried, followed by wash and rehydration in PBS, incubation with 3% H2 O2 , washed in PBS, incubated with 5% bovine serum albumin and 10% goat serum, and then with HRP-conjugated anti-rat Ig, control HRP-conjugated anti-rabbit Ig, or PBS, followed by DAB staining (DAKO).

    Techniques: Derivative Assay, Mouse Assay, Injection

    Antimicrobial activities. (A) Outer membrane permeability of 4 kinds of microbe (2 Gram-negative and 2 Gram-positive bacteria) was measured using PMAP36-P22 lysozyme fusion protein by EtBr influx assay. The activities were compared with PBS buffer as a control. The error bars show the standard deviation of the mean from three independent trials. Asterisks indicate statistically significant differences between groups. (****, Unpaired t -test, P

    Journal: Molecules and Cells

    Article Title: Enhanced Expression and Functional Characterization of the Recombinant Putative Lysozyme-PMAP36 Fusion Protein

    doi: 10.14348/molcells.2019.2365

    Figure Lengend Snippet: Antimicrobial activities. (A) Outer membrane permeability of 4 kinds of microbe (2 Gram-negative and 2 Gram-positive bacteria) was measured using PMAP36-P22 lysozyme fusion protein by EtBr influx assay. The activities were compared with PBS buffer as a control. The error bars show the standard deviation of the mean from three independent trials. Asterisks indicate statistically significant differences between groups. (****, Unpaired t -test, P

    Article Snippet: The cell cultures at mid-logarithmic phase, OD600 of 0.2, were mixed with PBS (GE Healthcare) and PMAP36-P22 lysozyme fusion protein (final concentration: 64 μM) and incubated for 10 min at 37°C.

    Techniques: Permeability, Standard Deviation

    Secondary structure CD spectra of the P22 lysozyme (straight line), PMAP36-P22 lysozyme fusion protein (dash) and PMAP36 peptide (dot) in PBS (A) and 1% SDS (B). The concentration of all proteins and peptide were 0.5 mg/ml.

    Journal: Molecules and Cells

    Article Title: Enhanced Expression and Functional Characterization of the Recombinant Putative Lysozyme-PMAP36 Fusion Protein

    doi: 10.14348/molcells.2019.2365

    Figure Lengend Snippet: Secondary structure CD spectra of the P22 lysozyme (straight line), PMAP36-P22 lysozyme fusion protein (dash) and PMAP36 peptide (dot) in PBS (A) and 1% SDS (B). The concentration of all proteins and peptide were 0.5 mg/ml.

    Article Snippet: The cell cultures at mid-logarithmic phase, OD600 of 0.2, were mixed with PBS (GE Healthcare) and PMAP36-P22 lysozyme fusion protein (final concentration: 64 μM) and incubated for 10 min at 37°C.

    Techniques: Concentration Assay

    Localization and expression level of MAD2 in fresh mouse oocytes with blocked microtubule polymerization. (A) Immunofluorescent staining of MAD2 (red) and α-tubulin (green). DNA (blue) was stained with DAPI. D-PBS, instead of the primary antibody, was used as the negative control. The inserted panel in the middle panel of the nocodazole group indicates only MAD2 signals. Scale bar = 10 μm. (B) Western blotting and expression levels of MAD2 in oocytes treated with nocodazole. β-actin was used as a loading control.

    Journal: The Journal of Reproduction and Development

    Article Title: Destabilization of spindle assembly checkpoint causes aneuploidy during meiosis II in murine post-ovulatory aged oocytes

    doi: 10.1262/jrd.2018-056

    Figure Lengend Snippet: Localization and expression level of MAD2 in fresh mouse oocytes with blocked microtubule polymerization. (A) Immunofluorescent staining of MAD2 (red) and α-tubulin (green). DNA (blue) was stained with DAPI. D-PBS, instead of the primary antibody, was used as the negative control. The inserted panel in the middle panel of the nocodazole group indicates only MAD2 signals. Scale bar = 10 μm. (B) Western blotting and expression levels of MAD2 in oocytes treated with nocodazole. β-actin was used as a loading control.

    Article Snippet: This was followed by incubation in Alexa Fluor® 594-conjugated anti-goat IgG secondary antibody (1:500 dilution, Invitrogen) for 60 min. After washing thrice with D-PBS, the oocytes were incubated with mouse anti-α-Tubulin monoclonal antibody (1:500 dilution, Sigma) as a primary antibody for 60 min at room temperature, washed thrice with D-PBS, then incubated in fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (1:100 dilution, Sigma) as a secondary antibody for 1 h at room temperature.

