p44 wild type  (Millipore)


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    Structured Review

    Millipore p44 wild type
    Immunoblot analysis of a panel of Hsp90- and Hsp70-related proteins. The results shown are from a representative analysis of testis extracts from wild-type and mutant animals from P12 until completion of the first wave of spermatogenesis at <t>P44.</t> GAPDH serves as loading control. Note that the order of the sample pairs is reversed for the P16 and P18 samples at the center of the blots (area highlighted by hairlines).
    P44 Wild Type, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 8723 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p44 wild type - by Bioz Stars, 2020-05
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    Images

    1) Product Images from "The Molecular Chaperone Hsp90? Is Required for Meiotic Progression of Spermatocytes beyond Pachytene in the Mouse"

    Article Title: The Molecular Chaperone Hsp90? Is Required for Meiotic Progression of Spermatocytes beyond Pachytene in the Mouse

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0015770

    Immunoblot analysis of a panel of Hsp90- and Hsp70-related proteins. The results shown are from a representative analysis of testis extracts from wild-type and mutant animals from P12 until completion of the first wave of spermatogenesis at P44. GAPDH serves as loading control. Note that the order of the sample pairs is reversed for the P16 and P18 samples at the center of the blots (area highlighted by hairlines).
    Figure Legend Snippet: Immunoblot analysis of a panel of Hsp90- and Hsp70-related proteins. The results shown are from a representative analysis of testis extracts from wild-type and mutant animals from P12 until completion of the first wave of spermatogenesis at P44. GAPDH serves as loading control. Note that the order of the sample pairs is reversed for the P16 and P18 samples at the center of the blots (area highlighted by hairlines).

    Techniques Used: Mutagenesis

    Meiotic arrest and apoptosis in hsp90α gt/gt mutant testis. (A) Histological sections of testes of of wild-type (WT) and mutant (gt/gt) mice at 15, 21, 44 and 111 days after birth. In the righthand panels, arrows point out apoptotic nuclei and the asterisk in the P111 section indicates degenerating tubules. (B) Flow cytometric analysis of the DNA contents of the whole testis cell population at P44. (C) TUNEL analysis of sections of P21 testes.
    Figure Legend Snippet: Meiotic arrest and apoptosis in hsp90α gt/gt mutant testis. (A) Histological sections of testes of of wild-type (WT) and mutant (gt/gt) mice at 15, 21, 44 and 111 days after birth. In the righthand panels, arrows point out apoptotic nuclei and the asterisk in the P111 section indicates degenerating tubules. (B) Flow cytometric analysis of the DNA contents of the whole testis cell population at P44. (C) TUNEL analysis of sections of P21 testes.

    Techniques Used: Mutagenesis, Mouse Assay, Flow Cytometry, TUNEL Assay

    Absence of Hsp90α leads to atrophic testis and azoospermia. (A) Morphology of seminal vesicles and testis of wild-type (WT) and hsp90α gt/gt mutant (gt/gt) mice at P44. (B) Time course analysis of testis weight in WT and mutant (gt/gt) animals. n≥4. (C) No mature sperm cells could be detected in the epididymis of one year old hsp90α gt/g t animals.
    Figure Legend Snippet: Absence of Hsp90α leads to atrophic testis and azoospermia. (A) Morphology of seminal vesicles and testis of wild-type (WT) and hsp90α gt/gt mutant (gt/gt) mice at P44. (B) Time course analysis of testis weight in WT and mutant (gt/gt) animals. n≥4. (C) No mature sperm cells could be detected in the epididymis of one year old hsp90α gt/g t animals.

    Techniques Used: Mutagenesis, Mouse Assay

    Quantitative RT-PCR analysis of specific markers indicates pachytene arrest in the germ cells of hsp90α gt/gt mutant mouse testis. RNA samples are from P44 mice. (A) Markers of testis compartments. Insl3 and Star, Amh, and Pou5f1 are for Leydig, Sertoli, and germ cells, respectively. (B) Complete absence of post-meiotic markers. (C) Markers of advanced stages of meiosis.
    Figure Legend Snippet: Quantitative RT-PCR analysis of specific markers indicates pachytene arrest in the germ cells of hsp90α gt/gt mutant mouse testis. RNA samples are from P44 mice. (A) Markers of testis compartments. Insl3 and Star, Amh, and Pou5f1 are for Leydig, Sertoli, and germ cells, respectively. (B) Complete absence of post-meiotic markers. (C) Markers of advanced stages of meiosis.

    Techniques Used: Quantitative RT-PCR, Mutagenesis, Mouse Assay

    2) Product Images from "Requirement of Cognate CD4+ T-Cell Recognition for the Regulation of Allospecific CTL by Human CD4+CD127−CD25+FOXP3+ Cells Generated in MLR"

    Article Title: Requirement of Cognate CD4+ T-Cell Recognition for the Regulation of Allospecific CTL by Human CD4+CD127−CD25+FOXP3+ Cells Generated in MLR

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0022450

    MLR-Tregs inhibit the expression of perforin, granzyme B and CD25 on responding CD8 + cells. 5×10 4 PKH26 labeled purified responder CD8 cells were cultured with the original stimulators (1×10 5 ) used in generating MLR-Tregs, in the presence of 1×10 4 autologous modulator MLR-Tregs (right) vs. autologous modulator controls (Ax; middle). After 7 days in culture, the expression of intracellular Perforin-A, Granzyme-B, and membrane CD25 was assessed by flow cytometry. The CD8 + responder cells were gated and the PKH26 high non-proliferating and PKH26-diluted proliferating cells were analyzed. It was observed that the irradiated stimulators and Ax died off by day 7 (not shown); even the few that remained were gated out on CFSE vs. CD8 density-plot during the analysis. Note that there was a profound inhibition of both proliferation (PKH26 dilution) and expression of Perforin-A, Granzyme-B and CD25 by MLR-Tregs. This experiment is representative of 4 similar ones. (** = p
    Figure Legend Snippet: MLR-Tregs inhibit the expression of perforin, granzyme B and CD25 on responding CD8 + cells. 5×10 4 PKH26 labeled purified responder CD8 cells were cultured with the original stimulators (1×10 5 ) used in generating MLR-Tregs, in the presence of 1×10 4 autologous modulator MLR-Tregs (right) vs. autologous modulator controls (Ax; middle). After 7 days in culture, the expression of intracellular Perforin-A, Granzyme-B, and membrane CD25 was assessed by flow cytometry. The CD8 + responder cells were gated and the PKH26 high non-proliferating and PKH26-diluted proliferating cells were analyzed. It was observed that the irradiated stimulators and Ax died off by day 7 (not shown); even the few that remained were gated out on CFSE vs. CD8 density-plot during the analysis. Note that there was a profound inhibition of both proliferation (PKH26 dilution) and expression of Perforin-A, Granzyme-B and CD25 by MLR-Tregs. This experiment is representative of 4 similar ones. (** = p

    Techniques Used: Expressing, Labeling, Purification, Cell Culture, Flow Cytometry, Cytometry, Irradiation, Inhibition

    3) Product Images from "Tetrameric Complexes of Human Histocompatibility Leukocyte Antigen (HLA)-G Bind to Peripheral Blood Myelomonocytic Cells "

    Article Title: Tetrameric Complexes of Human Histocompatibility Leukocyte Antigen (HLA)-G Bind to Peripheral Blood Myelomonocytic Cells

    Journal: The Journal of Experimental Medicine

    doi:

    HLA-G tetramers bind to peripheral blood myelomonocytic cells. PBMCs from a healthy individual were stained with PE-labeled HLA-G tetramers or ExtrAvidin-PE control and anti-CD3, -CD56, -CD19, or -CD14 directly labeled mAb. An electronic gate based on forward and side light scatter properties was set on lymphoid cells (A) or myelomonocytic cells (B). Patterns in A were not significantly different from ExtrAvidin-PE control.
    Figure Legend Snippet: HLA-G tetramers bind to peripheral blood myelomonocytic cells. PBMCs from a healthy individual were stained with PE-labeled HLA-G tetramers or ExtrAvidin-PE control and anti-CD3, -CD56, -CD19, or -CD14 directly labeled mAb. An electronic gate based on forward and side light scatter properties was set on lymphoid cells (A) or myelomonocytic cells (B). Patterns in A were not significantly different from ExtrAvidin-PE control.

    Techniques Used: Staining, Labeling

    HLA-G tetramer staining of monocytes is largely due to interaction with ILT4. PBMCs from a healthy individual were stained with anti-CD14-FITC, HLA-G tetramer-PE, and various unlabeled mAbs reactive with ILT receptors. Only cells within a myelomonocytic light scatter gate are shown. Based on flow cytometry stains of the ILT transfectants shown in Fig. 4 , 40H2 recognizes ILT2, ILT4, ILT5 and ILT8; 28C8 recognizes ILT2 and ILT4; and 42D1 and 27D6 recognize ILT4.
    Figure Legend Snippet: HLA-G tetramer staining of monocytes is largely due to interaction with ILT4. PBMCs from a healthy individual were stained with anti-CD14-FITC, HLA-G tetramer-PE, and various unlabeled mAbs reactive with ILT receptors. Only cells within a myelomonocytic light scatter gate are shown. Based on flow cytometry stains of the ILT transfectants shown in Fig. 4 , 40H2 recognizes ILT2, ILT4, ILT5 and ILT8; 28C8 recognizes ILT2 and ILT4; and 42D1 and 27D6 recognize ILT4.

    Techniques Used: Staining, Flow Cytometry, Cytometry

    HLA-G tetramers do not bind to NK receptors. Baf3 cells transfected (A) with CD94 and NKG2A or CD94, NKG2C, and DAP12, or (B) with several KIR receptors were stained with HLA-G, HLA-E*0101, or HLA-B*2705 tetramers, or ExtrAvidin-PE or Streptavidin-PE controls. Expression of transgenes was verified with mAbs recognizing CD94, KIRs, or the FLAG peptide epitope, as indicated.
    Figure Legend Snippet: HLA-G tetramers do not bind to NK receptors. Baf3 cells transfected (A) with CD94 and NKG2A or CD94, NKG2C, and DAP12, or (B) with several KIR receptors were stained with HLA-G, HLA-E*0101, or HLA-B*2705 tetramers, or ExtrAvidin-PE or Streptavidin-PE controls. Expression of transgenes was verified with mAbs recognizing CD94, KIRs, or the FLAG peptide epitope, as indicated.

    Techniques Used: Transfection, Staining, Expressing

    HLA-G tetramers bind to ILT2 and ILT4. HLA-G tetramers or ExtrAvidin-PE control were used to stain P815 cells transfected with ILT1; Baf3 cells transfected with ILT2; Jurkat cells transfected with ILT3; or RBL cells transfected with ILT4, ILT5, or ILT8. Expression was verified with ILT-reactive or anti-FLAG mAbs as shown.
    Figure Legend Snippet: HLA-G tetramers bind to ILT2 and ILT4. HLA-G tetramers or ExtrAvidin-PE control were used to stain P815 cells transfected with ILT1; Baf3 cells transfected with ILT2; Jurkat cells transfected with ILT3; or RBL cells transfected with ILT4, ILT5, or ILT8. Expression was verified with ILT-reactive or anti-FLAG mAbs as shown.

    Techniques Used: Staining, Transfection, Expressing

    HLA-G tetramers intensely stain a distinct CD16 + CD14 mid monocyte subset. PBMCs from a healthy individual were stained with PE-labeled HLA-G tetramers and several FITC- labeled or unconjugated mAbs as indicated. Only cells within a myelomonocytic light scatter gate are shown. Results with FITC- labeled or unconjugated IgG1 and IgG2a control mAbs were very similar to unlabeled IgG1 shown. Unlabeled mAbs were detected with FITC-conjugated F(ab′) 2 goat anti–mouse Ig. Additionally, an HLA-G tetramer created with a different peptide (KIPAQFYIL) displayed a very similar pattern of staining (data not shown).
    Figure Legend Snippet: HLA-G tetramers intensely stain a distinct CD16 + CD14 mid monocyte subset. PBMCs from a healthy individual were stained with PE-labeled HLA-G tetramers and several FITC- labeled or unconjugated mAbs as indicated. Only cells within a myelomonocytic light scatter gate are shown. Results with FITC- labeled or unconjugated IgG1 and IgG2a control mAbs were very similar to unlabeled IgG1 shown. Unlabeled mAbs were detected with FITC-conjugated F(ab′) 2 goat anti–mouse Ig. Additionally, an HLA-G tetramer created with a different peptide (KIPAQFYIL) displayed a very similar pattern of staining (data not shown).

    Techniques Used: Staining, Labeling

    4) Product Images from "Down-regulation of p21WAF1/CIP1 or p27Kip1 abrogates antiestrogen-mediated cell cycle arrest in human breast cancer cells"

    Article Title: Down-regulation of p21WAF1/CIP1 or p27Kip1 abrogates antiestrogen-mediated cell cycle arrest in human breast cancer cells

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Different patterns of KIP binding during cyclin D1–cdk4 and cyclin E–cdk2 activation. Cdk4 ( A ) and cyclin E ( C ) immunoprecipitates (IP) from cell lysates recovered at intervals after readdition of estradiol to steroid-depleted MCF-7 cells were resolved and analyzed by immunoblotting with the indicated antibodies. Cyclin D1 ( B ) and cyclin E ( D ) at intervals after estradiol stimulation of quiescent MCF-7.
    Figure Legend Snippet: Different patterns of KIP binding during cyclin D1–cdk4 and cyclin E–cdk2 activation. Cdk4 ( A ) and cyclin E ( C ) immunoprecipitates (IP) from cell lysates recovered at intervals after readdition of estradiol to steroid-depleted MCF-7 cells were resolved and analyzed by immunoblotting with the indicated antibodies. Cyclin D1 ( B ) and cyclin E ( D ) at intervals after estradiol stimulation of quiescent MCF-7.

    Techniques Used: Binding Assay, Activation Assay

    Requirement for p21 and p27 in G 1 arrest by ER-blocking drugs or estradiol-depletion. MCF-7 cells were arrested by estradiol depletion or by treatment with 4-OH TAM or ICI 182780. The graph indicates the percentage of S phase cells after a 21-h exposure to lipid only (black bars), antisense (white bars), or mismatch (hatched bars) oligonucleotides to either p21 or p27.
    Figure Legend Snippet: Requirement for p21 and p27 in G 1 arrest by ER-blocking drugs or estradiol-depletion. MCF-7 cells were arrested by estradiol depletion or by treatment with 4-OH TAM or ICI 182780. The graph indicates the percentage of S phase cells after a 21-h exposure to lipid only (black bars), antisense (white bars), or mismatch (hatched bars) oligonucleotides to either p21 or p27.

    Techniques Used: Blocking Assay

    Losses of p21 and p27 during estradiol stimulation of quiescent MCF-7 cells. Quiescent, estradiol-depleted MCF-7 cells were stimulated by readdition of 10 nM estradiol, and samples were taken at intervals thereafter. ( A ) Cell cycle synchrony was determined by dual BrdUrd/propidium iodide pulse labeling and flow cytometric analysis. ( B ) p21 and p27 immunoblots revealed levels of these proteins during cell cycle progression. ( C ).
    Figure Legend Snippet: Losses of p21 and p27 during estradiol stimulation of quiescent MCF-7 cells. Quiescent, estradiol-depleted MCF-7 cells were stimulated by readdition of 10 nM estradiol, and samples were taken at intervals thereafter. ( A ) Cell cycle synchrony was determined by dual BrdUrd/propidium iodide pulse labeling and flow cytometric analysis. ( B ) p21 and p27 immunoblots revealed levels of these proteins during cell cycle progression. ( C ).

    Techniques Used: Labeling, Flow Cytometry, Western Blot

    p21 and p27 proteins increase during G 0 /G 1 arrest by ER blockade. Asynchronously growing MCF-7 cells were treated with the ER-blocking drug ICI 182780 (Faslodex) at time 0 h, and samples were collected for flow cytometry or protein analysis at times indicated. ( A ) Cell cycle distribution before and 48 h after drug treatment. ( B . Similar results were obtained for arrests with 4-OH TAM or after transfer to estradiol-depleted, charcoal-stripped serum.
    Figure Legend Snippet: p21 and p27 proteins increase during G 0 /G 1 arrest by ER blockade. Asynchronously growing MCF-7 cells were treated with the ER-blocking drug ICI 182780 (Faslodex) at time 0 h, and samples were collected for flow cytometry or protein analysis at times indicated. ( A ) Cell cycle distribution before and 48 h after drug treatment. ( B . Similar results were obtained for arrests with 4-OH TAM or after transfer to estradiol-depleted, charcoal-stripped serum.

    Techniques Used: Blocking Assay, Flow Cytometry, Cytometry

    Requirement of p27 for cell cycle arrest by estradiol depletion. ( A ) Estradiol-depleted MCF-7 cells were lysed before (left lane) or 1 h after exposure to lipid only (L), ASp27 (AS), or MSMp27 (MSM) and were immunoblotted for p21 and p27. Before and at 1 and 21 h after ASp27 transfection, cells were metabolically pulse labeled with [ 35 S]methionine, and p21 and p27 were immunoprecipitated from lysates containing equal trichloroacetic acid incorporation. The positions of metabolically labeled p27 are indicated by arrows. A nonspecific band, migrating close to p27 was present in all lanes, including the control nonspecific IgG lane. ( B ) Immunoblotting shows cell cycle regulatory protein levels before (left lane) or 21 h after transfection of lipid alone (L), ASp27, or MSMp27. Cyclin E immunoprecipitates (IP) were recovered from the same lysates as in B above and immunoblotted to detect associated proteins ( C ) or assayed for cyclin E-associated histone H1 kinase activity ( D ). ( E ) Flow cytometry 21 h after transfection with ASp27, lipid alone, or MSMp27.
    Figure Legend Snippet: Requirement of p27 for cell cycle arrest by estradiol depletion. ( A ) Estradiol-depleted MCF-7 cells were lysed before (left lane) or 1 h after exposure to lipid only (L), ASp27 (AS), or MSMp27 (MSM) and were immunoblotted for p21 and p27. Before and at 1 and 21 h after ASp27 transfection, cells were metabolically pulse labeled with [ 35 S]methionine, and p21 and p27 were immunoprecipitated from lysates containing equal trichloroacetic acid incorporation. The positions of metabolically labeled p27 are indicated by arrows. A nonspecific band, migrating close to p27 was present in all lanes, including the control nonspecific IgG lane. ( B ) Immunoblotting shows cell cycle regulatory protein levels before (left lane) or 21 h after transfection of lipid alone (L), ASp27, or MSMp27. Cyclin E immunoprecipitates (IP) were recovered from the same lysates as in B above and immunoblotted to detect associated proteins ( C ) or assayed for cyclin E-associated histone H1 kinase activity ( D ). ( E ) Flow cytometry 21 h after transfection with ASp27, lipid alone, or MSMp27.

    Techniques Used: Transfection, Metabolic Labelling, Labeling, Immunoprecipitation, Activity Assay, Flow Cytometry, Cytometry

    5) Product Images from "From fighting depression to conquering tumors: a novel tricyclic thiazepine compound as a tubulin polymerization inhibitor"

    Article Title: From fighting depression to conquering tumors: a novel tricyclic thiazepine compound as a tubulin polymerization inhibitor

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.53

    TBPT induces time-dependent apoptosis in both H460 TaxR and H460 cells. ( a ) The cells were treated with DMSO or TBPT (2 μ M), stained with PI/Annexin V-FITC, and analyzed using flow cytometry. ( b ) The activities of caspases 8, 9 and 3/7 were examined using a fluorescence-based assay. *Student's t -test with the DMSO group, P
    Figure Legend Snippet: TBPT induces time-dependent apoptosis in both H460 TaxR and H460 cells. ( a ) The cells were treated with DMSO or TBPT (2 μ M), stained with PI/Annexin V-FITC, and analyzed using flow cytometry. ( b ) The activities of caspases 8, 9 and 3/7 were examined using a fluorescence-based assay. *Student's t -test with the DMSO group, P

    Techniques Used: Staining, Flow Cytometry, Cytometry, Fluorescence

    6) Product Images from "Distinct assembly profiles of HLA-B molecules"

    Article Title: Distinct assembly profiles of HLA-B molecules

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1301670

    Reduced variability of HLA-B expression in tapasin-sufficient cells A) Cells from one of the infections described in Fig. 1A were subsequently infected with a tapasin-encoding virus. Following subtractions of the vector alone backgrounds the MFI ratios in the presence and absence of tapasin (+tapasin/−tapasin MFI ratios) were calculated for each HLA-B-expressing cell line. B) Representative histograms of M553 cell infections with a tapasin-encoding or control retrovirus. Flow cytometric analyses with the W6/32 antibody of cell surface expression of the endogenous MHC class I of M553 cells that were infected with retroviruses encoding or lacking tapasin. C) A Pearson analysis was used to examine correlation between averaged cell surface MFI ratios (HLA-B/vector) in M553 (Fig. 1A) vs. Log MFI (+Tapasin/−Tapasin) ratios (Fig. 2A). D) CEM cells (a CD4 T cell line) were infected with retroviruses encoding indicated HLA-B allotypes or a control virus lacking HLA-B (Vector). MHC class I cell surface expression was analyzed by flow cytometry using the W6/32 antibody, and HLA-B/vector MFI ratios were determined. E ) A Pearson analysis was used to examine correlation between averaged MFI ratios (HLA-B/vector) in M553 and CEM cells. Data represent averaged (A and D) MFI values derived from 2–4 ( A ) or 10–12 ( D ) independent flow cytometric analyses from one ( A ) or three ( D ) independent infections. Statistical analyses (A and D) were done using a one-way ANOVA test, followed by a Tukey’s multiple comparisons procedure for all pairwise differences of means. Pearson R square (R 2 ) and significantly different values (P) are indicated on the graph. P
    Figure Legend Snippet: Reduced variability of HLA-B expression in tapasin-sufficient cells A) Cells from one of the infections described in Fig. 1A were subsequently infected with a tapasin-encoding virus. Following subtractions of the vector alone backgrounds the MFI ratios in the presence and absence of tapasin (+tapasin/−tapasin MFI ratios) were calculated for each HLA-B-expressing cell line. B) Representative histograms of M553 cell infections with a tapasin-encoding or control retrovirus. Flow cytometric analyses with the W6/32 antibody of cell surface expression of the endogenous MHC class I of M553 cells that were infected with retroviruses encoding or lacking tapasin. C) A Pearson analysis was used to examine correlation between averaged cell surface MFI ratios (HLA-B/vector) in M553 (Fig. 1A) vs. Log MFI (+Tapasin/−Tapasin) ratios (Fig. 2A). D) CEM cells (a CD4 T cell line) were infected with retroviruses encoding indicated HLA-B allotypes or a control virus lacking HLA-B (Vector). MHC class I cell surface expression was analyzed by flow cytometry using the W6/32 antibody, and HLA-B/vector MFI ratios were determined. E ) A Pearson analysis was used to examine correlation between averaged MFI ratios (HLA-B/vector) in M553 and CEM cells. Data represent averaged (A and D) MFI values derived from 2–4 ( A ) or 10–12 ( D ) independent flow cytometric analyses from one ( A ) or three ( D ) independent infections. Statistical analyses (A and D) were done using a one-way ANOVA test, followed by a Tukey’s multiple comparisons procedure for all pairwise differences of means. Pearson R square (R 2 ) and significantly different values (P) are indicated on the graph. P

    Techniques Used: Expressing, Infection, Plasmid Preparation, Flow Cytometry, Cytometry, Derivative Assay

    7) Product Images from "The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells"

    Article Title: The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

    Journal: The EMBO Journal

    doi: 10.15252/embj.201797479

    Analysis of doxycycline‐inducible expression of full‐length FLAG‐LGP2, FLAG‐LGP2 CTD, or FLAG‐LGP2 CTD K634E in Ifnar1 −/− MEFs Relative expression (RE) of LGP2 ( DHX58 ) in wild‐type MEFs treated for 24 or 48 h with type I IFN was assessed by qRT–PCR and normalised to β‐actin ( ACTB ) using the ∆∆ C t method. Verification of doxycycline‐dependent induction of FLAG‐LGP2 expression in Ifnar1 −/− MEFs by flow cytometry. Various Ifnar1 −/− iLGP2 clones were treated for 72 h with doxycycline (dox) and subsequently fixed, permeabilised and stained with a FLAG‐Cy3 antibody followed by flow cytometry. Verification of doxycycline‐dependent induction of FLAG‐LGP2 CTD and CTD K634E expression in Ifnar1 −/− MEFs. The indicated clones were treated for 72 h with dox and subsequently fixed, permeabilised, stained with a FLAG‐Cy3 antibody and analysed by flow cytometry. Immunoblot analysis of four clones of Ifnar1 −/− MEFs in which expression of FLAG‐tagged human LGP2 CTD or CTD K634E is induced following 72 h of dox treatment. β‐Actin serves as loading control. Northern blot analysis of dsRNA‐derived siRNAs in two individual clones of Ifnar1 −/− iLGP2 CTD and CTD K634E MEFs left untreated or treated with dox for 24 h prior to transfection with dsRNA‐GFP. Twenty‐four hours post‐transfection, cells were harvested and the generation of siRNAs was analysed by Northern blotting using a probe specific for dsRNA‐GFP. The arrow points to dsRNA‐GFP‐derived siRNAs. A miRNA ladder was used as a size marker and endogenous U6 served as loading control. Source data are available online for this figure.
    Figure Legend Snippet: Analysis of doxycycline‐inducible expression of full‐length FLAG‐LGP2, FLAG‐LGP2 CTD, or FLAG‐LGP2 CTD K634E in Ifnar1 −/− MEFs Relative expression (RE) of LGP2 ( DHX58 ) in wild‐type MEFs treated for 24 or 48 h with type I IFN was assessed by qRT–PCR and normalised to β‐actin ( ACTB ) using the ∆∆ C t method. Verification of doxycycline‐dependent induction of FLAG‐LGP2 expression in Ifnar1 −/− MEFs by flow cytometry. Various Ifnar1 −/− iLGP2 clones were treated for 72 h with doxycycline (dox) and subsequently fixed, permeabilised and stained with a FLAG‐Cy3 antibody followed by flow cytometry. Verification of doxycycline‐dependent induction of FLAG‐LGP2 CTD and CTD K634E expression in Ifnar1 −/− MEFs. The indicated clones were treated for 72 h with dox and subsequently fixed, permeabilised, stained with a FLAG‐Cy3 antibody and analysed by flow cytometry. Immunoblot analysis of four clones of Ifnar1 −/− MEFs in which expression of FLAG‐tagged human LGP2 CTD or CTD K634E is induced following 72 h of dox treatment. β‐Actin serves as loading control. Northern blot analysis of dsRNA‐derived siRNAs in two individual clones of Ifnar1 −/− iLGP2 CTD and CTD K634E MEFs left untreated or treated with dox for 24 h prior to transfection with dsRNA‐GFP. Twenty‐four hours post‐transfection, cells were harvested and the generation of siRNAs was analysed by Northern blotting using a probe specific for dsRNA‐GFP. The arrow points to dsRNA‐GFP‐derived siRNAs. A miRNA ladder was used as a size marker and endogenous U6 served as loading control. Source data are available online for this figure.

    Techniques Used: Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry, Clone Assay, Staining, Northern Blot, Derivative Assay, Transfection, Marker

    Expression of RIG‐I or MDA5, unlike that of LGP2, does not inhibit dsRNA‐mediated RNAi in Ifnar1 −/− cells Doxycycline treatment does not impact on dsRNAi in parental Ifnar1 −/− MEFs that lack inducible LGP2 expression constructs. Verification of doxycycline‐dependent induction of FLAG‐RIG‐I (iRIG‐I) and FLAG‐MDA5 (iMDA5) expression in Ifnar1 −/− MEFs by flow cytometry. Various Ifnar1 −/− iRIG‐I and iMDA5 clones were treated for 72 h with doxycycline (dox) and subsequently fixed, permeabilised and stained with a FLAG‐Cy3 antibody followed by flow cytometry. Immunoblot analysis of four clones of Ifnar1 −/− MEFs in which expression of FLAG‐RIG‐I or FLAG‐MDA5 is induced following 72 h of doxycycline (dox) treatment. β‐Actin serves as loading control. Expression of full‐length RIG‐I or MDA5 does not affect dsRNA‐mediated RNAi in Ifnar1 −/− cells. Ifnar1 −/− iRIG‐I and Ifnar1 −/− iMDA5 cells, which also express a destabilised form of GFP (d2GFP), were transfected with Cy5‐labelled long dsRNA corresponding to the first 200 nt of Renilla luciferase (dsRNA‐RL) or GFP (dsRNA‐GFP) in the absence or presence or doxycycline. Forty‐eight hours post‐transfection, cells were harvested and d2GFP expression in live, single, Cy5 + cells was analysed by flow cytometry. Histogram plots of one representative clone are shown and are representative of three independent experiments. Each histogram and bar represents a sample size of 10,000 cells. Bar graphs display the percentage of GFP median fluorescence intensity of dsRNA‐GFP‐transfected cells relative to dsRNA‐RL‐transfected cells in four independent clones. The median fluorescence values were normalised to those in Renilla‐transfected samples. Mean values and SD of three independent experiments are shown. Statistical analysis was performed using two‐way ANOVA with Sidak's multiple comparisons test as post‐test for pairwise comparisons. Significant differences with Sidak's multiple comparisons test are shown (ns, not significant). Source data are available online for this figure.
    Figure Legend Snippet: Expression of RIG‐I or MDA5, unlike that of LGP2, does not inhibit dsRNA‐mediated RNAi in Ifnar1 −/− cells Doxycycline treatment does not impact on dsRNAi in parental Ifnar1 −/− MEFs that lack inducible LGP2 expression constructs. Verification of doxycycline‐dependent induction of FLAG‐RIG‐I (iRIG‐I) and FLAG‐MDA5 (iMDA5) expression in Ifnar1 −/− MEFs by flow cytometry. Various Ifnar1 −/− iRIG‐I and iMDA5 clones were treated for 72 h with doxycycline (dox) and subsequently fixed, permeabilised and stained with a FLAG‐Cy3 antibody followed by flow cytometry. Immunoblot analysis of four clones of Ifnar1 −/− MEFs in which expression of FLAG‐RIG‐I or FLAG‐MDA5 is induced following 72 h of doxycycline (dox) treatment. β‐Actin serves as loading control. Expression of full‐length RIG‐I or MDA5 does not affect dsRNA‐mediated RNAi in Ifnar1 −/− cells. Ifnar1 −/− iRIG‐I and Ifnar1 −/− iMDA5 cells, which also express a destabilised form of GFP (d2GFP), were transfected with Cy5‐labelled long dsRNA corresponding to the first 200 nt of Renilla luciferase (dsRNA‐RL) or GFP (dsRNA‐GFP) in the absence or presence or doxycycline. Forty‐eight hours post‐transfection, cells were harvested and d2GFP expression in live, single, Cy5 + cells was analysed by flow cytometry. Histogram plots of one representative clone are shown and are representative of three independent experiments. Each histogram and bar represents a sample size of 10,000 cells. Bar graphs display the percentage of GFP median fluorescence intensity of dsRNA‐GFP‐transfected cells relative to dsRNA‐RL‐transfected cells in four independent clones. The median fluorescence values were normalised to those in Renilla‐transfected samples. Mean values and SD of three independent experiments are shown. Statistical analysis was performed using two‐way ANOVA with Sidak's multiple comparisons test as post‐test for pairwise comparisons. Significant differences with Sidak's multiple comparisons test are shown (ns, not significant). Source data are available online for this figure.

    Techniques Used: Expressing, Construct, Flow Cytometry, Cytometry, Clone Assay, Staining, Transfection, Luciferase, Fluorescence

    8) Product Images from "Changes in the Molecular and Functional Phenotype of Bovine Monocytes during Theileria parva Infection"

    Article Title: Changes in the Molecular and Functional Phenotype of Bovine Monocytes during Theileria parva Infection

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00703-19

    Differential response of monocytes from T. parva lethally and nonlethally infected cattle to T. parva infected cells and E. coli LPS. (A) Intracellular staining for the T. parva polymorphic immunodominant molecule in a representative schizont-infected cell line. (B) Intracellular staining for IFN-γ in a representative schizont-infected cell line. (C and D) Expression of mRNA for TNF-α (C) and IL-10 (D) in a representative schizont-infected cell line at 4 and 16 h after Histopaque centrifugation. (E) Levels of soluble IL-1β in CD172a + cell supernatant following stimulation of monocytes from uninfected ( n = 6), T. parva lethally infected ( n = 6), and T. parva nonlethally infected ( n = 4) cattle with T. parva schizont-infected cells (using a transwell format) or E. coli LPS (1 μg/ml). Amounts of soluble IL-1β were compared using ANOVA and Tukey’s post hoc test. (F) Expression of IL-10 mRNA in CD172a + cells from T. parva lethally infected ( n = 6) and nonlethally infected ( n = 4) cattle following stimulation with T. parva schizont-infected cells (using a transwell format) or E. coli LPS (1 μg/ml). IL-10 mRNA expression was compared using ANOVA and Tukey’s post hoc test. *, P
    Figure Legend Snippet: Differential response of monocytes from T. parva lethally and nonlethally infected cattle to T. parva infected cells and E. coli LPS. (A) Intracellular staining for the T. parva polymorphic immunodominant molecule in a representative schizont-infected cell line. (B) Intracellular staining for IFN-γ in a representative schizont-infected cell line. (C and D) Expression of mRNA for TNF-α (C) and IL-10 (D) in a representative schizont-infected cell line at 4 and 16 h after Histopaque centrifugation. (E) Levels of soluble IL-1β in CD172a + cell supernatant following stimulation of monocytes from uninfected ( n = 6), T. parva lethally infected ( n = 6), and T. parva nonlethally infected ( n = 4) cattle with T. parva schizont-infected cells (using a transwell format) or E. coli LPS (1 μg/ml). Amounts of soluble IL-1β were compared using ANOVA and Tukey’s post hoc test. (F) Expression of IL-10 mRNA in CD172a + cells from T. parva lethally infected ( n = 6) and nonlethally infected ( n = 4) cattle following stimulation with T. parva schizont-infected cells (using a transwell format) or E. coli LPS (1 μg/ml). IL-10 mRNA expression was compared using ANOVA and Tukey’s post hoc test. *, P

    Techniques Used: Infection, Staining, Expressing, Centrifugation

    9) Product Images from "Robust Phenotypic Activation of Eosinophils during Experimental Toxocara canis Infection"

    Article Title: Robust Phenotypic Activation of Eosinophils during Experimental Toxocara canis Infection

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00064

    Impact of in vitro stimulation with Toxocara canis antigens on bone marrow-derived eosinophils. In vitro T. canis antigen stimulation was performed on bone marrow - derived eosinophils from T. canis -infected as well as non-infected control groups. Flow cytometric evaluation of major histocompatibility complex (MHC-II), CD80, CD86, and CD69 within Siglec-F + -eosinophils was performed by ImageStream Mark II and analyzed by the Ideas software. Activation and co-stimulatory-related molecules were assessed in Siglec-F + -eosinophils by mean fluorescence intensity (MFI) assessed or percentage of positive cells as verified by histograms.
    Figure Legend Snippet: Impact of in vitro stimulation with Toxocara canis antigens on bone marrow-derived eosinophils. In vitro T. canis antigen stimulation was performed on bone marrow - derived eosinophils from T. canis -infected as well as non-infected control groups. Flow cytometric evaluation of major histocompatibility complex (MHC-II), CD80, CD86, and CD69 within Siglec-F + -eosinophils was performed by ImageStream Mark II and analyzed by the Ideas software. Activation and co-stimulatory-related molecules were assessed in Siglec-F + -eosinophils by mean fluorescence intensity (MFI) assessed or percentage of positive cells as verified by histograms.

    Techniques Used: In Vitro, Derivative Assay, Infection, Flow Cytometry, Software, Activation Assay, Fluorescence

    10) Product Images from "Necdin, a p53-Target Gene, Is an Inhibitor of p53-Mediated Growth Arrest"

    Article Title: Necdin, a p53-Target Gene, Is an Inhibitor of p53-Mediated Growth Arrest

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031916

    Necdin confers resistance to p53-dependent growth arrest. (A–B) NIHLT cells depleted in Necdin by shRNAs and exposed to nutlin-3 showed an increase in growth arrest (A) measured by DNA content analysis by flow cytometry (as described in figure 4A ) or (B) assessed by Wst-1 colorimetric assay. Results for Wst-1 represent normalized data according to the portion of arrested cells (O.D. untreated – O.D. treated) relative to arrested control NIH after 48 h of exposure to nutlin-3. (C–D) NIH and NIHLT cells overexpressing Necdin showed growth arrest resistance upon nutlin-3 treatment. (C) FACS analysis or (D) Wst-1 colorimetric assay (* P
    Figure Legend Snippet: Necdin confers resistance to p53-dependent growth arrest. (A–B) NIHLT cells depleted in Necdin by shRNAs and exposed to nutlin-3 showed an increase in growth arrest (A) measured by DNA content analysis by flow cytometry (as described in figure 4A ) or (B) assessed by Wst-1 colorimetric assay. Results for Wst-1 represent normalized data according to the portion of arrested cells (O.D. untreated – O.D. treated) relative to arrested control NIH after 48 h of exposure to nutlin-3. (C–D) NIH and NIHLT cells overexpressing Necdin showed growth arrest resistance upon nutlin-3 treatment. (C) FACS analysis or (D) Wst-1 colorimetric assay (* P

    Techniques Used: Flow Cytometry, Cytometry, Colorimetric Assay, FACS

    Nutlin-3 induces a p53-dependent growth arrest in NIH3T3 cells that is bypassed by PyLT expression. (A–B) Flow cytometry analysis of NIH or NIHLT populations treated with nutlin-3 (5 µM) demonstrate that nutlin-3 induces a growth arrest in NIH cells, but not in NIHLT cells. Results presented are from one representative experiment (A) Cell cycle arrest was represented by the variation of ratio of arrested cells (G1+G2 phases) over proliferating cells (S phase) in treated cells versus untreated controls. (B) No variation of the percentage of cells in Sub-G1 phase, representing cell death, was observed after nutlin-3 treatment. (C–D) The use of a p53 inhibitor peptide (GSE22) validates the p53-dependence of the growth arrest induced by Nuclin-3. (C) High efficiency of infection and functionality of the GSE22 peptide were demonstrated by the accumulation of non-functional p53 in the nucleus by immunocytochemistry detecting p53 in NIH transduced with GSE22 or control vector. The stabilization of non-functional p53 was also seen in Western blots of the corresponding infected cells. (D) FACS analysis on NIH transduced with GSE22 or vector with nutlin-3 treatment (* P
    Figure Legend Snippet: Nutlin-3 induces a p53-dependent growth arrest in NIH3T3 cells that is bypassed by PyLT expression. (A–B) Flow cytometry analysis of NIH or NIHLT populations treated with nutlin-3 (5 µM) demonstrate that nutlin-3 induces a growth arrest in NIH cells, but not in NIHLT cells. Results presented are from one representative experiment (A) Cell cycle arrest was represented by the variation of ratio of arrested cells (G1+G2 phases) over proliferating cells (S phase) in treated cells versus untreated controls. (B) No variation of the percentage of cells in Sub-G1 phase, representing cell death, was observed after nutlin-3 treatment. (C–D) The use of a p53 inhibitor peptide (GSE22) validates the p53-dependence of the growth arrest induced by Nuclin-3. (C) High efficiency of infection and functionality of the GSE22 peptide were demonstrated by the accumulation of non-functional p53 in the nucleus by immunocytochemistry detecting p53 in NIH transduced with GSE22 or control vector. The stabilization of non-functional p53 was also seen in Western blots of the corresponding infected cells. (D) FACS analysis on NIH transduced with GSE22 or vector with nutlin-3 treatment (* P

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Infection, Functional Assay, Immunocytochemistry, Transduction, Plasmid Preparation, Western Blot, FACS