    Techniques: Expressing, Staining, Negative Control, Western Blot

    In vivo biotinylated and trimyc-tagged sdAbs in bacterial lysate allow a sensitive antigenic detection on slide or beads A) Immobilization of sdAbs using trimyc tag allows the direct use of bacterial lysates. Epoxy bead were coated with 9E10 (1 μg/ml) and incubated with bacterial lysate (50 nl/wells) containing sdAbs with three (●), one (■) or no (▲) c-myc followed by serial dilutions of biotinylated Nef. The captured antigen was detected with HRP-conjugated streptavidin. B) Immobilization of sdAbs using trimyc allows direct spotting of bacterial lysate. Nitrocellulose slides were coated with 9E10 (9E10) or PBS (Ø). Bacterial lysates containing sdAbs fused to three, one or no c-myc were spotted. Serial dilutions of biotinylated Nef were incubated and the captured antigen was detected with Alexa705-conjugated streptavidin. C): Streptavidin beads were coated with bacterial lysate (50 nl/wells) containing anti-Nef sdAbs biotinylated in vivo (●, ▲, ◆) or not (■, ▼, ○) and incubated with serial dilutions of Nef or Alexa488-conjugated Nef (◆, ○). The captured antigen was detected with a mouse anti-Nef antibody followed by a goat anti-mouse HRP (●,■) or Alexa488-conjugated mAb (▲, ▼). D) Nitrocellulose slides were incubated with streptavidin (S) or PBS (Ø). Bacterial lysates containing sdAbs biotinylated in vivo or unbiotinylated were spotted. Serial dilutions of Nef were incubated and the captured antigen was detected with a mouse anti-Nef antibody followed by a goat anti-mouse Alexa705-conjugated mAb. Standard deviation represents two experiments performed in triplicates.

    Journal: Molecular Biosystems

    Article Title: Strong and oriented immobilization of single domain antibodies from crude bacterial lysates for high-throughput compatible cost-effective antibody array generation

    doi: 10.1039/c005279e

    Figure Lengend Snippet: In vivo biotinylated and trimyc-tagged sdAbs in bacterial lysate allow a sensitive antigenic detection on slide or beads A) Immobilization of sdAbs using trimyc tag allows the direct use of bacterial lysates. Epoxy bead were coated with 9E10 (1 μg/ml) and incubated with bacterial lysate (50 nl/wells) containing sdAbs with three (●), one (■) or no (▲) c-myc followed by serial dilutions of biotinylated Nef. The captured antigen was detected with HRP-conjugated streptavidin. B) Immobilization of sdAbs using trimyc allows direct spotting of bacterial lysate. Nitrocellulose slides were coated with 9E10 (9E10) or PBS (Ø). Bacterial lysates containing sdAbs fused to three, one or no c-myc were spotted. Serial dilutions of biotinylated Nef were incubated and the captured antigen was detected with Alexa705-conjugated streptavidin. C): Streptavidin beads were coated with bacterial lysate (50 nl/wells) containing anti-Nef sdAbs biotinylated in vivo (●, ▲, ◆) or not (■, ▼, ○) and incubated with serial dilutions of Nef or Alexa488-conjugated Nef (◆, ○). The captured antigen was detected with a mouse anti-Nef antibody followed by a goat anti-mouse HRP (●,■) or Alexa488-conjugated mAb (▲, ▼). D) Nitrocellulose slides were incubated with streptavidin (S) or PBS (Ø). Bacterial lysates containing sdAbs biotinylated in vivo or unbiotinylated were spotted. Serial dilutions of Nef were incubated and the captured antigen was detected with a mouse anti-Nef antibody followed by a goat anti-mouse Alexa705-conjugated mAb. Standard deviation represents two experiments performed in triplicates.

    Article Snippet: After three washes with PBS, plates were incubated with 9E10 mAb (against c-myc) (santa cruz biotechnology) (1 μg/ml) in 2% MPBS for one hour at RT.

    Techniques: In Vivo, Incubation, Standard Deviation

    Confocal images of TRPV1 (A–D) and SP immunolabeling (E–H) expression in (A, E) BA, (B, F) AICA, (C, G) SMA, and (D, H) RA. Arrows indicate labeled: longitudinal (a), circumferential (b) fibers. Insets in A–D, show negligible labeling in BA, AICA, SMA, and RA incubated with antigen-adsorbed TRPV1 antibodies and secondary antibodies. Insets in E–H show negligible labeling in the BA, AICA, SMA, and radiating arterioles incubated with 1% BSA-PBS and secondary antibodies. Scale bars in microns.