    PyLT-induced Necdin expression is p53-independent. Necdin is induced following activation of p53. (A) Dose response treatment with nutlin-3 increased Necdin protein level in NIH (left) and NIHLT (right). (B) Genotoxic stress induced by Actinomycin D and Camptothecin also stimulated Necdin protein expression. (C) In NIHLT cells, Necdin expression is not dependent on p53 activity as assessed by p53 inhibition. Mean of relative expression of Necdin, p21, p53 and GAPDH in NIHLT cells with or without the p53 inhibitor GSE22. Expression was measured by Q-PCR in three independent samples from each group. Expression is relative to actin (** P
    Figure Legend Snippet: PyLT-induced Necdin expression is p53-independent. Necdin is induced following activation of p53. (A) Dose response treatment with nutlin-3 increased Necdin protein level in NIH (left) and NIHLT (right). (B) Genotoxic stress induced by Actinomycin D and Camptothecin also stimulated Necdin protein expression. (C) In NIHLT cells, Necdin expression is not dependent on p53 activity as assessed by p53 inhibition. Mean of relative expression of Necdin, p21, p53 and GAPDH in NIHLT cells with or without the p53 inhibitor GSE22. Expression was measured by Q-PCR in three independent samples from each group. Expression is relative to actin (** P

    Techniques Used: Expressing, Activation Assay, Activity Assay, Inhibition, Polymerase Chain Reaction

    11) Product Images from "A metabolic synthetic lethal strategy with arginine deprivation and chloroquine leads to cell death in ASS1-deficient sarcomas"

    Article Title: A metabolic synthetic lethal strategy with arginine deprivation and chloroquine leads to cell death in ASS1-deficient sarcomas

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.232

    Induction of necroptosis upon simultaneous arginine deprivation and chloroquine treatment. ( a ) Cell death as measured by FACS analysis after propidium iodide uptake. MNNG/HOS (left) and SK-LMS-1 (right) cells treated with 1 μ g/ml ADI-PEG20, 20 μ M chloroquine, both, or in combination with 100 μ M ZVAD (an apoptosis inhibitor) or 10 μ M necrostatin (a necroptosis inhibitor). Protection of cell death was more effective with necrostatin, indicating cell death is occurring primarily via necroptosis. ( N =3). Data represented as mean±S.D. ( b ) Cell death as measured by FACS analysis after propidium iodide uptake in wild type, shRIP1 or shRIP3 SK-LMS-1 cells after treatment with 1 μ g/ml ADI-PEG20 with or without 20 μ M chloroquine. RIP kinase knockdown protected from induction of cell death, indicating necroptosis induction upon dual agent treatment. Data represented as mean±S.D. ( N =2). ( c ) Western blots of SK-LMS-1 and MNNG/HOS cells untreated, or treated with ADI-PEG20 and chloroquine for 24, 48 or 72 h. In the presence of chloroquine and ADI-PEG20, the loss of the proapoptotic cleaved caspase 3 and the anti-necroptotic cIAP1 increases the threshold for apoptosis signaling while priming cells for death by necroptosis. Decrease in levels of cleaved RIP1 further suggest necroptosis induction ( d ). RIP1 co-IP. SK-LMS-1 ASS1 low cells were treated with ADI-PEG20, chloroquine or both for 3 days. A significant RIP3 co-precipitation was observed upon exposure to 1 μ g/ml ADI-PEG20 and 20 μ M chloroquine, whereas caspase 8, which is negative regulator of ripoptosome formation, was reduced. Collectively, these observations are indicative of active ripoptosome formation and subsequent cell death executed preferentially by necroptosis
    Figure Legend Snippet: Induction of necroptosis upon simultaneous arginine deprivation and chloroquine treatment. ( a ) Cell death as measured by FACS analysis after propidium iodide uptake. MNNG/HOS (left) and SK-LMS-1 (right) cells treated with 1 μ g/ml ADI-PEG20, 20 μ M chloroquine, both, or in combination with 100 μ M ZVAD (an apoptosis inhibitor) or 10 μ M necrostatin (a necroptosis inhibitor). Protection of cell death was more effective with necrostatin, indicating cell death is occurring primarily via necroptosis. ( N =3). Data represented as mean±S.D. ( b ) Cell death as measured by FACS analysis after propidium iodide uptake in wild type, shRIP1 or shRIP3 SK-LMS-1 cells after treatment with 1 μ g/ml ADI-PEG20 with or without 20 μ M chloroquine. RIP kinase knockdown protected from induction of cell death, indicating necroptosis induction upon dual agent treatment. Data represented as mean±S.D. ( N =2). ( c ) Western blots of SK-LMS-1 and MNNG/HOS cells untreated, or treated with ADI-PEG20 and chloroquine for 24, 48 or 72 h. In the presence of chloroquine and ADI-PEG20, the loss of the proapoptotic cleaved caspase 3 and the anti-necroptotic cIAP1 increases the threshold for apoptosis signaling while priming cells for death by necroptosis. Decrease in levels of cleaved RIP1 further suggest necroptosis induction ( d ). RIP1 co-IP. SK-LMS-1 ASS1 low cells were treated with ADI-PEG20, chloroquine or both for 3 days. A significant RIP3 co-precipitation was observed upon exposure to 1 μ g/ml ADI-PEG20 and 20 μ M chloroquine, whereas caspase 8, which is negative regulator of ripoptosome formation, was reduced. Collectively, these observations are indicative of active ripoptosome formation and subsequent cell death executed preferentially by necroptosis

    Techniques Used: FACS, Western Blot, Co-Immunoprecipitation Assay

    12) Product Images from "Redeployment-based drug screening identifies the anti-helminthic niclosamide as anti-myeloma therapy that also reduces free light chain production"

    Article Title: Redeployment-based drug screening identifies the anti-helminthic niclosamide as anti-myeloma therapy that also reduces free light chain production

    Journal: Blood Cancer Journal

    doi: 10.1038/bcj.2011.38

    Niclosamide-treated myeloma cells die with markers of apoptosis and autophagy. H929, JJN3 and U226 were treated with different concentrations of niclosamide (Nic) for 48 h. Cells were analysed by flow cytometry for ( a ) annexin V/propidium iodide positivity ( b ) and caspase activity using Caspatag assay according to manufacturer's instructions. Data shown are the mean of three experiments±s.e.m. Representative dotplots are shown for each assay; * P
    Figure Legend Snippet: Niclosamide-treated myeloma cells die with markers of apoptosis and autophagy. H929, JJN3 and U226 were treated with different concentrations of niclosamide (Nic) for 48 h. Cells were analysed by flow cytometry for ( a ) annexin V/propidium iodide positivity ( b ) and caspase activity using Caspatag assay according to manufacturer's instructions. Data shown are the mean of three experiments±s.e.m. Representative dotplots are shown for each assay; * P

    Techniques Used: Flow Cytometry, Cytometry, Activity Assay

    13) Product Images from "MULTIVALENT PROTEOGLYCAN MODULATION OF FGF MITOGENIC RESPONSES IN PERIVASCULAR CELLS"

    Article Title: MULTIVALENT PROTEOGLYCAN MODULATION OF FGF MITOGENIC RESPONSES IN PERIVASCULAR CELLS

    Journal: Angiogenesis

    doi: 10.1007/s10456-012-9316-7

    (a) Flow cytometric FRET analyses of NG2-FGFR1/FGFR3 ligand-independent complex formation on the cell surface using pair-wise combinations of fluorescently-tagged anti-NG2 and anti-FGFR1 or anti-FGFR3 antibodies. Interaction between the individual subunits
    Figure Legend Snippet: (a) Flow cytometric FRET analyses of NG2-FGFR1/FGFR3 ligand-independent complex formation on the cell surface using pair-wise combinations of fluorescently-tagged anti-NG2 and anti-FGFR1 or anti-FGFR3 antibodies. Interaction between the individual subunits

    Techniques Used: Flow Cytometry

    Selectivity of the functional cooperation of NG2 with FGFR1 and FGFR3. FGF-2-induced mitogenic responses were compared in different fibroblastic cell lines selected on the basis of their diverse FGFR expression patterns ( a, b ) and
    Figure Legend Snippet: Selectivity of the functional cooperation of NG2 with FGFR1 and FGFR3. FGF-2-induced mitogenic responses were compared in different fibroblastic cell lines selected on the basis of their diverse FGFR expression patterns ( a, b ) and

    Techniques Used: Functional Assay, Expressing

    (a) FGF-2 responses in cells expressing altered FGFR1/FGFR3-NG2 ratios. Cells enriched by immunoselection for their constitutive NG2 expression (NG2 + ) and cells transduced to overexpress the rodent full-length NG2 (NG2 ++ ) were exposed to optimal stimulatory
    Figure Legend Snippet: (a) FGF-2 responses in cells expressing altered FGFR1/FGFR3-NG2 ratios. Cells enriched by immunoselection for their constitutive NG2 expression (NG2 + ) and cells transduced to overexpress the rodent full-length NG2 (NG2 ++ ) were exposed to optimal stimulatory

    Techniques Used: Expressing

    14) Product Images from "Loss of microRNA-27b contributes to breast cancer stem cell generation by activating ENPP1"

    Article Title: Loss of microRNA-27b contributes to breast cancer stem cell generation by activating ENPP1

    Journal: Nature Communications

    doi: 10.1038/ncomms8318

    MiR-27b regulates the resistance of breast cancer cells to docetaxel. ( a ) Overview of the method used to establish miR-27b knockdown MCF7-luc (MCF7-luc anti-miR-27b) cells. ( b , c ) Dose–response curves of MCF7-luc anti-NC, MCF7-luc anti-miR-27b and MCF7-luc miR-27b o.e. cells treated with docetaxel. Cell viability was normalized to that of the corresponding cells treated with dimethylsulphoxide (DMSO). The red dashed line indicates the IC 50 value. Data are represented as the mean±s.d. of n =3 replicates. ( d ) Morphologies of the MCF7-luc anti-NC, MCF7-luc miR-27b o.e. and MCF7-luc anti-miR-27b cells. Scale bar, 100 μm. ( e ) Flow cytometric analyses of the SP fraction of MCF7-luc derivatives in the presence and absence of Ko143. ( f ) Quantification of the SP fraction of MCF7-luc derivatives. The SP fraction was determined as the difference between the level of Hoechst 33342 staining in the presence and absence of Ko143. Data are represented as the mean±s.d. of n =3 replicates. Statistical significance was determined by Student's t -test.
    Figure Legend Snippet: MiR-27b regulates the resistance of breast cancer cells to docetaxel. ( a ) Overview of the method used to establish miR-27b knockdown MCF7-luc (MCF7-luc anti-miR-27b) cells. ( b , c ) Dose–response curves of MCF7-luc anti-NC, MCF7-luc anti-miR-27b and MCF7-luc miR-27b o.e. cells treated with docetaxel. Cell viability was normalized to that of the corresponding cells treated with dimethylsulphoxide (DMSO). The red dashed line indicates the IC 50 value. Data are represented as the mean±s.d. of n =3 replicates. ( d ) Morphologies of the MCF7-luc anti-NC, MCF7-luc miR-27b o.e. and MCF7-luc anti-miR-27b cells. Scale bar, 100 μm. ( e ) Flow cytometric analyses of the SP fraction of MCF7-luc derivatives in the presence and absence of Ko143. ( f ) Quantification of the SP fraction of MCF7-luc derivatives. The SP fraction was determined as the difference between the level of Hoechst 33342 staining in the presence and absence of Ko143. Data are represented as the mean±s.d. of n =3 replicates. Statistical significance was determined by Student's t -test.

    Techniques Used: Flow Cytometry, Staining

    Functional analysis of ENPP1 in MCF7-luc cells. ( a ) Flow cytometric analysis of the SP fractions of MCF7-luc cells overexpressing ENPP1-MF or GFP as a control, in the presence and absence of Ko143. ( b ) Quantification of the SP fractions shown in a , determined as the difference between the level of Hoechst 33342 staining in the presence and absence of Ko143. Data are represented as the mean±s.d. of n =3 replicates. ( c ) Flow cytometric analysis showing the cell surface localization of ABCG2 in the indicated 293T co-transfectants. ( d ) Flow cytometric analyses of the cell surface localization of ABCG2 in MCF7-luc anti-miR-27b cells transfected with a control (shNC) or ENPP1-specific (shENPP1) shRNA. ( e ) Dose–response curves of docetaxel-treated MCF7-luc anti-miR-27b-DR cells transfected with shNC or shENPP1. Cell viability was normalized to that of the corresponding cells treated with dimethylsulphoxide (DMSO). The red dashed line indicates the IC 50 value. Data are represented as the mean±s.d. of n =3 replicates. ( f ) Proximity ligation assay using MCF7-luc anti-NC or MCF7-luc anti-miR-27b cells transiently expressing ABCG2-HA. Scale bar, 50 μm. ( g ) In vitro binding assay using C-terminally Flag-tagged GFP or C-terminally Myc- and Flag-tagged ENPP1 purified from 293T cells and C-terminally HA-tagged ABCG2 purified from Sf21 insect cell extracts.
    Figure Legend Snippet: Functional analysis of ENPP1 in MCF7-luc cells. ( a ) Flow cytometric analysis of the SP fractions of MCF7-luc cells overexpressing ENPP1-MF or GFP as a control, in the presence and absence of Ko143. ( b ) Quantification of the SP fractions shown in a , determined as the difference between the level of Hoechst 33342 staining in the presence and absence of Ko143. Data are represented as the mean±s.d. of n =3 replicates. ( c ) Flow cytometric analysis showing the cell surface localization of ABCG2 in the indicated 293T co-transfectants. ( d ) Flow cytometric analyses of the cell surface localization of ABCG2 in MCF7-luc anti-miR-27b cells transfected with a control (shNC) or ENPP1-specific (shENPP1) shRNA. ( e ) Dose–response curves of docetaxel-treated MCF7-luc anti-miR-27b-DR cells transfected with shNC or shENPP1. Cell viability was normalized to that of the corresponding cells treated with dimethylsulphoxide (DMSO). The red dashed line indicates the IC 50 value. Data are represented as the mean±s.d. of n =3 replicates. ( f ) Proximity ligation assay using MCF7-luc anti-NC or MCF7-luc anti-miR-27b cells transiently expressing ABCG2-HA. Scale bar, 50 μm. ( g ) In vitro binding assay using C-terminally Flag-tagged GFP or C-terminally Myc- and Flag-tagged ENPP1 purified from 293T cells and C-terminally HA-tagged ABCG2 purified from Sf21 insect cell extracts.

    Techniques Used: Functional Assay, Flow Cytometry, Staining, Transfection, shRNA, Proximity Ligation Assay, Expressing, In Vitro, Binding Assay, Purification

    15) Product Images from "Cystatin SN Affects Cell Proliferation by Regulating the ERα/PI3K/AKT/ERα Loopback Pathway in Breast Cancer"

    Article Title: Cystatin SN Affects Cell Proliferation by Regulating the ERα/PI3K/AKT/ERα Loopback Pathway in Breast Cancer

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S234328

    CST1 affected the G1 to S phase transition in ER+ breast cancer cells. Flow cytometry analysis was conducted to assess the effects of CST1 on the cell cycle. Single-cell suspensions were prepared. We centrifuged cells at 1200 rpm for 5 mins, repeated twice, then fixed them in 75% ethanol (−20°C) for 2 hrs. The cells were then stained with propidium iodide in the dark and 100 μg/mL of DNase-free RNase A, and analyzed on a BD FACSCanto™ II (BD Biosciences, USA) 30 mins later. ( A ) The results showed that CST1 knockdown inhibited the G1 to S phase transition in MCF7 breast cancer cells. ( B ) Cell cycle analyses showed that CST1 knockdown inhibited the G1 to S phase transition in T47D breast cancer cells. ( C ) Cell cycle analyses showed that CST1 knockdown inhibited the G1 to S phase transition in BT474 breast cancer cells. Differences were considered statistically significant at **p
    Figure Legend Snippet: CST1 affected the G1 to S phase transition in ER+ breast cancer cells. Flow cytometry analysis was conducted to assess the effects of CST1 on the cell cycle. Single-cell suspensions were prepared. We centrifuged cells at 1200 rpm for 5 mins, repeated twice, then fixed them in 75% ethanol (−20°C) for 2 hrs. The cells were then stained with propidium iodide in the dark and 100 μg/mL of DNase-free RNase A, and analyzed on a BD FACSCanto™ II (BD Biosciences, USA) 30 mins later. ( A ) The results showed that CST1 knockdown inhibited the G1 to S phase transition in MCF7 breast cancer cells. ( B ) Cell cycle analyses showed that CST1 knockdown inhibited the G1 to S phase transition in T47D breast cancer cells. ( C ) Cell cycle analyses showed that CST1 knockdown inhibited the G1 to S phase transition in BT474 breast cancer cells. Differences were considered statistically significant at **p

    Techniques Used: Sublimation, Flow Cytometry, Cytometry, Staining

    16) Product Images from "Measurement of Separase Proteolytic Activity in Single Living Cells by a Fluorogenic Flow Cytometry Assay"

    Article Title: Measurement of Separase Proteolytic Activity in Single Living Cells by a Fluorogenic Flow Cytometry Assay

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0133769

    Separase activity during mitotic progression in U937 cells. (A) U937 cells arrested in G2/M by nocodazole were monitored by flow cytometry at 0, 90, 180, and 270 min after release from the nocodazole block. (B) Influence of FCS supplementation on Separase activity and mitotic progression. After serum starvation cells were incubated with increasing percentages (10%, 50%, 100%) of FCS for 90 min before flow cytometric analysis. Potential extracellular FCS-born Separase-unrelated proteolytic activity was inhibited by a cocktail of peptidase inhibitors (PI mix ) as measured by cell lysate-based assay. Separase proteolytic activity was monitored as released Rh110 fluorescence. Corresponding Western blot immunostaining experiments (below) illustrate the expression levels of main Separase regulatory proteins (i.e. CyclinB1, Securin) that reversely correlate with Separase activity. Actin served as loading control. Cell cycle profiles were analyzed by flow cytometry after propidium iodide staining. The percentage of cells in G2/M as a measure of mitotic progression is depicted by the square dotted line. All assays were performed at least in triplicates. (C) Comparative analysis of DNA content in Rh110-negative (upper panel) and positive (lower panel) U937 cells after Hoechst 33342 staining simultaneously performed within the Separase assay. Abbreviations: FCS, fetal calf serum; fluo/cell, fluorescence per cell; 1n, haploid cells; 2n, diploid cells.
    Figure Legend Snippet: Separase activity during mitotic progression in U937 cells. (A) U937 cells arrested in G2/M by nocodazole were monitored by flow cytometry at 0, 90, 180, and 270 min after release from the nocodazole block. (B) Influence of FCS supplementation on Separase activity and mitotic progression. After serum starvation cells were incubated with increasing percentages (10%, 50%, 100%) of FCS for 90 min before flow cytometric analysis. Potential extracellular FCS-born Separase-unrelated proteolytic activity was inhibited by a cocktail of peptidase inhibitors (PI mix ) as measured by cell lysate-based assay. Separase proteolytic activity was monitored as released Rh110 fluorescence. Corresponding Western blot immunostaining experiments (below) illustrate the expression levels of main Separase regulatory proteins (i.e. CyclinB1, Securin) that reversely correlate with Separase activity. Actin served as loading control. Cell cycle profiles were analyzed by flow cytometry after propidium iodide staining. The percentage of cells in G2/M as a measure of mitotic progression is depicted by the square dotted line. All assays were performed at least in triplicates. (C) Comparative analysis of DNA content in Rh110-negative (upper panel) and positive (lower panel) U937 cells after Hoechst 33342 staining simultaneously performed within the Separase assay. Abbreviations: FCS, fetal calf serum; fluo/cell, fluorescence per cell; 1n, haploid cells; 2n, diploid cells.

    Techniques Used: Activity Assay, Flow Cytometry, Cytometry, Blocking Assay, Incubation, Fluorescence, Western Blot, Immunostaining, Expressing, Staining

    Comparability of flow cytometric- (A) and lysate-based (B) Separase activity assays. Cell lines with high (MEG01) and low (BV173) levels of Separase activity and U937 cells at distinct time points (90, 180, 270 min) after release from the nocodazole block were comparatively analyzed in parallel. For (A) the mean Rh110 fluorescence was calculated for all Separase positive cells. Data derived from the lysate-based assay (B) were calculated with respect to levels of Actin that served as internal standard (lysate loading control). Abbreviations: RFU, relative fluorescence units.
    Figure Legend Snippet: Comparability of flow cytometric- (A) and lysate-based (B) Separase activity assays. Cell lines with high (MEG01) and low (BV173) levels of Separase activity and U937 cells at distinct time points (90, 180, 270 min) after release from the nocodazole block were comparatively analyzed in parallel. For (A) the mean Rh110 fluorescence was calculated for all Separase positive cells. Data derived from the lysate-based assay (B) were calculated with respect to levels of Actin that served as internal standard (lysate loading control). Abbreviations: RFU, relative fluorescence units.