    Journal: Neuroscience

    Article Title: CO-LOCALIZATION OF THE VANILLOID CAPSAICIN RECEPTOR AND SUBSTANCE P IN SENSORY NERVE FIBERS INNERVATING COCHLEAR AND VERTEBRO-BASILAR ARTERIES

    doi: 10.1016/j.neuroscience.2003.12.030

    Figure Lengend Snippet: Confocal images of TRPV1 (A–D) and SP immunolabeling (E–H) expression in (A, E) BA, (B, F) AICA, (C, G) SMA, and (D, H) RA. Arrows indicate labeled: longitudinal (a), circumferential (b) fibers. Insets in A–D, show negligible labeling in BA, AICA, SMA, and RA incubated with antigen-adsorbed TRPV1 antibodies and secondary antibodies. Insets in E–H show negligible labeling in the BA, AICA, SMA, and radiating arterioles incubated with 1% BSA-PBS and secondary antibodies. Scale bars in microns.

    Article Snippet: The tissues were washed in fresh 0.02 M PBS (pH 7.4), permeabilized in 0.5% Triton-X 100 (Sigma, St. Louis, MO, USA) in 0.02 M PBS (pH 7.3) for 30 min, and immunoblocked in 10% goat serum in 1% bovine serum albumin (BSA) in 0.02 M PBS (1% BSA-PBS) for 60 min. For immunocytochemistry, tissues were incubated with rabbit anti-TRPV1 antiserum (diluted 1:1000 in 1% BSA-PBS; gift from Dr. Michael J Caterina, John Hopkins University) for 48 h. After washing in 1% BSA-PBS, tissues were subsequently incubated in Alexa-488-conjugated goat anti-rabbit IgG for 3 h (Molecular Probes; 1:100 in BSA-PBS).

    Techniques: Immunolabeling, Expressing, Labeling, Incubation

    Confocal images of the DRG double-immunolabeled for TRPV1 (A), and for SP (B). (C) A subset of neuronal cell bodies co-express TRPV1 and SP (white, arrows) in the merged image. Inset in (A) shows negligible labeling in the DRG when incubated with antigen-adsorbed TRPV1 antibodies and secondary antibodies. Inset in B shows negligible labeling in the DRG when incubated with 1% BSA-PBS and secondary antibodies. Scale bar in microns.

    Journal: Neuroscience

    Article Title: CO-LOCALIZATION OF THE VANILLOID CAPSAICIN RECEPTOR AND SUBSTANCE P IN SENSORY NERVE FIBERS INNERVATING COCHLEAR AND VERTEBRO-BASILAR ARTERIES

    doi: 10.1016/j.neuroscience.2003.12.030

    Figure Lengend Snippet: Confocal images of the DRG double-immunolabeled for TRPV1 (A), and for SP (B). (C) A subset of neuronal cell bodies co-express TRPV1 and SP (white, arrows) in the merged image. Inset in (A) shows negligible labeling in the DRG when incubated with antigen-adsorbed TRPV1 antibodies and secondary antibodies. Inset in B shows negligible labeling in the DRG when incubated with 1% BSA-PBS and secondary antibodies. Scale bar in microns.

    Article Snippet: The tissues were washed in fresh 0.02 M PBS (pH 7.4), permeabilized in 0.5% Triton-X 100 (Sigma, St. Louis, MO, USA) in 0.02 M PBS (pH 7.3) for 30 min, and immunoblocked in 10% goat serum in 1% bovine serum albumin (BSA) in 0.02 M PBS (1% BSA-PBS) for 60 min. For immunocytochemistry, tissues were incubated with rabbit anti-TRPV1 antiserum (diluted 1:1000 in 1% BSA-PBS; gift from Dr. Michael J Caterina, John Hopkins University) for 48 h. After washing in 1% BSA-PBS, tissues were subsequently incubated in Alexa-488-conjugated goat anti-rabbit IgG for 3 h (Molecular Probes; 1:100 in BSA-PBS).

    Techniques: Immunolabeling, Labeling, Incubation

    In vitro release kinetics of entrapped FITC-dextran from PSN microparticles in PBS (pH 7.4) at 37 °C. Data is shown as mean ± S.D. (n = 3).

    Journal: Journal of microencapsulation

    Article Title: Microparticles prepared from sulfenamide-based polymers

    doi: 10.3109/02652048.2013.814728

    Figure Lengend Snippet: In vitro release kinetics of entrapped FITC-dextran from PSN microparticles in PBS (pH 7.4) at 37 °C. Data is shown as mean ± S.D. (n = 3).

    Article Snippet: Ten mg FITC-dextran loaded microparticles were suspended in 1 mL PBS (0.01 M, pH 7.4) containing 0.1 % w/v Tween® 80 (Fischer scientific, NJ).

    Techniques: In Vitro

    Interleukin 11 (IL-11)/IL-11R signaling pathway induced the chemo-resistance through JAK/STAT3 pathway. (A) Immunofluoresence of p-JAK2 in BGC823 and SGC7901 cells pre-treated with or without cultured medium of cancer-associated-fibroblasts (CAFs-CM)/rhIL-11 in the presence or absence of IL-11 neutralizing antibody. (B) Immunofluoresence of p-STAT3 in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (C) Western blotting of p-JAK2, total JAK2 and β-actin in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (D) Western blotting of p-STAT3, total STAT3, and β-actin in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (E) The cell viability of BGC823 cells treated with 6 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of ruxolitinib (5 μM). PBS, phosphate buffered saline; Dox, doxorubicin; Eto, etoposide. (F) The cell viability of SGC7901 cells treated with 4 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of ruxolitinib (5 μM). (G) The cell viability of BGC823 cells treated with 6 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the wild type or shSTAT3 cells. (H) The cell viability of SGC7901 cells treated with 4 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the wild type or shSTAT3 cells. The data was presented as the mean±standard error of mean from three independent experiments. ** p

    Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

    Article Title: Cancer-Associated Fibroblasts Promote the Chemo-resistance in Gastric Cancer through Secreting IL-11 Targeting JAK/STAT3/Bcl2 Pathway

    doi: 10.4143/crt.2018.031

    Figure Lengend Snippet: Interleukin 11 (IL-11)/IL-11R signaling pathway induced the chemo-resistance through JAK/STAT3 pathway. (A) Immunofluoresence of p-JAK2 in BGC823 and SGC7901 cells pre-treated with or without cultured medium of cancer-associated-fibroblasts (CAFs-CM)/rhIL-11 in the presence or absence of IL-11 neutralizing antibody. (B) Immunofluoresence of p-STAT3 in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (C) Western blotting of p-JAK2, total JAK2 and β-actin in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (D) Western blotting of p-STAT3, total STAT3, and β-actin in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (E) The cell viability of BGC823 cells treated with 6 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of ruxolitinib (5 μM). PBS, phosphate buffered saline; Dox, doxorubicin; Eto, etoposide. (F) The cell viability of SGC7901 cells treated with 4 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of ruxolitinib (5 μM). (G) The cell viability of BGC823 cells treated with 6 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the wild type or shSTAT3 cells. (H) The cell viability of SGC7901 cells treated with 4 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the wild type or shSTAT3 cells. The data was presented as the mean±standard error of mean from three independent experiments. ** p

    Article Snippet: Then the sections or gastric cancer cells (fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100) were blocked with 5% BSA in PBS and incubated with p-JAK (1:200, Abcam) or p-STAT3 (1:500, Abcam) at 4°C overnight, followed by secondary antibodies (Thermo Fisher) for 1 hour at room temperature.

    Techniques: Cell Culture, Western Blot

    Interleukin 11 (IL-11) triggers the JAK/STAT3 pathway to elevate Bcl2 expression. (A) The expression of Bcl2 in BGC823 and SGC7901 cells treated with cultured medium of cancer-associated-fibroblasts (CAFs-CM) or rhIL-11 in the presence or absence of ruxolitinib (5 μM) in mRNA level. (B) The expression of Bcl2 in BGC823 and SGC7901 cells treated with CAFs-CM or rhIL-11 (10 ng/mL) in the presence or absence of ruxolitinib (5 μM) in protein level. (C) The cell viability of BGC823 cells treated with CAFs-CM or rhIL-11 in wild type or Bcl2 silenced cells. (D) The cell viability of SGC7901 cells treated with CAFs-CM or rhIL-11 in wild type or Bcl2 silenced cells. PBS, phosphate buffered saline; Dox, doxorubicin; Eto, etoposide. The data was presented as the mean±standard error of mean from three independent experiments. ** p

    Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

    Article Title: Cancer-Associated Fibroblasts Promote the Chemo-resistance in Gastric Cancer through Secreting IL-11 Targeting JAK/STAT3/Bcl2 Pathway

    doi: 10.4143/crt.2018.031

    Figure Lengend Snippet: Interleukin 11 (IL-11) triggers the JAK/STAT3 pathway to elevate Bcl2 expression. (A) The expression of Bcl2 in BGC823 and SGC7901 cells treated with cultured medium of cancer-associated-fibroblasts (CAFs-CM) or rhIL-11 in the presence or absence of ruxolitinib (5 μM) in mRNA level. (B) The expression of Bcl2 in BGC823 and SGC7901 cells treated with CAFs-CM or rhIL-11 (10 ng/mL) in the presence or absence of ruxolitinib (5 μM) in protein level. (C) The cell viability of BGC823 cells treated with CAFs-CM or rhIL-11 in wild type or Bcl2 silenced cells. (D) The cell viability of SGC7901 cells treated with CAFs-CM or rhIL-11 in wild type or Bcl2 silenced cells. PBS, phosphate buffered saline; Dox, doxorubicin; Eto, etoposide. The data was presented as the mean±standard error of mean from three independent experiments. ** p

    Article Snippet: Then the sections or gastric cancer cells (fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100) were blocked with 5% BSA in PBS and incubated with p-JAK (1:200, Abcam) or p-STAT3 (1:500, Abcam) at 4°C overnight, followed by secondary antibodies (Thermo Fisher) for 1 hour at room temperature.