    Techniques Used: Flow Cytometry, Activity Assay, Blocking Assay, Fluorescence, Derivative Assay

    17) Product Images from "Necdin, a p53-Target Gene, Is an Inhibitor of p53-Mediated Growth Arrest"

    Article Title: Necdin, a p53-Target Gene, Is an Inhibitor of p53-Mediated Growth Arrest

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031916

    Necdin confers resistance to p53-dependent growth arrest. (A–B) NIHLT cells depleted in Necdin by shRNAs and exposed to nutlin-3 showed an increase in growth arrest (A) measured by DNA content analysis by flow cytometry (as described in figure 4A ) or (B) assessed by Wst-1 colorimetric assay. Results for Wst-1 represent normalized data according to the portion of arrested cells (O.D. untreated – O.D. treated) relative to arrested control NIH after 48 h of exposure to nutlin-3. (C–D) NIH and NIHLT cells overexpressing Necdin showed growth arrest resistance upon nutlin-3 treatment. (C) FACS analysis or (D) Wst-1 colorimetric assay (* P
    Figure Legend Snippet: Necdin confers resistance to p53-dependent growth arrest. (A–B) NIHLT cells depleted in Necdin by shRNAs and exposed to nutlin-3 showed an increase in growth arrest (A) measured by DNA content analysis by flow cytometry (as described in figure 4A ) or (B) assessed by Wst-1 colorimetric assay. Results for Wst-1 represent normalized data according to the portion of arrested cells (O.D. untreated – O.D. treated) relative to arrested control NIH after 48 h of exposure to nutlin-3. (C–D) NIH and NIHLT cells overexpressing Necdin showed growth arrest resistance upon nutlin-3 treatment. (C) FACS analysis or (D) Wst-1 colorimetric assay (* P

    Techniques Used: Flow Cytometry, Cytometry, Colorimetric Assay, FACS

    Nutlin-3 induces a p53-dependent growth arrest in NIH3T3 cells that is bypassed by PyLT expression. (A–B) Flow cytometry analysis of NIH or NIHLT populations treated with nutlin-3 (5 µM) demonstrate that nutlin-3 induces a growth arrest in NIH cells, but not in NIHLT cells. Results presented are from one representative experiment (A) Cell cycle arrest was represented by the variation of ratio of arrested cells (G1+G2 phases) over proliferating cells (S phase) in treated cells versus untreated controls. (B) No variation of the percentage of cells in Sub-G1 phase, representing cell death, was observed after nutlin-3 treatment. (C–D) The use of a p53 inhibitor peptide (GSE22) validates the p53-dependence of the growth arrest induced by Nuclin-3. (C) High efficiency of infection and functionality of the GSE22 peptide were demonstrated by the accumulation of non-functional p53 in the nucleus by immunocytochemistry detecting p53 in NIH transduced with GSE22 or control vector. The stabilization of non-functional p53 was also seen in Western blots of the corresponding infected cells. (D) FACS analysis on NIH transduced with GSE22 or vector with nutlin-3 treatment (* P
    Figure Legend Snippet: Nutlin-3 induces a p53-dependent growth arrest in NIH3T3 cells that is bypassed by PyLT expression. (A–B) Flow cytometry analysis of NIH or NIHLT populations treated with nutlin-3 (5 µM) demonstrate that nutlin-3 induces a growth arrest in NIH cells, but not in NIHLT cells. Results presented are from one representative experiment (A) Cell cycle arrest was represented by the variation of ratio of arrested cells (G1+G2 phases) over proliferating cells (S phase) in treated cells versus untreated controls. (B) No variation of the percentage of cells in Sub-G1 phase, representing cell death, was observed after nutlin-3 treatment. (C–D) The use of a p53 inhibitor peptide (GSE22) validates the p53-dependence of the growth arrest induced by Nuclin-3. (C) High efficiency of infection and functionality of the GSE22 peptide were demonstrated by the accumulation of non-functional p53 in the nucleus by immunocytochemistry detecting p53 in NIH transduced with GSE22 or control vector. The stabilization of non-functional p53 was also seen in Western blots of the corresponding infected cells. (D) FACS analysis on NIH transduced with GSE22 or vector with nutlin-3 treatment (* P

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Infection, Functional Assay, Immunocytochemistry, Transduction, Plasmid Preparation, Western Blot, FACS

    PyLT-induced Necdin expression is p53-independent. Necdin is induced following activation of p53. (A) Dose response treatment with nutlin-3 increased Necdin protein level in NIH (left) and NIHLT (right). (B) Genotoxic stress induced by Actinomycin D and Camptothecin also stimulated Necdin protein expression. (C) In NIHLT cells, Necdin expression is not dependent on p53 activity as assessed by p53 inhibition. Mean of relative expression of Necdin, p21, p53 and GAPDH in NIHLT cells with or without the p53 inhibitor GSE22. Expression was measured by Q-PCR in three independent samples from each group. Expression is relative to actin (** P
    Figure Legend Snippet: PyLT-induced Necdin expression is p53-independent. Necdin is induced following activation of p53. (A) Dose response treatment with nutlin-3 increased Necdin protein level in NIH (left) and NIHLT (right). (B) Genotoxic stress induced by Actinomycin D and Camptothecin also stimulated Necdin protein expression. (C) In NIHLT cells, Necdin expression is not dependent on p53 activity as assessed by p53 inhibition. Mean of relative expression of Necdin, p21, p53 and GAPDH in NIHLT cells with or without the p53 inhibitor GSE22. Expression was measured by Q-PCR in three independent samples from each group. Expression is relative to actin (** P

    Techniques Used: Expressing, Activation Assay, Activity Assay, Inhibition, Polymerase Chain Reaction

    18) Product Images from "The Effect of Lentivirus-Mediated PSPN Genetic Engineering Bone Marrow Mesenchymal Stem Cells on Parkinson’s Disease Rat Model"

    Article Title: The Effect of Lentivirus-Mediated PSPN Genetic Engineering Bone Marrow Mesenchymal Stem Cells on Parkinson’s Disease Rat Model

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0105118

    Attenuation of 6-OHDA-induced cell death by Lv-PSPN-MSC in MES23.5 cells. (A) Effect of different concentrations of 6-OHDA (1–200 µM) on metabolic rate. (B) The apoptotic rates of the MES23.5 cells treated in (C, D, E, F) were assessed at the indicated time periods by annexin V/7-AAD staining. (C) MES23.5. (D) MES23.5+6-OHDA. (E) 6-OHDA+MES23.5+MSCs. (F) 6-OHDA+MES23.5+ Lv-PSPN-MSCs. Cells were analyzed for annexin V/7-AAD staining by flow cytometry. Events in each of the four quadrants are as follows: Lower left, viable cells; lower right, cells in the early-to mid-stages of apoptosis; upper right, cells in the late-stages of apoptosis; upper left, mostly nuclear debris.* P
    Figure Legend Snippet: Attenuation of 6-OHDA-induced cell death by Lv-PSPN-MSC in MES23.5 cells. (A) Effect of different concentrations of 6-OHDA (1–200 µM) on metabolic rate. (B) The apoptotic rates of the MES23.5 cells treated in (C, D, E, F) were assessed at the indicated time periods by annexin V/7-AAD staining. (C) MES23.5. (D) MES23.5+6-OHDA. (E) 6-OHDA+MES23.5+MSCs. (F) 6-OHDA+MES23.5+ Lv-PSPN-MSCs. Cells were analyzed for annexin V/7-AAD staining by flow cytometry. Events in each of the four quadrants are as follows: Lower left, viable cells; lower right, cells in the early-to mid-stages of apoptosis; upper right, cells in the late-stages of apoptosis; upper left, mostly nuclear debris.* P

    Techniques Used: Staining, Flow Cytometry, Cytometry

    19) Product Images from "Ectopic Pregnancy-Derived Human Trophoblastic Stem Cells Regenerate Dopaminergic Nigrostriatal Pathway to Treat Parkinsonian Rats"

    Article Title: Ectopic Pregnancy-Derived Human Trophoblastic Stem Cells Regenerate Dopaminergic Nigrostriatal Pathway to Treat Parkinsonian Rats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0052491

    Regulation of Pluripotency Transcription Factors by LIF. ( A ) Expressions of Nanog, Cdx2, Sox2 and Oct4 mRNAs after treating different concentrations of LIF ( B ) By flow cytomery, withdrawal of LIF overexpressed Nanog and ( C ) enhanced expression of Nanog and Cdx2 but suppressed Oct4 and Sox2. ( D ) Physiological gradient of LIF levels from ampulla toward isthmus with an increased Nanog/Cdx2 ratio, but a decreased Oct4/Cdx2 ratio in a dose-dependent manner. ( E ) A reciprocal relationship between Nanog and Cdx2 evidenced by pretreatment with siRNAs (10 −8 M, Sigma) and ( F ) with shRNAs (10 −8 M, Sigma), respectively.
    Figure Legend Snippet: Regulation of Pluripotency Transcription Factors by LIF. ( A ) Expressions of Nanog, Cdx2, Sox2 and Oct4 mRNAs after treating different concentrations of LIF ( B ) By flow cytomery, withdrawal of LIF overexpressed Nanog and ( C ) enhanced expression of Nanog and Cdx2 but suppressed Oct4 and Sox2. ( D ) Physiological gradient of LIF levels from ampulla toward isthmus with an increased Nanog/Cdx2 ratio, but a decreased Oct4/Cdx2 ratio in a dose-dependent manner. ( E ) A reciprocal relationship between Nanog and Cdx2 evidenced by pretreatment with siRNAs (10 −8 M, Sigma) and ( F ) with shRNAs (10 −8 M, Sigma), respectively.

    Techniques Used: Flow Cytometry, Expressing

    20) Product Images from "Transgenic Fluorescent Plasmodium cynomolgi Liver Stages Enable Live Imaging and Purification of Malaria Hypnozoite-Forms"

    Article Title: Transgenic Fluorescent Plasmodium cynomolgi Liver Stages Enable Live Imaging and Purification of Malaria Hypnozoite-Forms

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0054888

    Flow cytometry and cell sorting of P. cynomolgi liver stage parasites, including hypnozoite-forms. (A) Liver stage parasites used for flowcytometry as detected by anti-HSP70 antibodies 3 days and (B) 6 days post hepatocyte infection. White bars correspond to 50 µm. Note that day 3 cultures contain uniform small parasites while day 6 cultures contain both small and large liver stages (arrows). Flow cytometric plots of PcyC-PAC-GFP hsp70 -mCherry ef1α (PcyC-PAC) P. cynomolgi liver stage parasites show a single GFP positive population compared to wild type parasites 3 days post hepatocyte infection (A, Gate 1) and two GFP positive populations 6 days post hepatocyte infection (B, Gates 2 and 3). The y-axis represents the PE-Texas Red Channel (for detection of autofluorescence), while the x-axis represents the GFP signal. (C) Post-sorting images of PcyC-PAC-GFP hsp70 -mCherry ef1α P. cynomolgi liver stage parasites ‘GFPlow’ (Gate 2) and ‘GFPhigh’ (Gate 3) parasites sorted at day 6 post hepatocyte infection. The upper panel shows a GFP/Brightfield overlay while the lower panel shows mCherry/Brightfield overlay. The panels below show close-ups of the sorted parasites revealing the size differences between the ‘GFPlow’ and ‘GFPhigh’ populations. White bars correspond to 50 µm.
    Figure Legend Snippet: Flow cytometry and cell sorting of P. cynomolgi liver stage parasites, including hypnozoite-forms. (A) Liver stage parasites used for flowcytometry as detected by anti-HSP70 antibodies 3 days and (B) 6 days post hepatocyte infection. White bars correspond to 50 µm. Note that day 3 cultures contain uniform small parasites while day 6 cultures contain both small and large liver stages (arrows). Flow cytometric plots of PcyC-PAC-GFP hsp70 -mCherry ef1α (PcyC-PAC) P. cynomolgi liver stage parasites show a single GFP positive population compared to wild type parasites 3 days post hepatocyte infection (A, Gate 1) and two GFP positive populations 6 days post hepatocyte infection (B, Gates 2 and 3). The y-axis represents the PE-Texas Red Channel (for detection of autofluorescence), while the x-axis represents the GFP signal. (C) Post-sorting images of PcyC-PAC-GFP hsp70 -mCherry ef1α P. cynomolgi liver stage parasites ‘GFPlow’ (Gate 2) and ‘GFPhigh’ (Gate 3) parasites sorted at day 6 post hepatocyte infection. The upper panel shows a GFP/Brightfield overlay while the lower panel shows mCherry/Brightfield overlay. The panels below show close-ups of the sorted parasites revealing the size differences between the ‘GFPlow’ and ‘GFPhigh’ populations. White bars correspond to 50 µm.

    Techniques Used: Flow Cytometry, Cytometry, FACS, Infection

    21) Product Images from "Multiparametric Analysis of Circulating Exosomes and Other Small Extracellular Vesicles by Advanced Imaging Flow Cytometry"

    Article Title: Multiparametric Analysis of Circulating Exosomes and Other Small Extracellular Vesicles by Advanced Imaging Flow Cytometry

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01583

    Submicron particle detection and multiparametric characterization of circulating “small EV” (sEV) by ImageStream x (ISx). Gating sEV populations is achieved by assessment of the Scatter intensity of particles of known size, and through confirmation by single-event visual interrogation. (A) Fluorescently labeled polystyrene beads and liposomes which have a refractive index closer to that of EV are used and contrasted to CFDA-SE labeled circulating human extracellular vesicles (EVs) derived by ultracentrifugation. CFDA-SE-mediated labeling of intact vesicles is demonstrated by detergent lysis (0.1% Triton™ X-100) of UC-derived EVs prior to labeling. (B) The acquisition of appropriate control samples is an important step prior to running experimental samples if the common pitfalls of flow-cytometric profiling of EVs are to be avoided. Representative ISx dot-plots of buffer alone (filtered phosphate-buffered saline), unstained EVs, and buffer plus reagents without EVs are shown. (C) Representative dot-plot of CFDA-SE Vs. Scatter intensity to demonstrate the heterogeneity of acquired events and the principle of visual interrogation in gate-setting. The difference in properties of the two CFDA-SE-positive populations (G1 and G2) is clear by visual interrogation, showing G2 to be predominantly composed of cellular debris or particle aggregates as opposed to the uniform particles in G1—the sEV gate. (D) Multiparametric phenotyping of these gated sEV (G1) is demonstrated by fluorescent labeling with EV markers, the tetraspanins CD9, CD63, and CD81 combined (PE, Ch03) and HLA-DR (PECy7, Ch06). Fluorescence minus one and isotype controls are used to set gating. A buffer + reagent + antibody control should be performed and is demonstrated here.
    Figure Legend Snippet: Submicron particle detection and multiparametric characterization of circulating “small EV” (sEV) by ImageStream x (ISx). Gating sEV populations is achieved by assessment of the Scatter intensity of particles of known size, and through confirmation by single-event visual interrogation. (A) Fluorescently labeled polystyrene beads and liposomes which have a refractive index closer to that of EV are used and contrasted to CFDA-SE labeled circulating human extracellular vesicles (EVs) derived by ultracentrifugation. CFDA-SE-mediated labeling of intact vesicles is demonstrated by detergent lysis (0.1% Triton™ X-100) of UC-derived EVs prior to labeling. (B) The acquisition of appropriate control samples is an important step prior to running experimental samples if the common pitfalls of flow-cytometric profiling of EVs are to be avoided. Representative ISx dot-plots of buffer alone (filtered phosphate-buffered saline), unstained EVs, and buffer plus reagents without EVs are shown. (C) Representative dot-plot of CFDA-SE Vs. Scatter intensity to demonstrate the heterogeneity of acquired events and the principle of visual interrogation in gate-setting. The difference in properties of the two CFDA-SE-positive populations (G1 and G2) is clear by visual interrogation, showing G2 to be predominantly composed of cellular debris or particle aggregates as opposed to the uniform particles in G1—the sEV gate. (D) Multiparametric phenotyping of these gated sEV (G1) is demonstrated by fluorescent labeling with EV markers, the tetraspanins CD9, CD63, and CD81 combined (PE, Ch03) and HLA-DR (PECy7, Ch06). Fluorescence minus one and isotype controls are used to set gating. A buffer + reagent + antibody control should be performed and is demonstrated here.

    Techniques Used: Labeling, Derivative Assay, Lysis, Flow Cytometry, Fluorescence

    22) Product Images from "Itk is required for Th9 differentiation via TCR-mediated induction of IL-2 and IRF4"

    Article Title: Itk is required for Th9 differentiation via TCR-mediated induction of IL-2 and IRF4

    Journal: Nature Communications

    doi: 10.1038/ncomms10857

    IL-9 expression correlates with strength of TCR signals. ( a ) Sorted naive WT CD4 + T cells were differentiated under Th9 conditions plus TL1A with 0.01, 0.1 or 1 μg ml −1 of anti-CD3, then restimulated with PMA and Ionomycin and IL-9 production analysed by intracellular staining. ( b ) Itk-deficient CD4 + T cells were transduced with retroviruses expressing constitutively active NFATc1 (ca-NFATc1), or a control (MIGR), differentiated under Th9 plus TL1A or Th17 conditions, and cytokine production determined by intracellular staining after PMA and ionomycin restimulation. Results in a and b are from one representative of three independent experiments.
    Figure Legend Snippet: IL-9 expression correlates with strength of TCR signals. ( a ) Sorted naive WT CD4 + T cells were differentiated under Th9 conditions plus TL1A with 0.01, 0.1 or 1 μg ml −1 of anti-CD3, then restimulated with PMA and Ionomycin and IL-9 production analysed by intracellular staining. ( b ) Itk-deficient CD4 + T cells were transduced with retroviruses expressing constitutively active NFATc1 (ca-NFATc1), or a control (MIGR), differentiated under Th9 plus TL1A or Th17 conditions, and cytokine production determined by intracellular staining after PMA and ionomycin restimulation. Results in a and b are from one representative of three independent experiments.