    Techniques: Expressing, Cell Culture

    Cell electrotransfection and viability rates using Bio-Rad buffer, OptiMEM-GlutaMAX, and PBS. Electrotransfection efficiency of mouse iPS ( a and b ) and MEF cells ( c and d ). In each electroporation reaction, 20 µg (1.5–2.5 µg/µl) of the reporter plasmid (encoding mCherry) was pre-mixed with cells and underwent electroporation using the square-wave protocol consisted of 250 V for iPS and 300 V for MEF cells, 2 pulses, each 10 ms length, 10 s interval, and 4 mm cuvette. The reporter expression was assessed 36 h after electroporation under a fluorescence microscope. White and black bars are electrotransfection efficiency and cell viability, respectively. Bars with different A, B, C or a, b, c letters are significantly different ( p value

    Journal: Scientific Reports

    Article Title: A versatile bulk electrotransfection protocol for murine embryonic fibroblasts and iPS cells

    doi: 10.1038/s41598-020-70258-w

    Figure Lengend Snippet: Cell electrotransfection and viability rates using Bio-Rad buffer, OptiMEM-GlutaMAX, and PBS. Electrotransfection efficiency of mouse iPS ( a and b ) and MEF cells ( c and d ). In each electroporation reaction, 20 µg (1.5–2.5 µg/µl) of the reporter plasmid (encoding mCherry) was pre-mixed with cells and underwent electroporation using the square-wave protocol consisted of 250 V for iPS and 300 V for MEF cells, 2 pulses, each 10 ms length, 10 s interval, and 4 mm cuvette. The reporter expression was assessed 36 h after electroporation under a fluorescence microscope. White and black bars are electrotransfection efficiency and cell viability, respectively. Bars with different A, B, C or a, b, c letters are significantly different ( p value

    Article Snippet: Viability of iPS cells was significantly lower in Bio-Rad (22.0%) and PBS (3.2%) compared to the OptiMEM-GlutaMAX medium (78.2%).

    Techniques: Electroporation, Plasmid Preparation, Expressing, Fluorescence, Microscopy

    High-sensitivity flow-cytometry analysis of fractionated IDG revealed the peculiar enrichment of EVs in the 12,000 g pellet. (a) A pool of all the IDG fractions was labelled with deep red CellTracker fluorescent dye and the gates set for fluorescent EV (green) detection, compared with unlabelled EVs (Red) and CellTracker in PBS-loaded (empty) IDG fractions (PBS, Blue). (b–c) Each IDG fraction of the 12,000 g (b) and 35,000 g (c) was labelled and analysed for the presence of esterase-positive EVs. Results are expressed as a count of events (mean ± SD; n = 3). (d) After setting the gates for size, using beads of known diameter, we analysed the CellTracker positive population in each fraction to determine the approximate size of the observed events. (e–f) Representative results obtained for IDG fractions F7 and F8 from 12,000 g (e) and 35,000 g (f) EVs are displayed. A green colour represents CellTracker positive events. The associated quantification results are available as Supplementary File 5.

    Journal: Journal of Extracellular Vesicles

    Article Title: A subset of extracellular vesicles carries the bulk of microRNAs in commercial dairy cow’s milk

    doi: 10.1080/20013078.2017.1401897

    Figure Lengend Snippet: High-sensitivity flow-cytometry analysis of fractionated IDG revealed the peculiar enrichment of EVs in the 12,000 g pellet. (a) A pool of all the IDG fractions was labelled with deep red CellTracker fluorescent dye and the gates set for fluorescent EV (green) detection, compared with unlabelled EVs (Red) and CellTracker in PBS-loaded (empty) IDG fractions (PBS, Blue). (b–c) Each IDG fraction of the 12,000 g (b) and 35,000 g (c) was labelled and analysed for the presence of esterase-positive EVs. Results are expressed as a count of events (mean ± SD; n = 3). (d) After setting the gates for size, using beads of known diameter, we analysed the CellTracker positive population in each fraction to determine the approximate size of the observed events. (e–f) Representative results obtained for IDG fractions F7 and F8 from 12,000 g (e) and 35,000 g (f) EVs are displayed. A green colour represents CellTracker positive events. The associated quantification results are available as Supplementary File 5.