    Techniques Used: Expressing, Staining, Transduction

    23) Product Images from "Requirement of Cognate CD4+ T-Cell Recognition for the Regulation of Allospecific CTL by Human CD4+CD127−CD25+FOXP3+ Cells Generated in MLR"

    Article Title: Requirement of Cognate CD4+ T-Cell Recognition for the Regulation of Allospecific CTL by Human CD4+CD127−CD25+FOXP3+ Cells Generated in MLR

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0022450

    MLR-Tregs suppress purified CD8 allospecific proliferation. 5×10 4 CFSE labeled purified CD8 cells were cultured with 1×10 5 irradiated PBMC from the original stimulator (used in generating MLR-Tregs) together with indicated numbers of autologous MLR-Treg modulators or autologous irradiated controls (Ax), either the presence (top row) or absence (bottom row) of IL-2 (10 U/ml). After 7 days in culture flow cytometric assays were performed and the percentage of CFSE-diluted cells was estimated after gating on viable lymphocytes followed by CD8 + cells. It was observed that the irradiated stimulators and Ax died off by day 7 (not shown); even the few that remained were gated out on CFSE vs. CD8 density-plot during the analysis. Note the increasing percentages of (CFSE diluted) proliferating cells with decreasing concentrations of the MLR-Treg modulators. In the left column are depicted the results of negative control cultures (CD8 responders) in the absence of allogeneic stimulators or modulators. The figure is representative of 3 such experiments. (** = p
    Figure Legend Snippet: MLR-Tregs suppress purified CD8 allospecific proliferation. 5×10 4 CFSE labeled purified CD8 cells were cultured with 1×10 5 irradiated PBMC from the original stimulator (used in generating MLR-Tregs) together with indicated numbers of autologous MLR-Treg modulators or autologous irradiated controls (Ax), either the presence (top row) or absence (bottom row) of IL-2 (10 U/ml). After 7 days in culture flow cytometric assays were performed and the percentage of CFSE-diluted cells was estimated after gating on viable lymphocytes followed by CD8 + cells. It was observed that the irradiated stimulators and Ax died off by day 7 (not shown); even the few that remained were gated out on CFSE vs. CD8 density-plot during the analysis. Note the increasing percentages of (CFSE diluted) proliferating cells with decreasing concentrations of the MLR-Treg modulators. In the left column are depicted the results of negative control cultures (CD8 responders) in the absence of allogeneic stimulators or modulators. The figure is representative of 3 such experiments. (** = p

    Techniques Used: Purification, Labeling, Cell Culture, Irradiation, Flow Cytometry, Negative Control

    Regulatory effects of MLR-Tregs in micro-CML assays of responding purified CD8 + cells . Micro-CML inhibition assays were performed as described in Figure 4 , except that 5×10 4 purified CD8 + cells rather than whole PBMC (1×10 5 ) were used as responders. The data are depicted as: (A) Percent specific lysis: against the specific stimulator used both in MLR-Treg generation and the micro-CML readout. Similar to the data in Figure 4A , the lysis of target cells was decreasingly inhibited by decreasing concentrations of MLR-Tregs (** = p
    Figure Legend Snippet: Regulatory effects of MLR-Tregs in micro-CML assays of responding purified CD8 + cells . Micro-CML inhibition assays were performed as described in Figure 4 , except that 5×10 4 purified CD8 + cells rather than whole PBMC (1×10 5 ) were used as responders. The data are depicted as: (A) Percent specific lysis: against the specific stimulator used both in MLR-Treg generation and the micro-CML readout. Similar to the data in Figure 4A , the lysis of target cells was decreasingly inhibited by decreasing concentrations of MLR-Tregs (** = p

    Techniques Used: Purification, Inhibition, Lysis

    24) Product Images from "Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing"

    Article Title: Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.101136398

    G47Δ precludes down-regulation of MHC class I expression in infected host cells. ( a ) Flow cytometric analyses of MHC class I expression in Detroit 551 human fibroblast cells 48 h after infection with HSV-1 (moi = 3). Whereas all HSVs with an intact α47 gene (wild-type strain F and G207) significantly down-regulated MHC class I expression, G47Δ completely precluded the down-regulation. ( b ) Time course of MHC class I down-regulation in Detroit 551 cells infected with HSV-1. For each virus, the peak value of MHC class I expression at 6, 24, or 48 h after infection, as analyzed by flow cytometry, was expressed as a percentage of the peak value of mock-infected cells (Control) at each time point. MHC class I down-regulation by G207 and R3616 occurred in a time-dependent fashion. Dissociation of MHC class I expression between α47 -deleted mutants (G47Δ and R47Δ) and α47 -intact viruses became apparent at 24–48 h after infection. ( c ) Flow cytometric analyses of MHC class I expression in human melanoma cell lines 24 h after infection with G207 and G47Δ. G47Δ caused a partial preclusion of MHC class I down-regulation in melanomas 1102 and 938, resulting in greater MHC class I expression than G207.
    Figure Legend Snippet: G47Δ precludes down-regulation of MHC class I expression in infected host cells. ( a ) Flow cytometric analyses of MHC class I expression in Detroit 551 human fibroblast cells 48 h after infection with HSV-1 (moi = 3). Whereas all HSVs with an intact α47 gene (wild-type strain F and G207) significantly down-regulated MHC class I expression, G47Δ completely precluded the down-regulation. ( b ) Time course of MHC class I down-regulation in Detroit 551 cells infected with HSV-1. For each virus, the peak value of MHC class I expression at 6, 24, or 48 h after infection, as analyzed by flow cytometry, was expressed as a percentage of the peak value of mock-infected cells (Control) at each time point. MHC class I down-regulation by G207 and R3616 occurred in a time-dependent fashion. Dissociation of MHC class I expression between α47 -deleted mutants (G47Δ and R47Δ) and α47 -intact viruses became apparent at 24–48 h after infection. ( c ) Flow cytometric analyses of MHC class I expression in human melanoma cell lines 24 h after infection with G207 and G47Δ. G47Δ caused a partial preclusion of MHC class I down-regulation in melanomas 1102 and 938, resulting in greater MHC class I expression than G207.

    Techniques Used: Expressing, Infection, Flow Cytometry, Cytometry

    25) Product Images from "Tumor cell–intrinsic EPHA2 suppresses antitumor immunity by regulating PTGS2 (COX-2)"

    Article Title: Tumor cell–intrinsic EPHA2 suppresses antitumor immunity by regulating PTGS2 (COX-2)

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI127755

    Tumor cell–intrinsic Ptgs2 promotes T cell–low TME and resistance to immunotherapy in PDA. ( A ) Relative expression of Ptgs2 mRNA measured by qPCR in T cell–high PDA tumor cell clones transduced with either empty vector (EV) or pCDH-FHC vector carrying mouse Ptgs2 gene for overexpression ( Ptgs2 -OE). Representative data from n = 3 independent experiments. ( B and C ) Representative immunofluorescent staining images ( B ) and quantification ( C ) of CD3 + T cells in 2838c3-EV and 2838c3- Ptgs2 -OE subcutaneous tumors ( n = 5/group): CD3 (red), YFP (green), and DAPI (blue). Original magnification, ×20. ( D – F ) Flow cytometric analysis of subcutaneously implanted empty vector or Ptgs2 -OE tumors from indicated clones ( n = 5-7/group). ( G ) Parental and Ptgs2 -OE T cell–high tumor growth and mouse survival with or without GAFCP treatment. Tumor cells implanted subcutaneously into C57BL/6 mice ( n = 5–8/group). GAFCP treatment started 9 days after implantation at 3–5 mm tumor diameter. ( A , C – F ) Data are presented as boxplots, with horizontal lines and error bars indicating mean and range, respectively. Statistical analysis between 2 groups calculated using Student’s unpaired t test ( C – F ). The log-rank P values for Kaplan-Meier curves in G were calculated using GraphPad Prism. Statistical analysis of tumor growth curves performed using linear mixed-effects model with Tukey’s HSD post test using lme4 and the survival package in R ( G ). * P
    Figure Legend Snippet: Tumor cell–intrinsic Ptgs2 promotes T cell–low TME and resistance to immunotherapy in PDA. ( A ) Relative expression of Ptgs2 mRNA measured by qPCR in T cell–high PDA tumor cell clones transduced with either empty vector (EV) or pCDH-FHC vector carrying mouse Ptgs2 gene for overexpression ( Ptgs2 -OE). Representative data from n = 3 independent experiments. ( B and C ) Representative immunofluorescent staining images ( B ) and quantification ( C ) of CD3 + T cells in 2838c3-EV and 2838c3- Ptgs2 -OE subcutaneous tumors ( n = 5/group): CD3 (red), YFP (green), and DAPI (blue). Original magnification, ×20. ( D – F ) Flow cytometric analysis of subcutaneously implanted empty vector or Ptgs2 -OE tumors from indicated clones ( n = 5-7/group). ( G ) Parental and Ptgs2 -OE T cell–high tumor growth and mouse survival with or without GAFCP treatment. Tumor cells implanted subcutaneously into C57BL/6 mice ( n = 5–8/group). GAFCP treatment started 9 days after implantation at 3–5 mm tumor diameter. ( A , C – F ) Data are presented as boxplots, with horizontal lines and error bars indicating mean and range, respectively. Statistical analysis between 2 groups calculated using Student’s unpaired t test ( C – F ). The log-rank P values for Kaplan-Meier curves in G were calculated using GraphPad Prism. Statistical analysis of tumor growth curves performed using linear mixed-effects model with Tukey’s HSD post test using lme4 and the survival package in R ( G ). * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Clone Assay, Transduction, Plasmid Preparation, Over Expression, Staining, Flow Cytometry, Mouse Assay

    Tumor cell–intrinsic Epha2 regulates tumor T cell infiltration. ( A and B ) Representative images of immunofluorescent stainings for CD3 (red), YFP (green), and nuclei (DAPI, blue) ( A ) and quantification ( B ) of CD3 + T cells in Epha2 -WT and Epha2 -KO tumors from indicated clones ( n = 5–10/group). Original magnification, ×20. ( C – F ) Flow cytometric analysis of subcutaneously implanted Epha2 -WT and Epha2 -KO tumors from indicated clones ( n = 12–25/group). ( G and H ) Flow cytometric analysis of orthotopically implanted Epha2 -WT and Epha2 -KO tumors from indicated clones ( n = 5–10/group). ( B – H ) Data are presented as box plots, with horizontal lines and error bars indicating mean and range, respectively. Statistical differences between groups calculated using Students’ unpaired t test. * P
    Figure Legend Snippet: Tumor cell–intrinsic Epha2 regulates tumor T cell infiltration. ( A and B ) Representative images of immunofluorescent stainings for CD3 (red), YFP (green), and nuclei (DAPI, blue) ( A ) and quantification ( B ) of CD3 + T cells in Epha2 -WT and Epha2 -KO tumors from indicated clones ( n = 5–10/group). Original magnification, ×20. ( C – F ) Flow cytometric analysis of subcutaneously implanted Epha2 -WT and Epha2 -KO tumors from indicated clones ( n = 12–25/group). ( G and H ) Flow cytometric analysis of orthotopically implanted Epha2 -WT and Epha2 -KO tumors from indicated clones ( n = 5–10/group). ( B – H ) Data are presented as box plots, with horizontal lines and error bars indicating mean and range, respectively. Statistical differences between groups calculated using Students’ unpaired t test. * P

    Techniques Used: Clone Assay, Flow Cytometry

    PTGS2 is a potential tumor cell–intrinsic regulator of TME. ( A ) Heatmap of differentially expressed genes in Epha2 -WT and Epha2 -KO tumor cells from subcutaneously implanted tumors from indicated clones ( n = 3–8/group). ( B ) Overlap of the genes enriched in Epha2 -WT (vs. Epha2 -KO) and T cell–low (vs. T cell–high) tumors. ( C ) Seventeen coenriched genes identified ( B ) in Epha2 -WT and Epha2 -KO tumor cells from subcutaneously implanted mouse tumors (clone 6694c2). ( D ) Boxplot of Ptgs2 gene expression (tpm) in mouse tumor cells of subcutaneously implanted T cell–low and T cell–high tumors ( n = 8/group). ( E ) PTGS2 expression in human PDA samples in the upper and lower deciles of cytolytic index ( n = 14/group). PDA samples retrieved from the TCGA data portal. ( F ) Survival of patients in the upper and lower deciles of PTGS2 expression ( n = 17/group, TCGA PDA data set). ( G ) HOMER analysis of promoter regions of differentially expressed genes. Prediction of transcriptional regulators enriched in Epha2 -WT and Epha2- KO tumor cells ( n = 3–8/group). ( H ) TGF-β signaling GSEA in Epha2 -WT versus Epha2- KO tumor cells ( n = 3–8/group). ( I ) Relative expression of Ptgs2 mRNA in PDA tumor cell clones treated with either PBS or TGF-β for 72 hours. Data from n = 5 independent experiments. Color key represents the normalized Z score. ( J ) Relative expression of Ptgs2 mRNA in control (ctrl, empty vector transduced) and Smad3- and Smad4- KO 6419c5 PDA tumor cell lines. Data from n = 4 independent experiments. EV, empty vector. ( K and L ) Flow cytometric analysis of immune cell populations in control and Smad3 -KO and Smad4 -KO tumors ( n = 8–10/group). ( D , J – L ) Data are presented as boxplots, with horizontal lines and error bars indicating mean and range, respectively. ( E ) Data are presented as mean with error bars indicating SEM. Statistical differences determined by Students’ t test ( D and E ) or 1-way ANOVA with Tukey’s HSD post test ( J – L ). The log-rank P value was calculated using GraphPad Prism ( F ). * P
    Figure Legend Snippet: PTGS2 is a potential tumor cell–intrinsic regulator of TME. ( A ) Heatmap of differentially expressed genes in Epha2 -WT and Epha2 -KO tumor cells from subcutaneously implanted tumors from indicated clones ( n = 3–8/group). ( B ) Overlap of the genes enriched in Epha2 -WT (vs. Epha2 -KO) and T cell–low (vs. T cell–high) tumors. ( C ) Seventeen coenriched genes identified ( B ) in Epha2 -WT and Epha2 -KO tumor cells from subcutaneously implanted mouse tumors (clone 6694c2). ( D ) Boxplot of Ptgs2 gene expression (tpm) in mouse tumor cells of subcutaneously implanted T cell–low and T cell–high tumors ( n = 8/group). ( E ) PTGS2 expression in human PDA samples in the upper and lower deciles of cytolytic index ( n = 14/group). PDA samples retrieved from the TCGA data portal. ( F ) Survival of patients in the upper and lower deciles of PTGS2 expression ( n = 17/group, TCGA PDA data set). ( G ) HOMER analysis of promoter regions of differentially expressed genes. Prediction of transcriptional regulators enriched in Epha2 -WT and Epha2- KO tumor cells ( n = 3–8/group). ( H ) TGF-β signaling GSEA in Epha2 -WT versus Epha2- KO tumor cells ( n = 3–8/group). ( I ) Relative expression of Ptgs2 mRNA in PDA tumor cell clones treated with either PBS or TGF-β for 72 hours. Data from n = 5 independent experiments. Color key represents the normalized Z score. ( J ) Relative expression of Ptgs2 mRNA in control (ctrl, empty vector transduced) and Smad3- and Smad4- KO 6419c5 PDA tumor cell lines. Data from n = 4 independent experiments. EV, empty vector. ( K and L ) Flow cytometric analysis of immune cell populations in control and Smad3 -KO and Smad4 -KO tumors ( n = 8–10/group). ( D , J – L ) Data are presented as boxplots, with horizontal lines and error bars indicating mean and range, respectively. ( E ) Data are presented as mean with error bars indicating SEM. Statistical differences determined by Students’ t test ( D and E ) or 1-way ANOVA with Tukey’s HSD post test ( J – L ). The log-rank P value was calculated using GraphPad Prism ( F ). * P

    Techniques Used: Clone Assay, Expressing, Plasmid Preparation, Flow Cytometry

    Ptgs2 ablation in PDA tumor cells increases tumor antigenicity and decreases immunosuppressive potential of the tumor. ( A ) Individual (left) and average (right) weights of subcutaneously implanted control and Ptgs2 -KO tumors 12 days after subcutaneous implantation. ( B – E ) Flow cytometric analysis of control and Ptgs2 -KO tumors 12 to 13 days after subcutaneous implantation in WT mice. Representative experiments of 3 presented ( n . Data presented as box plots, with horizontal lines and error bars indicating mean and range, respectively. Statistical analysis between 2 groups performed using Student’s unpaired t test in GraphPad Prism. * P
    Figure Legend Snippet: Ptgs2 ablation in PDA tumor cells increases tumor antigenicity and decreases immunosuppressive potential of the tumor. ( A ) Individual (left) and average (right) weights of subcutaneously implanted control and Ptgs2 -KO tumors 12 days after subcutaneous implantation. ( B – E ) Flow cytometric analysis of control and Ptgs2 -KO tumors 12 to 13 days after subcutaneous implantation in WT mice. Representative experiments of 3 presented ( n . Data presented as box plots, with horizontal lines and error bars indicating mean and range, respectively. Statistical analysis between 2 groups performed using Student’s unpaired t test in GraphPad Prism. * P

    Techniques Used: Flow Cytometry, Mouse Assay

    26) Product Images from "Cascade enzymes within self-assembled hybrid nanogel mimicked neutrophil lysosomes for singlet oxygen elevated cancer therapy"

    Article Title: Cascade enzymes within self-assembled hybrid nanogel mimicked neutrophil lysosomes for singlet oxygen elevated cancer therapy

    Journal: Nature Communications

    doi: 10.1038/s41467-018-08234-2

    Mechanism of tumour cell apoptosis induced by EDT. a Representative CLSM images of different groups of HepG2 cells using γH2AX as a DNA damage biomarker. Scale bar, 20 µm. The red fluorescence indicates the DNA damage of HepG2 cells caused by 1 O 2 after SCMGs treatment. b Representative fluorescent images of DNA damages in different groups of HepG2 cells using the neutral comet assays. Scale bars, 50 μm. The appearance of longer comet tails in HepG2 cells indicates the damaged DNA doubles-strand breaks. c Effects of SCNGs on cell cycles by flow cytometry assays on different groups of HepG2 cells with PI staining (left), and the corresponding data analysis (right). The control, NGs and SCNGs groups are referring to the HepG2 cells after incubation with PBS, NGs and SCNGs respectively at the IC 50 value for 24 h. The all experiments were performed three times, and the representative results are displayed. Data are presented as mean ± s.d. ( n = 3). p values were analyzed by Student’s two-sided t -test (* P
    Figure Legend Snippet: Mechanism of tumour cell apoptosis induced by EDT. a Representative CLSM images of different groups of HepG2 cells using γH2AX as a DNA damage biomarker. Scale bar, 20 µm. The red fluorescence indicates the DNA damage of HepG2 cells caused by 1 O 2 after SCMGs treatment. b Representative fluorescent images of DNA damages in different groups of HepG2 cells using the neutral comet assays. Scale bars, 50 μm. The appearance of longer comet tails in HepG2 cells indicates the damaged DNA doubles-strand breaks. c Effects of SCNGs on cell cycles by flow cytometry assays on different groups of HepG2 cells with PI staining (left), and the corresponding data analysis (right). The control, NGs and SCNGs groups are referring to the HepG2 cells after incubation with PBS, NGs and SCNGs respectively at the IC 50 value for 24 h. The all experiments were performed three times, and the representative results are displayed. Data are presented as mean ± s.d. ( n = 3). p values were analyzed by Student’s two-sided t -test (* P

    Techniques Used: Confocal Laser Scanning Microscopy, Biomarker Assay, Fluorescence, Flow Cytometry, Cytometry, Staining, Next-Generation Sequencing, Incubation

    In vivo EDT on HepG2 cell derived mouse model. a Photographs on the 0, 2nd, 6th, 14th day of tumour-bearing mice after various treatments by intravenous injection. Dose: PBS (100 μL), NGs (100 μL, 5 mg mL −1 ), SCNGs (100 μL, 5 mg mL −1 ). The relative tumour volume ( b ) and body weight ( c ) change curves of each group of mice in 14 days after various treatments. Date are the means ± s.d. ( n = 3). p values were analyzed by Student’s two-sided t -test (* P
    Figure Legend Snippet: In vivo EDT on HepG2 cell derived mouse model. a Photographs on the 0, 2nd, 6th, 14th day of tumour-bearing mice after various treatments by intravenous injection. Dose: PBS (100 μL), NGs (100 μL, 5 mg mL −1 ), SCNGs (100 μL, 5 mg mL −1 ). The relative tumour volume ( b ) and body weight ( c ) change curves of each group of mice in 14 days after various treatments. Date are the means ± s.d. ( n = 3). p values were analyzed by Student’s two-sided t -test (* P

    Techniques Used: In Vivo, Derivative Assay, Mouse Assay, Injection, Next-Generation Sequencing

    Safety, efficiency and mechanism of EDT. a Cytotoxicity of NGs and SCNGs by CCK8 assays after incubated with HepG2 cells for 24 h with the concentration of 1 μg mL -1 to 500 μg mL -1 . NGs exhibited no significant effect on the cell survival, while SCNGs had increased cytotoxicity to HepG2 cells with the increase of concentration. Data are presented as mean ± s.d. ( n = 3). b Cytotoxicity of NGs and SCNGs by CCK8 assays after incubated with HL-7702 cells for 24 h with the concentration of 1 μg mL -1 to 500 μg mL −1 . Both NGs and SCNGs exhibited no significant effect on the cell survival. Data are presented as mean ± s.d. ( n = 3). c The flow cytometry results of the apoptosis of HepG2 cells staining with Annexin V-FITC/PI after incubated with NGs and SCNGs at the IC 50 value for 24 h (left), and the corresponding data analysis (right). The enhanced apoptosis promoted by SCNGs confirms the significant effect in cancer-cell killing of SCNGs. Data are presented as mean ± s.d. ( n = 3). p values were analyzed by Student’s two-sided t -test (** P ≤ 0.01, n.s. represents no significant differences). d Time-dependent CLSM images (top) and corresponding flow-cytometry analysis (bottom) of HepG2 cells treated with NGs and SCNGs at the IC 50 value for 24 h using carboxy-H 2 DCFDA as a ROS detector (left), and the corresponding data analysis (right). The experiment was conducted three times, and representative results are present. Scale bar, 20 µm. Data are presented as mean ± s.d. ( n = 3). p values were analyzed by Student’s two-sided t -test (** P
    Figure Legend Snippet: Safety, efficiency and mechanism of EDT. a Cytotoxicity of NGs and SCNGs by CCK8 assays after incubated with HepG2 cells for 24 h with the concentration of 1 μg mL -1 to 500 μg mL -1 . NGs exhibited no significant effect on the cell survival, while SCNGs had increased cytotoxicity to HepG2 cells with the increase of concentration. Data are presented as mean ± s.d. ( n = 3). b Cytotoxicity of NGs and SCNGs by CCK8 assays after incubated with HL-7702 cells for 24 h with the concentration of 1 μg mL -1 to 500 μg mL −1 . Both NGs and SCNGs exhibited no significant effect on the cell survival. Data are presented as mean ± s.d. ( n = 3). c The flow cytometry results of the apoptosis of HepG2 cells staining with Annexin V-FITC/PI after incubated with NGs and SCNGs at the IC 50 value for 24 h (left), and the corresponding data analysis (right). The enhanced apoptosis promoted by SCNGs confirms the significant effect in cancer-cell killing of SCNGs. Data are presented as mean ± s.d. ( n = 3). p values were analyzed by Student’s two-sided t -test (** P ≤ 0.01, n.s. represents no significant differences). d Time-dependent CLSM images (top) and corresponding flow-cytometry analysis (bottom) of HepG2 cells treated with NGs and SCNGs at the IC 50 value for 24 h using carboxy-H 2 DCFDA as a ROS detector (left), and the corresponding data analysis (right). The experiment was conducted three times, and representative results are present. Scale bar, 20 µm. Data are presented as mean ± s.d. ( n = 3). p values were analyzed by Student’s two-sided t -test (** P

    Techniques Used: Next-Generation Sequencing, Incubation, Concentration Assay, Flow Cytometry, Cytometry, Staining, Confocal Laser Scanning Microscopy

    Characterization of the generation of 1 O 2 . a Time-dependent electron paramagnetic resonance (EPR) signals of 1 O 2 from SCNGs in the presence of 2,2,6,6-tetramethylpiperidine (TEMP). TEMP was served as a 1 O 2 trapper. b Fluorescence spectra of the singlet oxygen sensor green (SOSG) with SCNGs in PBS buffer (20 mM, pH 6.8). c Time-dependent fluorescence spectra of SOSG with free CPO and SCNGs in PBS buffer (20 mM, pH 6.8). a.u., arbitrary units. d Three dimensional confocal laser scanning microscopy (3D-CLSM) images of living HepG2 cells after co-incubation with SCNGs at 300 μg mL -1 for 2 h and treated with SOSG probe (5 µM) from 0 min to 30 min. Scale bar, X -axis: 140 μm, Y -axis: 140 μm, Z -axis: 12 μm. e The corresponding CLSM photomicrograph with YZ (cells on the red line) and XZ (cells on the orange line) planes of living HepG2 cells and cells after co-incubation with NGs and SCNGs at 300 μg mL -1 for 2 h and treated with SOSG probe (5 µM) at 18 min. Scale bar, 20 μm. Source data are provided as a Source Data file
    Figure Legend Snippet: Characterization of the generation of 1 O 2 . a Time-dependent electron paramagnetic resonance (EPR) signals of 1 O 2 from SCNGs in the presence of 2,2,6,6-tetramethylpiperidine (TEMP). TEMP was served as a 1 O 2 trapper. b Fluorescence spectra of the singlet oxygen sensor green (SOSG) with SCNGs in PBS buffer (20 mM, pH 6.8). c Time-dependent fluorescence spectra of SOSG with free CPO and SCNGs in PBS buffer (20 mM, pH 6.8). a.u., arbitrary units. d Three dimensional confocal laser scanning microscopy (3D-CLSM) images of living HepG2 cells after co-incubation with SCNGs at 300 μg mL -1 for 2 h and treated with SOSG probe (5 µM) from 0 min to 30 min. Scale bar, X -axis: 140 μm, Y -axis: 140 μm, Z -axis: 12 μm. e The corresponding CLSM photomicrograph with YZ (cells on the red line) and XZ (cells on the orange line) planes of living HepG2 cells and cells after co-incubation with NGs and SCNGs at 300 μg mL -1 for 2 h and treated with SOSG probe (5 µM) at 18 min. Scale bar, 20 μm. Source data are provided as a Source Data file

    Techniques Used: Electron Paramagnetic Resonance, Fluorescence, Confocal Laser Scanning Microscopy, Incubation, Next-Generation Sequencing

    27) Product Images from "Combination of interferon-alpha and 5-fluorouracil inhibits endothelial cell growth directly and by regulation of angiogenic factors released by tumor cells"

    Article Title: Combination of interferon-alpha and 5-fluorouracil inhibits endothelial cell growth directly and by regulation of angiogenic factors released by tumor cells

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-9-361

    Flow cytometric analysis of cell cycle progression in HUVEC cells treated with or without IFN-α (500 units/ml) and/or 5-FU (0.5 μg/ml) . To synchronize the cell cycle in G0-G1, HUVEC cells were first pre-treated with 2 μM aphidicolin for 16 h. Cells were collected 12, 24, 48 and 72 h later. After pre-treatment by aphidicolin, the majority of cells (86.3%) were in G0-G1. At 24 h, IFN-α alone and IFN/5-FU increased the number of cells with S-phase DNA content. At 48 h and 72 h, IFN/5-FU still resulted in S-phase accumulation.
    Figure Legend Snippet: Flow cytometric analysis of cell cycle progression in HUVEC cells treated with or without IFN-α (500 units/ml) and/or 5-FU (0.5 μg/ml) . To synchronize the cell cycle in G0-G1, HUVEC cells were first pre-treated with 2 μM aphidicolin for 16 h. Cells were collected 12, 24, 48 and 72 h later. After pre-treatment by aphidicolin, the majority of cells (86.3%) were in G0-G1. At 24 h, IFN-α alone and IFN/5-FU increased the number of cells with S-phase DNA content. At 48 h and 72 h, IFN/5-FU still resulted in S-phase accumulation.

    Techniques Used: Flow Cytometry

    28) Product Images from "Measurement of Separase Proteolytic Activity in Single Living Cells by a Fluorogenic Flow Cytometry Assay"

    Article Title: Measurement of Separase Proteolytic Activity in Single Living Cells by a Fluorogenic Flow Cytometry Assay

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0133769

    Kinetics and flow cytometric detection of [Cy5-D-R-E-I-M-R] 2 -Rh110 peptide uptake (Cy5 fluorescence) and peptide cleavage (Rh110 fluorescence) in U937 cells. (A) U937 cells were incubated with 10 μM peptide in complete RPMI-1640 medium for increasing time periods (1–240 min) followed by flow cytometric analysis according to the standard protocol as described in Materials Methods. (B) Visualization of peptide uptake by Cy5 fluorescence microscopy. Equal amounts of peptide-treated (10μM, 90 min) and untreated cells were mixed and subjected to fluorescence microscopic analysis. Cy5 fluorescence is shown in pink, nuclei are counterstained with DAPI (blue). (C) Time kinetics of peptide uptake was calculated as the percentage of peptide (Cy5) positive cells (triangular dots) and the mean fluorescence per cell (round dots). (D) Time kinetics of Separase-related peptide cleavage was calculated as the percentage of Rh110 positive cells (triangular dots) and the mean fluorescence per cell (round dots). (E) Peptide dose (0–20 μM) dependency of Separase-related peptide cleavage. In all experiments, peptide alone (Ac-D-R-E-I-M-R) without dye (Cy5, Rh110) conjugation was used as non-fluorogenic control substrate and cellular autofluorescence gating control. Abbreviations: RFU, relative fluorescence units; fluor., fluorescence; conc., concentration; min, minutes.
    Figure Legend Snippet: Kinetics and flow cytometric detection of [Cy5-D-R-E-I-M-R] 2 -Rh110 peptide uptake (Cy5 fluorescence) and peptide cleavage (Rh110 fluorescence) in U937 cells. (A) U937 cells were incubated with 10 μM peptide in complete RPMI-1640 medium for increasing time periods (1–240 min) followed by flow cytometric analysis according to the standard protocol as described in Materials Methods. (B) Visualization of peptide uptake by Cy5 fluorescence microscopy. Equal amounts of peptide-treated (10μM, 90 min) and untreated cells were mixed and subjected to fluorescence microscopic analysis. Cy5 fluorescence is shown in pink, nuclei are counterstained with DAPI (blue). (C) Time kinetics of peptide uptake was calculated as the percentage of peptide (Cy5) positive cells (triangular dots) and the mean fluorescence per cell (round dots). (D) Time kinetics of Separase-related peptide cleavage was calculated as the percentage of Rh110 positive cells (triangular dots) and the mean fluorescence per cell (round dots). (E) Peptide dose (0–20 μM) dependency of Separase-related peptide cleavage. In all experiments, peptide alone (Ac-D-R-E-I-M-R) without dye (Cy5, Rh110) conjugation was used as non-fluorogenic control substrate and cellular autofluorescence gating control. Abbreviations: RFU, relative fluorescence units; fluor., fluorescence; conc., concentration; min, minutes.

    Techniques Used: Flow Cytometry, Fluorescence, Incubation, Microscopy, Conjugation Assay, Concentration Assay

    Influence of unspecific extracellular proteases on Separase assay results. Rh110 fluorescence in U937 cells (A) and in PBMC of a patient with chronic myeloid leukemia (B) as well as in the corresponding cell supernatants were assayed by flow cytometry and lysate-based assay, respectively. Three protease inhibitors alone (Pefabloc SC, soybean trypsin-chymotrypsin inhibitor, MMP-2/MMP-9 inhibitor III) or in combination (PI mix ) were used to suppress extracellular Separase-unrelated proteolytic activities according to the recommendations of the manufacturers. Proteolytic activity was measured as released Rh110 fluorescence. Abbreviations: RPMI, RPMI-1640 cell culture medium; PI, protease inhibitor; MMP9, matrix metalloproteinase 9, Pefa, Pefabloc SC; Tryp/Chym, trypsin-chymotrypsin inhibitor; PI mix , cocktail of all combined peptidase inhibitors. All assays were performed at least in triplicates.
    Figure Legend Snippet: Influence of unspecific extracellular proteases on Separase assay results. Rh110 fluorescence in U937 cells (A) and in PBMC of a patient with chronic myeloid leukemia (B) as well as in the corresponding cell supernatants were assayed by flow cytometry and lysate-based assay, respectively. Three protease inhibitors alone (Pefabloc SC, soybean trypsin-chymotrypsin inhibitor, MMP-2/MMP-9 inhibitor III) or in combination (PI mix ) were used to suppress extracellular Separase-unrelated proteolytic activities according to the recommendations of the manufacturers. Proteolytic activity was measured as released Rh110 fluorescence. Abbreviations: RPMI, RPMI-1640 cell culture medium; PI, protease inhibitor; MMP9, matrix metalloproteinase 9, Pefa, Pefabloc SC; Tryp/Chym, trypsin-chymotrypsin inhibitor; PI mix , cocktail of all combined peptidase inhibitors. All assays were performed at least in triplicates.

    Techniques Used: Fluorescence, Flow Cytometry, Cytometry, Activity Assay, Cell Culture, Protease Inhibitor

    29) Product Images from "Escherichia coli induces DNA damage in vivo and triggers genomic instability in mammalian cells"

    Article Title: Escherichia coli induces DNA damage in vivo and triggers genomic instability in mammalian cells

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1001261107

    DNA damage repair, cell death, and division after low-dose infection with pks + E. coli . CHO cells were infected for 4 h with live pks + or pks − E. coli with an MOI of 5–20 bacteria per cell or were left uninfected (Ctrl). At the end of the infection, the cells were washed and grown with gentamicin. ( A ) Cell-cycle analysis 16 and 30 h after infection. ( B ) γH2AX levels were quantified by flow cytometry 16 or 30 h after infection. ( C ) CHO or xrs-6 Ku80-defective cells were infected; 24 h later, apoptotic cells were labeled with a carboxyfluorescein fluoromethyl ketone peptide inhibitor of caspases (FLICA) for 1 h and quantified by flow cytometry. Error bars represent SE from three experiments. ( D ) The cells were examined by confocal microscopy for DNA (blue), Ser10-phosphorylated histone H3 (pH3, red), and γH2AX (green) 24 h after infection. (Scale bars, 10 μm.) ( E ) Quantification of γH2AX foci in mitotic cells. Error bars represent SEs from three experiments.
    Figure Legend Snippet: DNA damage repair, cell death, and division after low-dose infection with pks + E. coli . CHO cells were infected for 4 h with live pks + or pks − E. coli with an MOI of 5–20 bacteria per cell or were left uninfected (Ctrl). At the end of the infection, the cells were washed and grown with gentamicin. ( A ) Cell-cycle analysis 16 and 30 h after infection. ( B ) γH2AX levels were quantified by flow cytometry 16 or 30 h after infection. ( C ) CHO or xrs-6 Ku80-defective cells were infected; 24 h later, apoptotic cells were labeled with a carboxyfluorescein fluoromethyl ketone peptide inhibitor of caspases (FLICA) for 1 h and quantified by flow cytometry. Error bars represent SE from three experiments. ( D ) The cells were examined by confocal microscopy for DNA (blue), Ser10-phosphorylated histone H3 (pH3, red), and γH2AX (green) 24 h after infection. (Scale bars, 10 μm.) ( E ) Quantification of γH2AX foci in mitotic cells. Error bars represent SEs from three experiments.

    Techniques Used: Infection, Cell Cycle Assay, Flow Cytometry, Cytometry, Labeling, Confocal Microscopy

    30) Product Images from "New Monoclonal Antibodies to Defined Cell Surface Proteins on Human Pluripotent Stem Cells"

    Article Title: New Monoclonal Antibodies to Defined Cell Surface Proteins on Human Pluripotent Stem Cells

    Journal: Stem Cells (Dayton, Ohio)

    doi: 10.1002/stem.2558

    Expression of pluripotency‐associated antigens in human breast tissue and on normal and cancerous intestinal epithelial cells. (A) : Human CD31 − CD45 − CD235a − cell populations isolated from mammary tissue were (i) fluorescence activated cell sorting (FACS)‐delineated using EpCAM and CD49f to resolve each of the four breast epithelial cell subsets:‐ luminal progenitor (CD49f + EpCAM + ), MaSC and basal (CD49f + EpCAM − ), mature luminal (CD49f − EpCAM + ), and fibroblast‐enriched stromal (CD49f − EpCAM − ); (ii) Representative flow histogram plots show the presence or absence of antigens detected by the purified monoclonal antibodies (mAbs) anti‐hGRP64, anti‐hCDCP1, anti‐hF11R, anti‐hDSG2, anti‐hCDH2, anti‐hNLGN4X, and anti‐hPCDH1 in these cell subsets, compared with isotype controls. (B) : Bar graphs show the detection of pluripotency‐associated antigens for mammary cell subsets from four donor specimens as mean +/− SEM across two technical replicates compared with isotype controls and NHF1‐3 hiPS positive control cells. (C) : (i) Human intestinal epithelial cells were isolated from normal and cancerous colorectal tissues by FACS selection for EpCAM + CD31 − CD45 − cells. (ii) Bar graphs show the variation in detection of pluripotency‐associated antigens by the panel of mAbs (A‐B) on normal and cancerous intestinal epithelial cells from three patient donor samples compared with isotype controls. Abbreviation: hiPSCs, human iPS cells.
    Figure Legend Snippet: Expression of pluripotency‐associated antigens in human breast tissue and on normal and cancerous intestinal epithelial cells. (A) : Human CD31 − CD45 − CD235a − cell populations isolated from mammary tissue were (i) fluorescence activated cell sorting (FACS)‐delineated using EpCAM and CD49f to resolve each of the four breast epithelial cell subsets:‐ luminal progenitor (CD49f + EpCAM + ), MaSC and basal (CD49f + EpCAM − ), mature luminal (CD49f − EpCAM + ), and fibroblast‐enriched stromal (CD49f − EpCAM − ); (ii) Representative flow histogram plots show the presence or absence of antigens detected by the purified monoclonal antibodies (mAbs) anti‐hGRP64, anti‐hCDCP1, anti‐hF11R, anti‐hDSG2, anti‐hCDH2, anti‐hNLGN4X, and anti‐hPCDH1 in these cell subsets, compared with isotype controls. (B) : Bar graphs show the detection of pluripotency‐associated antigens for mammary cell subsets from four donor specimens as mean +/− SEM across two technical replicates compared with isotype controls and NHF1‐3 hiPS positive control cells. (C) : (i) Human intestinal epithelial cells were isolated from normal and cancerous colorectal tissues by FACS selection for EpCAM + CD31 − CD45 − cells. (ii) Bar graphs show the variation in detection of pluripotency‐associated antigens by the panel of mAbs (A‐B) on normal and cancerous intestinal epithelial cells from three patient donor samples compared with isotype controls. Abbreviation: hiPSCs, human iPS cells.

    Techniques Used: Expressing, Isolation, Fluorescence, FACS, Flow Cytometry, Purification, Positive Control, Selection

    31) Product Images from "The Role of cis Dimerization of Signal Regulatory Protein ? (SIRP?) in Binding to CD47 *"

    Article Title: The Role of cis Dimerization of Signal Regulatory Protein ? (SIRP?) in Binding to CD47 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.180018

    SIRPα dimerization is disrupted after treatment with tunicamycin. A , flow cytometric analysis of tunicamycin-treated ( solid line ) and untreated ( dashed line ) CHO-SIRPα transfectants labeled with PHA-L-FITC ( top left panel ) to measure the
    Figure Legend Snippet: SIRPα dimerization is disrupted after treatment with tunicamycin. A , flow cytometric analysis of tunicamycin-treated ( solid line ) and untreated ( dashed line ) CHO-SIRPα transfectants labeled with PHA-L-FITC ( top left panel ) to measure the

    Techniques Used: Flow Cytometry, Labeling

    32) Product Images from "Endothelial colony forming cells and mesenchymal progenitor cells form blood vessels and increase blood flow in ischemic muscle"

    Article Title: Endothelial colony forming cells and mesenchymal progenitor cells form blood vessels and increase blood flow in ischemic muscle

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-00809-1

    Depletion of host myeloid cells reduces blood flow recovery mediated by human ECFC + MPC in ischemic hind limb muscles. ( A ) Experimental schedule: Anti-Gr-1 or IgG control antibody (200 μg each) was given by intraperitoneal injection every 2 days. ECFC + MPC or Matrigel were injected into the ischemic hind limb muscles at day 0 (one day after hind limb ischemia induction). ( B ) Representative flow cytometry analyses of ECFC + MPC-injected ischemic hind limb muscles at day 2. ( C ) Quantitative cytometric analyses of myeloid lineage cells obtained from ischemic hind limb muscles (n = 3; means ± SEM). ○ Shows cell number obtained from ECFC + MPC-injected ischemic hind limb muscles from mice treated with the IgG control antibody. ● Shows cell number obtained from ECFC + MPC-injected ischemic hind limb muscles from mice treated with anti-Gr-1 antibody. *Significant difference (P ≤ 0.05) between groups. ( D ) Representative laser Doppler images and photographs of ischemic hind limbs on day 7 after animals were injected with Matrigel or ECFC + MPC and treated with IgG control or anti-Gr-1 antibody administration. ( E ) Graph of blood flow presented as the ligated/non-ligated leg ratio (n = 5–6; means ± SEM.). *Significant difference (P ≤ 0.05) from the group of Matrigel injection with no antibody administration. ( F ) Dot graph expressed as single values for each mouse (n = 5–6; means shown by horizontal bars). *Significant difference (P ≤ 0.05) between groups. ( G ) Representative confocal images of intravenously injected lectin-labeled vessels in the ECFC + MPC-injected ischemic hind limb muscles with IgG control or anti-Gr-1 antibody administration at day 7 (Scale bars represent 50 μm). ( H ) Graph of total, human (red), and murine green) vessels in the ECFC + MPC-injected ischemic hind limb muscles with IgG control or anti-Gr-1 antibody administration at day 7 (n = 3; means ± SEM.). *Significant difference (P ≤ 0.05) between groups for total microvessel density. † Significant difference (P ≤ 0.05) between groups for human microvessel density. ( I ) Quantitative cytometric analyses of human and murine vascular cells and murine stromal cells obtained from ischemic hind limb muscles at day 0, 2, and 7 (n = 3; means ± SEM). *Significant difference (P ≤ 0.05) between groups.
    Figure Legend Snippet: Depletion of host myeloid cells reduces blood flow recovery mediated by human ECFC + MPC in ischemic hind limb muscles. ( A ) Experimental schedule: Anti-Gr-1 or IgG control antibody (200 μg each) was given by intraperitoneal injection every 2 days. ECFC + MPC or Matrigel were injected into the ischemic hind limb muscles at day 0 (one day after hind limb ischemia induction). ( B ) Representative flow cytometry analyses of ECFC + MPC-injected ischemic hind limb muscles at day 2. ( C ) Quantitative cytometric analyses of myeloid lineage cells obtained from ischemic hind limb muscles (n = 3; means ± SEM). ○ Shows cell number obtained from ECFC + MPC-injected ischemic hind limb muscles from mice treated with the IgG control antibody. ● Shows cell number obtained from ECFC + MPC-injected ischemic hind limb muscles from mice treated with anti-Gr-1 antibody. *Significant difference (P ≤ 0.05) between groups. ( D ) Representative laser Doppler images and photographs of ischemic hind limbs on day 7 after animals were injected with Matrigel or ECFC + MPC and treated with IgG control or anti-Gr-1 antibody administration. ( E ) Graph of blood flow presented as the ligated/non-ligated leg ratio (n = 5–6; means ± SEM.). *Significant difference (P ≤ 0.05) from the group of Matrigel injection with no antibody administration. ( F ) Dot graph expressed as single values for each mouse (n = 5–6; means shown by horizontal bars). *Significant difference (P ≤ 0.05) between groups. ( G ) Representative confocal images of intravenously injected lectin-labeled vessels in the ECFC + MPC-injected ischemic hind limb muscles with IgG control or anti-Gr-1 antibody administration at day 7 (Scale bars represent 50 μm). ( H ) Graph of total, human (red), and murine green) vessels in the ECFC + MPC-injected ischemic hind limb muscles with IgG control or anti-Gr-1 antibody administration at day 7 (n = 3; means ± SEM.). *Significant difference (P ≤ 0.05) between groups for total microvessel density. † Significant difference (P ≤ 0.05) between groups for human microvessel density. ( I ) Quantitative cytometric analyses of human and murine vascular cells and murine stromal cells obtained from ischemic hind limb muscles at day 0, 2, and 7 (n = 3; means ± SEM). *Significant difference (P ≤ 0.05) between groups.