    Article Snippet: To this end, 2 µL of each IDG fraction was diluted in 98 µL of 0.2 µm filtered sterile PBS (Gibco, ThermoFisher scientific) containing the fluorescent CellTracker™ deep red dye [ ] (C34565; Life Technologies, ThermoFisher Scientific) at a final concentration of 1 µM, for 20 min at 37°C, in the dark, following the manufacturer’s recommendations.

    Techniques: Flow Cytometry, Cytometry

    Quantitative analysis of neurofilament protein accumulation proximal and distal to the constriction site after constriction for 2 h in the presence or absence of metabolic substrates. Each column represents the mean accumulation ratio for the number of cells indicated and the error bars represent the standard deviation about the mean as described in the legend to Fig. 5 . For these experiments, the L-15-based culture me dium was replaced with a simpler Dulbecco's PBS-based medium with or without 0.6% glucose and 0.055% sodium pyruvate (fed and starved, respectively). The mean proximal accumulation ratio in the presence of glucose and pyruvate was lower than for axons constricted for the same period of time in L-15-based medium (Fig. 5 A) but it was, nevertheless, reduced significantly when these two substrates were omitted ( P = 0.01, t test).

    Journal: The Journal of Cell Biology

    Article Title: Slow Axonal Transport of Neurofilament Protein in Cultured Neurons

    doi:

    Figure Lengend Snippet: Quantitative analysis of neurofilament protein accumulation proximal and distal to the constriction site after constriction for 2 h in the presence or absence of metabolic substrates. Each column represents the mean accumulation ratio for the number of cells indicated and the error bars represent the standard deviation about the mean as described in the legend to Fig. 5 . For these experiments, the L-15-based culture me dium was replaced with a simpler Dulbecco's PBS-based medium with or without 0.6% glucose and 0.055% sodium pyruvate (fed and starved, respectively). The mean proximal accumulation ratio in the presence of glucose and pyruvate was lower than for axons constricted for the same period of time in L-15-based medium (Fig. 5 A) but it was, nevertheless, reduced significantly when these two substrates were omitted ( P = 0.01, t test).

    Article Snippet: For experiments on axons in the presence and absence of metabolic substrates, the above medium was rinsed and replaced with Dulbecco's PBS ( GIBCO BRL ) supplemented with 0.5% Methocel™ and 50 ng/ml 2.5S nerve growth factor, with or without 0.6% glucose ( Sigma Chemical Co. ) and 0.055% sodium pyruvate ( Sigma Chemical Co. ).

    Techniques: Standard Deviation

    Tagging of CDCA8 in HeLa cells with GFP-3xFLAG. (A) HeLa cells were arrested in mitosis by overnight treatment with STLC and collected via mitotic shake-off, followed by washing with PBS. The indicated drugs were added to the final wash of the cells, after which the cells were lysed with freeze–thaw cycles. Cell lysates were then immunoblotted for pT232-AURK (recognizing pAURKA, pAURKB, and pAURKC) and α-tubulin. The p value between the barasertib- and okadaic acid–treated cells is 0.069; n = 2. (B) Lysates of the parent and tagged cell line were prepared as in A. Magnetic beads preloaded with an antibody against FLAG were used to immunoprecipitate (IP) tagged proteins from the lysates, and the resulting proteins were blotted for FLAG after separation via SDS–PAGE gel electrophoresis; n = 2. (C) Lysates of the tagged cell line were prepared as in A, and IPs were done with beads loaded with antibodies against random IgG or FLAG, followed by blotting for FLAG as in A; n = 2. (D) HeLa cells expressing GFP-tagged CPC were fixed with 4% paraformaldehyde, stained with an antibody against tubulin, and imaged with a spinning-disk confocal microscope at 60× magnification. Scale bars, 10 μm. Cells were imaged on three separate occasions.

    Journal: Molecular Biology of the Cell

    Article Title: Chromosomal passenger complex hydrodynamics suggests chaperoning of the inactive state by nucleoplasmin/nucleophosmin

    doi: 10.1091/mbc.E16-12-0860

    Figure Lengend Snippet: Tagging of CDCA8 in HeLa cells with GFP-3xFLAG. (A) HeLa cells were arrested in mitosis by overnight treatment with STLC and collected via mitotic shake-off, followed by washing with PBS. The indicated drugs were added to the final wash of the cells, after which the cells were lysed with freeze–thaw cycles. Cell lysates were then immunoblotted for pT232-AURK (recognizing pAURKA, pAURKB, and pAURKC) and α-tubulin. The p value between the barasertib- and okadaic acid–treated cells is 0.069; n = 2. (B) Lysates of the parent and tagged cell line were prepared as in A. Magnetic beads preloaded with an antibody against FLAG were used to immunoprecipitate (IP) tagged proteins from the lysates, and the resulting proteins were blotted for FLAG after separation via SDS–PAGE gel electrophoresis; n = 2. (C) Lysates of the tagged cell line were prepared as in A, and IPs were done with beads loaded with antibodies against random IgG or FLAG, followed by blotting for FLAG as in A; n = 2. (D) HeLa cells expressing GFP-tagged CPC were fixed with 4% paraformaldehyde, stained with an antibody against tubulin, and imaged with a spinning-disk confocal microscope at 60× magnification. Scale bars, 10 μm. Cells were imaged on three separate occasions.

    Article Snippet: The mitotic cells were washed three times with cold PBS (Corning) supplemented with STLC.

    Techniques: Magnetic Beads, SDS Page, Nucleic Acid Electrophoresis, Expressing, Staining, Microscopy

    Localisation of the hBD2 peptide in epithelial bronchial 16HBE cells . 16HBE cells were seeded at 5 × 10 5 cells per well in 1 ml of DMEM/F12 on 18-mm-diameter cover slips in 12 well plates in triplicate and grown for 16 h at 37°C. After washing the cover slips with PBS-BSA, the cells were exposed to either latex beads, ethanol fixed conidia or ethanol fixed HF for 18 hours. Il-1β was used as a positive control. Following washing with PBS, the cells were fixed with a paraformaldehyde solution for 30 min at 37°C. The slides were then incubated in 1% BSA/PBS-Triton 0.05%, followed by a solution of 10% normal goat serum. After washing, polyclonal rabbit anti-human hBD2 at a dilution of 1:250 was applied as primary antibody overnight at 4°C, followed by incubation with FITC-labelled goat anti-rabbit secondary antibody at a dilution of 1:300 for 4 hours at room temperature. After washing, the cover slips were mounted on slides with ProLong antifade Vectashield. Samples were viewed with a Zeiss fluorescence microscope using ×400 magnification. The arrows indicate the cells stained with anti-hBD2 antibody. The percentage of stained cells was computed from triplicates of four experiments. Means followed by the same letter are not significantly different. +, presence; -, absence of Il-1β, A. fumigatus fixed organisms and latex beads. The punctuated localisation of the signal, which is concentrated adjacent to the nucleus (arrow), was observed. The data shown are representative of four independent experiments.

    Journal: BMC Microbiology

    Article Title: Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms

    doi: 10.1186/1471-2180-9-33

    Figure Lengend Snippet: Localisation of the hBD2 peptide in epithelial bronchial 16HBE cells . 16HBE cells were seeded at 5 × 10 5 cells per well in 1 ml of DMEM/F12 on 18-mm-diameter cover slips in 12 well plates in triplicate and grown for 16 h at 37°C. After washing the cover slips with PBS-BSA, the cells were exposed to either latex beads, ethanol fixed conidia or ethanol fixed HF for 18 hours. Il-1β was used as a positive control. Following washing with PBS, the cells were fixed with a paraformaldehyde solution for 30 min at 37°C. The slides were then incubated in 1% BSA/PBS-Triton 0.05%, followed by a solution of 10% normal goat serum. After washing, polyclonal rabbit anti-human hBD2 at a dilution of 1:250 was applied as primary antibody overnight at 4°C, followed by incubation with FITC-labelled goat anti-rabbit secondary antibody at a dilution of 1:300 for 4 hours at room temperature. After washing, the cover slips were mounted on slides with ProLong antifade Vectashield. Samples were viewed with a Zeiss fluorescence microscope using ×400 magnification. The arrows indicate the cells stained with anti-hBD2 antibody. The percentage of stained cells was computed from triplicates of four experiments. Means followed by the same letter are not significantly different. +, presence; -, absence of Il-1β, A. fumigatus fixed organisms and latex beads. The punctuated localisation of the signal, which is concentrated adjacent to the nucleus (arrow), was observed. The data shown are representative of four independent experiments.

    Article Snippet: After washing the cover slips with 5% BSA/PBS (BSA, Fraction V, Sigma), the cells were exposed to either 106 fixed conidia or to 20 μl of the fixed HF solution (20 mg of dry weight/ml), or 5 × 106 latex beads for 24 hours.