    Techniques Used: Flow Cytometry, Injection, Cytometry, Mouse Assay, Labeling

    Myeloid cells recruited into murine ischemic hind limb muscle after human ECFC + MPC injection. Ischemic hind limb muscles with/without cell injection were harvested, fixed, and sectioned for confocal microscopy analysis. Sections were incubated with biotinylated UEA I followed by Fluorescein-Streptavidin to detect human ECFC (green). After that, sections were incubated with primary antibodies (rat anti-mouse CD11b, rat anti-mouse Ly-6G, or rat anti-mouse F4/80 antibodies) followed by goat anti-rat Alexa Fluor-568 (red). In parallel experimental sets, ischemic hind limb muscles were harvested, digested and analyzed by flow cytometry. ( A ) Representative confocal images of myeloid cells in the ischemic hind limb muscles injected with Matrigel or ECFC + MPC at day 2 and 7 (scale bars represent 50 μm). ( B ) Representative flow cytometry analyses of contralateral hind limb muscles and ischemic hind limb muscles injected with Matrigel or ECFC + MPC at day 2. ( C ) Quantitative cytometric analyses of myeloid lineage cells obtained from ischemic hind limb muscles (n = 3; means ± SEM). ○ Shows cell number obtained from ischemic hind limb muscles injected with Matrigel alone. ● Shows cell number obtained from ischemic hind limb muscles injected with ECFC + MPC in Matrigel. *Significant difference (P ≤ 0.05) between groups. ( D ) Quantitative cytometric analyses of human and murine vascular cells and murine stromal cells obtained from ischemic hind limb muscles at day 0, 2, and 7 (n = 3; means ± SEM). *Significant difference (P ≤ 0.05) between groups.
    Figure Legend Snippet: Myeloid cells recruited into murine ischemic hind limb muscle after human ECFC + MPC injection. Ischemic hind limb muscles with/without cell injection were harvested, fixed, and sectioned for confocal microscopy analysis. Sections were incubated with biotinylated UEA I followed by Fluorescein-Streptavidin to detect human ECFC (green). After that, sections were incubated with primary antibodies (rat anti-mouse CD11b, rat anti-mouse Ly-6G, or rat anti-mouse F4/80 antibodies) followed by goat anti-rat Alexa Fluor-568 (red). In parallel experimental sets, ischemic hind limb muscles were harvested, digested and analyzed by flow cytometry. ( A ) Representative confocal images of myeloid cells in the ischemic hind limb muscles injected with Matrigel or ECFC + MPC at day 2 and 7 (scale bars represent 50 μm). ( B ) Representative flow cytometry analyses of contralateral hind limb muscles and ischemic hind limb muscles injected with Matrigel or ECFC + MPC at day 2. ( C ) Quantitative cytometric analyses of myeloid lineage cells obtained from ischemic hind limb muscles (n = 3; means ± SEM). ○ Shows cell number obtained from ischemic hind limb muscles injected with Matrigel alone. ● Shows cell number obtained from ischemic hind limb muscles injected with ECFC + MPC in Matrigel. *Significant difference (P ≤ 0.05) between groups. ( D ) Quantitative cytometric analyses of human and murine vascular cells and murine stromal cells obtained from ischemic hind limb muscles at day 0, 2, and 7 (n = 3; means ± SEM). *Significant difference (P ≤ 0.05) between groups.

    Techniques Used: Injection, Confocal Microscopy, Incubation, Flow Cytometry, Cytometry

    33) Product Images from "The Extracellular Matrix Protein TGFBI Induces Microtubule Stabilization and Sensitizes Ovarian Cancers to Paclitaxel"

    Article Title: The Extracellular Matrix Protein TGFBI Induces Microtubule Stabilization and Sensitizes Ovarian Cancers to Paclitaxel

    Journal: Cancer Cell

    doi: 10.1016/j.ccr.2007.11.014

    Loss of TGFBI Is Sufficient to Induce Paclitaxel Resistance (A) Volcano plot shows log fold change in gene expression in the paclitaxel-resistant cell line SKOV-3TR compared to the sensitive parental line SKOV-3 and plotted against the likelihood of differential expression. Note that negative log2 expression ratios indicate underexpression in SKOV-3TR. Data points represent the probability value for differential gene expression and data shown is from four replicate experiments. (B) Relative expression levels of TGFBI in different cell lines using real time PCR. (C) Immunocytochemistry of stained sections from embedded cell pellets using anti-TGFBI antibody. Scale bars, 10 μm. (D) Western blotting of culture medium from SKOV-3TR, mock-transfected SKOV-3, and TGFBI siRNA-transfected SKOV3-K cell lines probed with anti-TGFBI antibody. (E) Effect of stable KD of TGFBI (SKOV3-A, SKOV3-K, and SKOV3-AK) on paclitaxel-induced apoptosis measured by FITC-annexin V and 7-AAD staining at 48 hr following paclitaxel treatment (150, 300, 600, 1200, and 2000 nM) compared to SKOV-3, mock-transfected SKOV-3 (mtSKOV3), and SKOV-3TR cells. Filled triangle indicates increasing paclitaxel dose across each group of bars. (F) Effect of stable KD of TGFBI on caspase 3/7 activation 48 hr following paclitaxel treatment. (G and H) Transient TGFBI -KD in OVCAR3 and TR175 lines induces paclitaxel resistance. Caspase 3/7 activation was estimated 48 hr following transfection using either a pool of 4 siRNAs targeting TGFBI or nontargeting scrambled controls (sc). OVCAR3 cells (G) or TR175 cells (H) were treated with paclitaxel for 48 hr. Immunoblot confirming knockdown of TGFBI protein is shown in (G). Error bars show mean ± SD.
    Figure Legend Snippet: Loss of TGFBI Is Sufficient to Induce Paclitaxel Resistance (A) Volcano plot shows log fold change in gene expression in the paclitaxel-resistant cell line SKOV-3TR compared to the sensitive parental line SKOV-3 and plotted against the likelihood of differential expression. Note that negative log2 expression ratios indicate underexpression in SKOV-3TR. Data points represent the probability value for differential gene expression and data shown is from four replicate experiments. (B) Relative expression levels of TGFBI in different cell lines using real time PCR. (C) Immunocytochemistry of stained sections from embedded cell pellets using anti-TGFBI antibody. Scale bars, 10 μm. (D) Western blotting of culture medium from SKOV-3TR, mock-transfected SKOV-3, and TGFBI siRNA-transfected SKOV3-K cell lines probed with anti-TGFBI antibody. (E) Effect of stable KD of TGFBI (SKOV3-A, SKOV3-K, and SKOV3-AK) on paclitaxel-induced apoptosis measured by FITC-annexin V and 7-AAD staining at 48 hr following paclitaxel treatment (150, 300, 600, 1200, and 2000 nM) compared to SKOV-3, mock-transfected SKOV-3 (mtSKOV3), and SKOV-3TR cells. Filled triangle indicates increasing paclitaxel dose across each group of bars. (F) Effect of stable KD of TGFBI on caspase 3/7 activation 48 hr following paclitaxel treatment. (G and H) Transient TGFBI -KD in OVCAR3 and TR175 lines induces paclitaxel resistance. Caspase 3/7 activation was estimated 48 hr following transfection using either a pool of 4 siRNAs targeting TGFBI or nontargeting scrambled controls (sc). OVCAR3 cells (G) or TR175 cells (H) were treated with paclitaxel for 48 hr. Immunoblot confirming knockdown of TGFBI protein is shown in (G). Error bars show mean ± SD.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Immunocytochemistry, Staining, Western Blot, Transfection, Activation Assay

    34) Product Images from "Serglycin Proteoglycan Is Required for Multiple Myeloma Cell Adhesion, in Vivo Growth, and Vascularization *"

    Article Title: Serglycin Proteoglycan Is Required for Multiple Myeloma Cell Adhesion, in Vivo Growth, and Vascularization *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.532143

    Serglycin is expressed by human myeloma cells. A , immunohistochemistry demonstrating serglycin expression on bone marrow biopsy sections from myeloma patients (nos.1–3) ( upper panel ). Scale bars, 200 μm. Lower panel represents immunohistochemistry
    Figure Legend Snippet: Serglycin is expressed by human myeloma cells. A , immunohistochemistry demonstrating serglycin expression on bone marrow biopsy sections from myeloma patients (nos.1–3) ( upper panel ). Scale bars, 200 μm. Lower panel represents immunohistochemistry

    Techniques Used: Immunohistochemistry, Expressing

    Knockdown of serglycin expression diminishes subcutaneous growth of myeloma cells. A , knockdown of serglycin expression does not alter the in vitro growth of myeloma cells. Control or serglycin-knockdown CAG cells were seeded at equal density, and the
    Figure Legend Snippet: Knockdown of serglycin expression diminishes subcutaneous growth of myeloma cells. A , knockdown of serglycin expression does not alter the in vitro growth of myeloma cells. Control or serglycin-knockdown CAG cells were seeded at equal density, and the

    Techniques Used: Expressing, In Vitro

    Tumors formed by serglycin-knockdown cells have a poorly developed vasculature and decreased levels of human HGF. A , immunohistochemistry of tumors formed by control ( left panel ) or serglycin-knockdown ( right panel ) cells. Tumor tissue was stained with
    Figure Legend Snippet: Tumors formed by serglycin-knockdown cells have a poorly developed vasculature and decreased levels of human HGF. A , immunohistochemistry of tumors formed by control ( left panel ) or serglycin-knockdown ( right panel ) cells. Tumor tissue was stained with

    Techniques Used: Immunohistochemistry, Staining

    Serglycin is secreted by myeloma cells and is present on the cell surface. A , Western blot analysis for the presence of serglycin in the conditioned medium from five different human myeloma cell lines. Culture medium without cells is used as the control.
    Figure Legend Snippet: Serglycin is secreted by myeloma cells and is present on the cell surface. A , Western blot analysis for the presence of serglycin in the conditioned medium from five different human myeloma cell lines. Culture medium without cells is used as the control.

    Techniques Used: Western Blot

    Activin enhances C4ST1 and serglycin expression in myeloma cells. CAG or RPMI 8226 myeloma cells were treated with recombinant activin (500 ng/ml) overnight and the expression of C4ST1 ( A ) and serglycin ( B ) was assessed by real time PCR and normalized
    Figure Legend Snippet: Activin enhances C4ST1 and serglycin expression in myeloma cells. CAG or RPMI 8226 myeloma cells were treated with recombinant activin (500 ng/ml) overnight and the expression of C4ST1 ( A ) and serglycin ( B ) was assessed by real time PCR and normalized

    Techniques Used: Expressing, Recombinant, Real-time Polymerase Chain Reaction

    Serglycin promotes myeloma cell adhesion to bone marrow stromal cells and collagen I. A , serglycin secreted by myeloma cells binds to CD44. Recombinant soluble CD44 (CD44-IgG1) was added to the culture supernatants from 6 different human myeloma cell
    Figure Legend Snippet: Serglycin promotes myeloma cell adhesion to bone marrow stromal cells and collagen I. A , serglycin secreted by myeloma cells binds to CD44. Recombinant soluble CD44 (CD44-IgG1) was added to the culture supernatants from 6 different human myeloma cell

    Techniques Used: Recombinant

    35) Product Images from "Different tissue phagocytes sample apoptotic cells to direct distinct homeostasis programs"

    Article Title: Different tissue phagocytes sample apoptotic cells to direct distinct homeostasis programs

    Journal: Nature

    doi: 10.1038/nature20138

    Ileum lamina propria CD11c + phagocytes extend dendrites towards apoptotic intestinal epithelial cells a – h , Conventional ( a – e ) and confocal ( f – h ) whole-mount microscopy of excised ileum from B6 or VDTR mice following PBS or diphtheria toxin as indicated. b , c , e , eGFP not overlaid. Insets from f and h depicted in bottom and right panels, respectively, without eGFP overlay. L, lumen. Arrowhead in a points to a CC3 + IEC; in d , to a CD11c + dendrite. Scale bars, 25 μm ( a , e ), 50 μm ( b , d , h ), 100 μm ( c , f , g ).
    Figure Legend Snippet: Ileum lamina propria CD11c + phagocytes extend dendrites towards apoptotic intestinal epithelial cells a – h , Conventional ( a – e ) and confocal ( f – h ) whole-mount microscopy of excised ileum from B6 or VDTR mice following PBS or diphtheria toxin as indicated. b , c , e , eGFP not overlaid. Insets from f and h depicted in bottom and right panels, respectively, without eGFP overlay. L, lumen. Arrowhead in a points to a CC3 + IEC; in d , to a CD11c + dendrite. Scale bars, 25 μm ( a , e ), 50 μm ( b , d , h ), 100 μm ( c , f , g ).

    Techniques Used: Microscopy, Mouse Assay

    36) Product Images from "The Role of CD133 in Normal Human Prostate Stem Cells and Malignant Cancer-Initiating Cells"

    Article Title: The Role of CD133 in Normal Human Prostate Stem Cells and Malignant Cancer-Initiating Cells

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-08-3084

    Expression of stem cell markers in PrEC cultures. A, ABCG2-FITC and CD133-PE (AC141) expression in commercially available PrECs. A nonspecific IgG was used as an isotype control. B, dual-label flow cytometry showing that CD133 + cells are also ABCG2 + . C, CD133 + PrECs do not express the intermediate cell marker PSCA.
    Figure Legend Snippet: Expression of stem cell markers in PrEC cultures. A, ABCG2-FITC and CD133-PE (AC141) expression in commercially available PrECs. A nonspecific IgG was used as an isotype control. B, dual-label flow cytometry showing that CD133 + cells are also ABCG2 + . C, CD133 + PrECs do not express the intermediate cell marker PSCA.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Marker

    37) Product Images from "MULTIVALENT PROTEOGLYCAN MODULATION OF FGF MITOGENIC RESPONSES IN PERIVASCULAR CELLS"

    Article Title: MULTIVALENT PROTEOGLYCAN MODULATION OF FGF MITOGENIC RESPONSES IN PERIVASCULAR CELLS

    Journal: Angiogenesis

    doi: 10.1007/s10456-012-9316-7

    (a) Flow cytometric FRET analyses of NG2-FGFR1/FGFR3 ligand-independent complex formation on the cell surface using pair-wise combinations of fluorescently-tagged anti-NG2 and anti-FGFR1 or anti-FGFR3 antibodies. Interaction between the individual subunits
    Figure Legend Snippet: (a) Flow cytometric FRET analyses of NG2-FGFR1/FGFR3 ligand-independent complex formation on the cell surface using pair-wise combinations of fluorescently-tagged anti-NG2 and anti-FGFR1 or anti-FGFR3 antibodies. Interaction between the individual subunits

    Techniques Used: Flow Cytometry

    Selectivity of the functional cooperation of NG2 with FGFR1 and FGFR3. FGF-2-induced mitogenic responses were compared in different fibroblastic cell lines selected on the basis of their diverse FGFR expression patterns ( a, b ) and
    Figure Legend Snippet: Selectivity of the functional cooperation of NG2 with FGFR1 and FGFR3. FGF-2-induced mitogenic responses were compared in different fibroblastic cell lines selected on the basis of their diverse FGFR expression patterns ( a, b ) and

    Techniques Used: Functional Assay, Expressing

    (a) FGF-2 responses in cells expressing altered FGFR1/FGFR3-NG2 ratios. Cells enriched by immunoselection for their constitutive NG2 expression (NG2 + ) and cells transduced to overexpress the rodent full-length NG2 (NG2 ++ ) were exposed to optimal stimulatory
    Figure Legend Snippet: (a) FGF-2 responses in cells expressing altered FGFR1/FGFR3-NG2 ratios. Cells enriched by immunoselection for their constitutive NG2 expression (NG2 + ) and cells transduced to overexpress the rodent full-length NG2 (NG2 ++ ) were exposed to optimal stimulatory

    Techniques Used: Expressing

    38) Product Images from "Targeting MARCO can lead to enhanced dendritic cell motility and anti-melanoma activity"

    Article Title: Targeting MARCO can lead to enhanced dendritic cell motility and anti-melanoma activity

    Journal: Cancer immunology, immunotherapy : CII

    doi: 10.1007/s00262-009-0813-5

    Exposure of B16 melanoma lysate-loaded DC ( Upper ) to anti-MARCO mAb in vitro leads to cell rounding and loss of dendritic processes ( Middle ). Such morphologic changes were not observed following incubation of the DC to antibody directed toward a second
    Figure Legend Snippet: Exposure of B16 melanoma lysate-loaded DC ( Upper ) to anti-MARCO mAb in vitro leads to cell rounding and loss of dendritic processes ( Middle ). Such morphologic changes were not observed following incubation of the DC to antibody directed toward a second

    Techniques Used: In Vitro, Incubation

    Enhancement of MARCO transcript expression in DC loaded pulsed with LPS or loaded with tumor lysate ( a ). Total RNA from unpulsed (UP)-DC and DC pulsed with LPS or loaded with B16 melanoma lysate as indicated, were analyzed by RT-PCR for MARCO transcript
    Figure Legend Snippet: Enhancement of MARCO transcript expression in DC loaded pulsed with LPS or loaded with tumor lysate ( a ). Total RNA from unpulsed (UP)-DC and DC pulsed with LPS or loaded with B16 melanoma lysate as indicated, were analyzed by RT-PCR for MARCO transcript

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    Enhanced trafficking of skin injected, B16 melanoma lysate-loaded DC to lymph node following exposure to anti-MARCO mAb ( a ). DC were first stained with PKH26-red dye, and after staining > 95% were confirmed positive by both flow cytometry ( Upper
    Figure Legend Snippet: Enhanced trafficking of skin injected, B16 melanoma lysate-loaded DC to lymph node following exposure to anti-MARCO mAb ( a ). DC were first stained with PKH26-red dye, and after staining > 95% were confirmed positive by both flow cytometry ( Upper

    Techniques Used: Injection, Staining, Flow Cytometry, Cytometry

    39) Product Images from "Itk is required for Th9 differentiation via TCR-mediated induction of IL-2 and IRF4"

    Article Title: Itk is required for Th9 differentiation via TCR-mediated induction of IL-2 and IRF4

    Journal: Nature Communications

    doi: 10.1038/ncomms10857

    IL-9 expression correlates with strength of TCR signals. ( a ) Sorted naive WT CD4 + T cells were differentiated under Th9 conditions plus TL1A with 0.01, 0.1 or 1 μg ml −1 of anti-CD3, then restimulated with PMA and Ionomycin and IL-9 production analysed by intracellular staining. ( b ) Itk-deficient CD4 + T cells were transduced with retroviruses expressing constitutively active NFATc1 (ca-NFATc1), or a control (MIGR), differentiated under Th9 plus TL1A or Th17 conditions, and cytokine production determined by intracellular staining after PMA and ionomycin restimulation. Results in a and b are from one representative of three independent experiments.
    Figure Legend Snippet: IL-9 expression correlates with strength of TCR signals. ( a ) Sorted naive WT CD4 + T cells were differentiated under Th9 conditions plus TL1A with 0.01, 0.1 or 1 μg ml −1 of anti-CD3, then restimulated with PMA and Ionomycin and IL-9 production analysed by intracellular staining. ( b ) Itk-deficient CD4 + T cells were transduced with retroviruses expressing constitutively active NFATc1 (ca-NFATc1), or a control (MIGR), differentiated under Th9 plus TL1A or Th17 conditions, and cytokine production determined by intracellular staining after PMA and ionomycin restimulation. Results in a and b are from one representative of three independent experiments.