    Techniques: Positive Control, Incubation, Fluorescence, Microscopy, Staining

    Analysis of the defensin expression and its localisation in pneumocytes A549 exposed to live A. fumigatus . A . RT-PCR analysis of defensin mRNA expression by human pneumocyte A549 cells exposed to live A. fumigatus . A549 human epithelial bronchial cells (5 × 10 6 ) were grown in six well plates for 24 hours. The cells were then exposed either to live A. fumigatus conidia or latex beads. After 18 hours of incubation, the cells were washed with PBS, mRNA was isolated by TRIzol Reagent, and RT-PCR was performed as described above in Methods. Specific primer pairs (Table 1) were used for RNA amplification. The size of the amplified product is indicated and was as predicted. Cells were cultivated in a control well in the absence of A. fumigatus . As an additional control, the cells were exposed to 10 6 latex beads for the same period. GAPDH was uniformly expressed. One of the four results is shown. B . Immunofluorescence detection of hBD2 in the A549 exposed to live A. fumigatus conidia. A549 cells were seeded at 5 × 10 5 cells per well in 1 ml of DMEM/F12 on 18-mm-diameter cover slips in 12 well plates in triplicate and grown for 16 h at 37°C. After washing the cover slips with 1%BSA/PBS, the cells were exposed to either latex beads or live A. fumigatus conidia for 18 hours. Il-1β was used as a positive control. Some cells were treated with TNF-α. Following washing with PBS, the cells were fixed with a paraformaldehyde solution for 30 min at 37°C. The slides were then incubated in 1% BSA/PBS, followed by a solution of 10% normal goat serum. After washing, polyclonal rabbit anti-human hBD2 at a dilution of 1:250 was applied as primary antibody overnight at 4°C, followed by incubation with FITC-labelled goat anti-rabbit secondary antibody at a dilution of 1:300 for 4 hours at room temperature. After washing, the cover slips were mounted on slides with ProLong antifade Vectashield. Samples were viewed with a Zeiss fluorescence microscope using ×400 magnification. The arrows indicate the cells stained with anti-hBD2 antibody. The percentage of stained cells was computed from triplicates of four experiments. Means followed by the same letter are not significantly different. +, presence; -, absence of Il-1β, TNF-α, live A. fumigatus organism and latex beads. The punctuated localisation of the signal, which is concentrated adjacent to the nucleus (arrow), was observed. The data shown are representative of four independent experiments.

    Journal: BMC Microbiology

    Article Title: Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms

    doi: 10.1186/1471-2180-9-33

    Figure Lengend Snippet: Analysis of the defensin expression and its localisation in pneumocytes A549 exposed to live A. fumigatus . A . RT-PCR analysis of defensin mRNA expression by human pneumocyte A549 cells exposed to live A. fumigatus . A549 human epithelial bronchial cells (5 × 10 6 ) were grown in six well plates for 24 hours. The cells were then exposed either to live A. fumigatus conidia or latex beads. After 18 hours of incubation, the cells were washed with PBS, mRNA was isolated by TRIzol Reagent, and RT-PCR was performed as described above in Methods. Specific primer pairs (Table 1) were used for RNA amplification. The size of the amplified product is indicated and was as predicted. Cells were cultivated in a control well in the absence of A. fumigatus . As an additional control, the cells were exposed to 10 6 latex beads for the same period. GAPDH was uniformly expressed. One of the four results is shown. B . Immunofluorescence detection of hBD2 in the A549 exposed to live A. fumigatus conidia. A549 cells were seeded at 5 × 10 5 cells per well in 1 ml of DMEM/F12 on 18-mm-diameter cover slips in 12 well plates in triplicate and grown for 16 h at 37°C. After washing the cover slips with 1%BSA/PBS, the cells were exposed to either latex beads or live A. fumigatus conidia for 18 hours. Il-1β was used as a positive control. Some cells were treated with TNF-α. Following washing with PBS, the cells were fixed with a paraformaldehyde solution for 30 min at 37°C. The slides were then incubated in 1% BSA/PBS, followed by a solution of 10% normal goat serum. After washing, polyclonal rabbit anti-human hBD2 at a dilution of 1:250 was applied as primary antibody overnight at 4°C, followed by incubation with FITC-labelled goat anti-rabbit secondary antibody at a dilution of 1:300 for 4 hours at room temperature. After washing, the cover slips were mounted on slides with ProLong antifade Vectashield. Samples were viewed with a Zeiss fluorescence microscope using ×400 magnification. The arrows indicate the cells stained with anti-hBD2 antibody. The percentage of stained cells was computed from triplicates of four experiments. Means followed by the same letter are not significantly different. +, presence; -, absence of Il-1β, TNF-α, live A. fumigatus organism and latex beads. The punctuated localisation of the signal, which is concentrated adjacent to the nucleus (arrow), was observed. The data shown are representative of four independent experiments.

    Article Snippet: After washing the cover slips with 5% BSA/PBS (BSA, Fraction V, Sigma), the cells were exposed to either 106 fixed conidia or to 20 μl of the fixed HF solution (20 mg of dry weight/ml), or 5 × 106 latex beads for 24 hours.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Isolation, Amplification, Immunofluorescence, Positive Control, Fluorescence, Microscopy, Staining