    Techniques Used: Expressing, Staining, Transduction

    IL-2 rescues pSTAT5 and pS6 in Itk −/− CD4 T cells. ( a - c ) Sorted naive CD4 + T cells from WT and Itk −/− mice were differentiated for 3 days under Th9 or Th9 plus TL1A conditions in absence or presence of blocking anti-IL-2 plus hIL-2 and analysed for pSTAT5 (MFI are indicated) ( a ) CD25 ( b ) and pS6 ( c ). ( d ) Itk-deficient CD4 + T cells were transduced with control (MIGR) or constitutively active STAT5-expressing retroviruses, differentiated under Th9 plus TL1A conditions and IL-9 production determined by intracellular staining after PMA and Ionomycin restimulation. Results in a - d are representative of one out of at least 3 experiments.
    Figure Legend Snippet: IL-2 rescues pSTAT5 and pS6 in Itk −/− CD4 T cells. ( a - c ) Sorted naive CD4 + T cells from WT and Itk −/− mice were differentiated for 3 days under Th9 or Th9 plus TL1A conditions in absence or presence of blocking anti-IL-2 plus hIL-2 and analysed for pSTAT5 (MFI are indicated) ( a ) CD25 ( b ) and pS6 ( c ). ( d ) Itk-deficient CD4 + T cells were transduced with control (MIGR) or constitutively active STAT5-expressing retroviruses, differentiated under Th9 plus TL1A conditions and IL-9 production determined by intracellular staining after PMA and Ionomycin restimulation. Results in a - d are representative of one out of at least 3 experiments.

    Techniques Used: Mouse Assay, Blocking Assay, Transduction, Expressing, Staining

    IL-2 rescues Th9 differentiation in Itk −/− CD4 + T cells. ( a - c ) Sorted naïve CD4 + T cells from WT and Itk −/− mice were differentiated under Th9 conditions plus TL1A for 3 days and pSTAT5 ( a ) and CD25 ( b ) were determined by flow cytometry: WT (black), Itk −/− (grey) lines. ( c ) Sorted naive CD4 + T cells were differentiated as in a or under Th17 conditions and IL-2 in supernatants were determined at 48 and 72 h by Luminex. Th9 conditions: black bars. Th17 conditions: hatched bars. ( d , e ) Sorted naïve CD4 + T cells were differentiated for 3 days under Th9 ( d ) or Th9 plus TL1A ( e ) conditions in absence or presence of blocking anti-IL-2 plus hIL-2, restimulated with PMA and Ionomycin and IL-9 analysed by flow cytometry. ( f ) Sorted naive CD4 + T cells from WT and Itk −/− mice were stained with CSFE, differentiated, and restimulated with PMA and Ionomycin to evaluate IL-9 expression. Data in a - f are representative of one out of at least three independent experiments.
    Figure Legend Snippet: IL-2 rescues Th9 differentiation in Itk −/− CD4 + T cells. ( a - c ) Sorted naïve CD4 + T cells from WT and Itk −/− mice were differentiated under Th9 conditions plus TL1A for 3 days and pSTAT5 ( a ) and CD25 ( b ) were determined by flow cytometry: WT (black), Itk −/− (grey) lines. ( c ) Sorted naive CD4 + T cells were differentiated as in a or under Th17 conditions and IL-2 in supernatants were determined at 48 and 72 h by Luminex. Th9 conditions: black bars. Th17 conditions: hatched bars. ( d , e ) Sorted naïve CD4 + T cells were differentiated for 3 days under Th9 ( d ) or Th9 plus TL1A ( e ) conditions in absence or presence of blocking anti-IL-2 plus hIL-2, restimulated with PMA and Ionomycin and IL-9 analysed by flow cytometry. ( f ) Sorted naive CD4 + T cells from WT and Itk −/− mice were stained with CSFE, differentiated, and restimulated with PMA and Ionomycin to evaluate IL-9 expression. Data in a - f are representative of one out of at least three independent experiments.

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Luminex, Blocking Assay, Staining, Expressing

    IL-2 rescues IRF4 expression in Itk −/− CD4 T cells. ( a ) Sorted naive CD4 + T cells differentiated for 3 days under Th9 plus TL1A in absence or presence of blocking anti-IL-2 plus hIL-2, were stained for IRF4 and analysed by flow cytometry. ( b ) mRNA of Irf4 and Maf in cells differentiated as in a evaluated by qRT-PCR. ( c ) Itk-deficient CD4 + T cells were transduced with constitutively active STAT5 (ca-STAT5) or control retroviruses, differentiated under Th9 plus TL1A conditions and intracellular IRF4 determined. ( d ) Sorted naive WT CD4 + T cells were differentiated under Th9 plus TL1A with 0.003 or 0.01 μg ml −1 of anti-CD3, in absence or presence of blocking anti-IL-2 or blocking anti-IL-2 plus hIL-2, then restimulated with PMA and Ionomycin and IL-9 and IRF4 production were analysed by intracellular staining, MFI values for IRF4 are indicated. Data in figures a - d are representative examples from one of at least three independent experiments.
    Figure Legend Snippet: IL-2 rescues IRF4 expression in Itk −/− CD4 T cells. ( a ) Sorted naive CD4 + T cells differentiated for 3 days under Th9 plus TL1A in absence or presence of blocking anti-IL-2 plus hIL-2, were stained for IRF4 and analysed by flow cytometry. ( b ) mRNA of Irf4 and Maf in cells differentiated as in a evaluated by qRT-PCR. ( c ) Itk-deficient CD4 + T cells were transduced with constitutively active STAT5 (ca-STAT5) or control retroviruses, differentiated under Th9 plus TL1A conditions and intracellular IRF4 determined. ( d ) Sorted naive WT CD4 + T cells were differentiated under Th9 plus TL1A with 0.003 or 0.01 μg ml −1 of anti-CD3, in absence or presence of blocking anti-IL-2 or blocking anti-IL-2 plus hIL-2, then restimulated with PMA and Ionomycin and IL-9 and IRF4 production were analysed by intracellular staining, MFI values for IRF4 are indicated. Data in figures a - d are representative examples from one of at least three independent experiments.

    Techniques Used: Expressing, Blocking Assay, Staining, Flow Cytometry, Cytometry, Quantitative RT-PCR, Transduction

    Itk is required for Th9 differentiation. ( a – d ) Sorted naive CD4 + T cells from WT and Itk −/− mice were differentiated under Th9 conditions (1 μg ml −1 anti-CD3, 3 μg ml −1 anti-CD28, 20 ng ml −1 IL-4, 5 ng ml −1 TGFβ1 with or without 10 ng ml −1 TL1A, in presence of T-depleted splenocytes as APCs) for 3 days, ( a ) cells were restimulated with PMA and Ionomycin and IL-9 and IFN-γ production were analysed by intracellular staining. Representative flow plots from one out of 10 experiments. ( b ) Mean±s.e.m. of 10 independent experiments ** P
    Figure Legend Snippet: Itk is required for Th9 differentiation. ( a – d ) Sorted naive CD4 + T cells from WT and Itk −/− mice were differentiated under Th9 conditions (1 μg ml −1 anti-CD3, 3 μg ml −1 anti-CD28, 20 ng ml −1 IL-4, 5 ng ml −1 TGFβ1 with or without 10 ng ml −1 TL1A, in presence of T-depleted splenocytes as APCs) for 3 days, ( a ) cells were restimulated with PMA and Ionomycin and IL-9 and IFN-γ production were analysed by intracellular staining. Representative flow plots from one out of 10 experiments. ( b ) Mean±s.e.m. of 10 independent experiments ** P

    Techniques Used: Mouse Assay, Staining, Flow Cytometry

    ITK is required for Th9 differentiation of human cells. Purified naive human CD4 + T cells were differentiated under Th9 conditions for 5 days in presence of increasing concentrations of ITK inhibitor. Cells were restimulated with PMA and Ionomycin and stained for IL-9. Data are representative of one of two independent experiments.
    Figure Legend Snippet: ITK is required for Th9 differentiation of human cells. Purified naive human CD4 + T cells were differentiated under Th9 conditions for 5 days in presence of increasing concentrations of ITK inhibitor. Cells were restimulated with PMA and Ionomycin and stained for IL-9. Data are representative of one of two independent experiments.

    Techniques Used: Purification, Staining

    40) Product Images from "NO2 inhalation induces maturation of pulmonary CD11c+ cells that promote antigenspecific CD4+ T cell polarization"

    Article Title: NO2 inhalation induces maturation of pulmonary CD11c+ cells that promote antigenspecific CD4+ T cell polarization

    Journal: Respiratory Research

    doi: 10.1186/1465-9921-11-102

    Effects of depleting CD11c + cells during sensitization in an NO 2 -promoted allergic asthma model . Eight- to fifteen-week-old female CD11c-DTR Tg + and Tg - mice were administered 4 ng DT/g of body weight via i.p. injection on day -1. All mice then underwent inhalation of 15 ppm NO 2 for 1 hour followed by 30 minutes of aerosolized ova on day 0. All mice were challenged with aerosolized ova on days 14, 15, and 16. Differential cell counts were measured from the BAL fluid 48 hours after the final ova challenge (A). Values shown are mean ± SEM with 8 Tg- and 5 Tg+ mice per group. CD4 + cells were isolated from spleens by positive selection on day 18 and co-cultured with antigen presenting cells (APCs) from naïve C57BL/6J mice and 100 μg/ml ova. Conditioned medium was collected at 96 hours and analyzed for the Th2 cytokines IL-4 (B), IL-5 (C), and IL-13 (D), and the Th17 cytokine IL-17 (E) by ELISA. No cytokines were detected in medium from APCs cultured alone or from CD4 + T cells cultured with APCs in the absence of ova. Values shown are mean ± SEM with 4-5 mice per group. The ova-specific immunoglobulins IgE (F) and IgG 1 (G) were measured from serum collected 48 hours after the final ova challenge (day 18) by ELISA using serum from Alum/ova-immunized mice to generate standard curves. Values shown are mean ± SEM with 7-10 mice per group. Statistics were computed by unpaired Student's t test. * denotes p
    Figure Legend Snippet: Effects of depleting CD11c + cells during sensitization in an NO 2 -promoted allergic asthma model . Eight- to fifteen-week-old female CD11c-DTR Tg + and Tg - mice were administered 4 ng DT/g of body weight via i.p. injection on day -1. All mice then underwent inhalation of 15 ppm NO 2 for 1 hour followed by 30 minutes of aerosolized ova on day 0. All mice were challenged with aerosolized ova on days 14, 15, and 16. Differential cell counts were measured from the BAL fluid 48 hours after the final ova challenge (A). Values shown are mean ± SEM with 8 Tg- and 5 Tg+ mice per group. CD4 + cells were isolated from spleens by positive selection on day 18 and co-cultured with antigen presenting cells (APCs) from naïve C57BL/6J mice and 100 μg/ml ova. Conditioned medium was collected at 96 hours and analyzed for the Th2 cytokines IL-4 (B), IL-5 (C), and IL-13 (D), and the Th17 cytokine IL-17 (E) by ELISA. No cytokines were detected in medium from APCs cultured alone or from CD4 + T cells cultured with APCs in the absence of ova. Values shown are mean ± SEM with 4-5 mice per group. The ova-specific immunoglobulins IgE (F) and IgG 1 (G) were measured from serum collected 48 hours after the final ova challenge (day 18) by ELISA using serum from Alum/ova-immunized mice to generate standard curves. Values shown are mean ± SEM with 7-10 mice per group. Statistics were computed by unpaired Student's t test. * denotes p

    Techniques Used: Mouse Assay, Injection, Isolation, Selection, Cell Culture, Enzyme-linked Immunosorbent Assay

    Diphtheria toxin-mediated depletion of CD11c + cells in the lung . Eight- to fifteen-week-old female CD11c-DTR Tg + and Tg - mice were administered 4ng DT/g of body weight via i.p. injection. Twenty-four hours later, lungs were harvested, digested into single-cell suspensions, and immunostained. Total lung cells were visualized by flow cytometry (A) and CD11c + cells from the parent gate were identified (B). Graphs show percent CD11c + cells from the lung (C) and total number of CD11c + cells in the lung, calculated based on the % CD11c + cells multiplied by the number of cells in the single-cell suspension from the lungs (D). Values are mean ± SEM with 4 animals per group. ** denotes p
    Figure Legend Snippet: Diphtheria toxin-mediated depletion of CD11c + cells in the lung . Eight- to fifteen-week-old female CD11c-DTR Tg + and Tg - mice were administered 4ng DT/g of body weight via i.p. injection. Twenty-four hours later, lungs were harvested, digested into single-cell suspensions, and immunostained. Total lung cells were visualized by flow cytometry (A) and CD11c + cells from the parent gate were identified (B). Graphs show percent CD11c + cells from the lung (C) and total number of CD11c + cells in the lung, calculated based on the % CD11c + cells multiplied by the number of cells in the single-cell suspension from the lungs (D). Values are mean ± SEM with 4 animals per group. ** denotes p

    Techniques Used: Mouse Assay, Injection, Flow Cytometry, Cytometry

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    Millipore celastrol flow cytometric analysis
    <t>Celastrol</t> inhibits cell proliferation and induces apoptosis in HeLa cells. ( a ) Structure of the celastrol molecule. ( b ) Cell viability (MTT assay) of HeLa cells treated with various concentrations of celastrol for 48 h. ( c ) Representative results of flow <t>cytometric</t> analysis of apoptosis in cells treated with various concentrations of celastrol for 48 h. ( d ) Statistical analysis result of flow cytometric analysis of apoptosis. Annexin V-positive cells were accepted as apoptotic cells. ( e ) Levels of caspase-9, caspase-3, poly(ADP-ribose) polymerases (PARP) and X-linked inhibitor of apoptosis protein (XIAP) in cells treated with 1 μ M celastrol for 48 h. * P
    Celastrol Flow Cytometric Analysis, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Celastrol inhibits cell proliferation and induces apoptosis in HeLa cells. ( a ) Structure of the celastrol molecule. ( b ) Cell viability (MTT assay) of HeLa cells treated with various concentrations of celastrol for 48 h. ( c ) Representative results of flow cytometric analysis of apoptosis in cells treated with various concentrations of celastrol for 48 h. ( d ) Statistical analysis result of flow cytometric analysis of apoptosis. Annexin V-positive cells were accepted as apoptotic cells. ( e ) Levels of caspase-9, caspase-3, poly(ADP-ribose) polymerases (PARP) and X-linked inhibitor of apoptosis protein (XIAP) in cells treated with 1 μ M celastrol for 48 h. * P

    Journal: Cell Death & Disease

    Article Title: ER stress-mediated apoptosis induced by celastrol in cancer cells and important role of glycogen synthase kinase-3β in the signal network

    doi: 10.1038/cddis.2013.222

    Figure Lengend Snippet: Celastrol inhibits cell proliferation and induces apoptosis in HeLa cells. ( a ) Structure of the celastrol molecule. ( b ) Cell viability (MTT assay) of HeLa cells treated with various concentrations of celastrol for 48 h. ( c ) Representative results of flow cytometric analysis of apoptosis in cells treated with various concentrations of celastrol for 48 h. ( d ) Statistical analysis result of flow cytometric analysis of apoptosis. Annexin V-positive cells were accepted as apoptotic cells. ( e ) Levels of caspase-9, caspase-3, poly(ADP-ribose) polymerases (PARP) and X-linked inhibitor of apoptosis protein (XIAP) in cells treated with 1 μ M celastrol for 48 h. * P

    Article Snippet: Flow cytometric analysis of apoptosis induced by celastrol Flow cytometric analysis of cell apoptosis was conducted using apoptosis detection kit (Calbiochem, Merck KGaA, Darmstadt, Germany) according to the manufacturer's instructions.

    Techniques: MTT Assay, Flow Cytometry

    Role of GSK3 β in signal network of celastrol and celastrol-induced apoptosis. ( a ) The constructed minimum PPI network of celastrol containing 152 experimental identified proteins found in 2-DE and LC-MS and 34 intermediate proteins. The 152 proteins (red diamonds for LC-MS and cyan box dots for 2-DE) can link together into one network through direct interaction or only one intermediate partner (green ellipse dots). ( b ) Degree distribution map of the proteins in PPI network of celastrol. Experimental identified proteins are shown as diamonds, whereas the intermediate proteins are shown as round dots. In this graph, the colors were only related to degree in the network. GSK3 β exhibited to have the biggest degree in all experimental identified proteins. ( c ) Results of western blotting assay of time-dependent change in protein levels of GSK3 β and p-GSK3 β (ser9) in cells treated with 1 μ M celastrol. ( d ) Statistical analysis result of flow cytometric analysis of apoptosis in celastrol-treated cells (0.5, 1, 2, 4 μ M celastrol for 48 h) with or without pretreatment of LiCL (1 mM for 1 h). Annexin V-positive cells were accepted as apoptotic cells. * P

    Journal: Cell Death & Disease

    Article Title: ER stress-mediated apoptosis induced by celastrol in cancer cells and important role of glycogen synthase kinase-3β in the signal network

    doi: 10.1038/cddis.2013.222

    Figure Lengend Snippet: Role of GSK3 β in signal network of celastrol and celastrol-induced apoptosis. ( a ) The constructed minimum PPI network of celastrol containing 152 experimental identified proteins found in 2-DE and LC-MS and 34 intermediate proteins. The 152 proteins (red diamonds for LC-MS and cyan box dots for 2-DE) can link together into one network through direct interaction or only one intermediate partner (green ellipse dots). ( b ) Degree distribution map of the proteins in PPI network of celastrol. Experimental identified proteins are shown as diamonds, whereas the intermediate proteins are shown as round dots. In this graph, the colors were only related to degree in the network. GSK3 β exhibited to have the biggest degree in all experimental identified proteins. ( c ) Results of western blotting assay of time-dependent change in protein levels of GSK3 β and p-GSK3 β (ser9) in cells treated with 1 μ M celastrol. ( d ) Statistical analysis result of flow cytometric analysis of apoptosis in celastrol-treated cells (0.5, 1, 2, 4 μ M celastrol for 48 h) with or without pretreatment of LiCL (1 mM for 1 h). Annexin V-positive cells were accepted as apoptotic cells. * P

    Article Snippet: Flow cytometric analysis of apoptosis induced by celastrol Flow cytometric analysis of cell apoptosis was conducted using apoptosis detection kit (Calbiochem, Merck KGaA, Darmstadt, Germany) according to the manufacturer's instructions.

    Techniques: Construct, Liquid Chromatography with Mass Spectroscopy, Western Blot, Flow Cytometry