Structured Review

Millipore ionomycin
Islet-infiltrating B cells show no enrichment of regulatory cytokines. Pancreatic islets were isolated from mice aged 6–8 weeks ( a – c ) and 12–15 weeks ( d – f ) treated with control IgG (black lines/circles) or 2H7 anti-CD20 depleting antibody (grey lines/circles). ( a , d ) Number of CD19 + B cells. Data are expressed as mean ± SEM and numbers represent all islets recovered from individual pancreases. ( b , c , e , f ) Frequency of islet B cells stimulated with <t>PMA/ionomycin</t> expressing the intracellular cytokines IL-10 ( b , e ) and TGF-β ( c , f ). Horizontal lines represent medians. Black circles, control IgG; white squares, 2H7. ( g – l ) Multivariable analysis of B cells performed by SPICE software at 12 weeks ( g , h ) and 30 weeks ( i , j ) post depletion; ( g , i ) pie charts for controls; ( h , j ) pie charts for 2H7. Pie charts indicate different heterogeneous subsets; the coloured arcs correspond to the fraction of cells that express specific markers shown in the key. p = 0.2566 at 12 weeks and p = 0.1486 at 30 weeks (permutation test to compare the pie charts, performed by SPICE software). Graphical representations of heterogeneous subsets in pie slices are shown for 12 weeks ( k ) and 30 weeks ( l ) post depletion. Horizontal lines represent medians. Black circles, control IgG; white squares, 2H7. Cells were gated on CD8 − CD4 − CD19 + . B cell combinations where the frequency did not exceed 1% are not included in SPICE analysis. Data are representative of a minimum of four mice in each group, from at least two independent experiments. * p
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1) Product Images from "B cell depletion reduces T cell activation in pancreatic islets in a murine autoimmune diabetes model"

Article Title: B cell depletion reduces T cell activation in pancreatic islets in a murine autoimmune diabetes model

Journal: Diabetologia

doi: 10.1007/s00125-018-4597-z

Islet-infiltrating B cells show no enrichment of regulatory cytokines. Pancreatic islets were isolated from mice aged 6–8 weeks ( a – c ) and 12–15 weeks ( d – f ) treated with control IgG (black lines/circles) or 2H7 anti-CD20 depleting antibody (grey lines/circles). ( a , d ) Number of CD19 + B cells. Data are expressed as mean ± SEM and numbers represent all islets recovered from individual pancreases. ( b , c , e , f ) Frequency of islet B cells stimulated with PMA/ionomycin expressing the intracellular cytokines IL-10 ( b , e ) and TGF-β ( c , f ). Horizontal lines represent medians. Black circles, control IgG; white squares, 2H7. ( g – l ) Multivariable analysis of B cells performed by SPICE software at 12 weeks ( g , h ) and 30 weeks ( i , j ) post depletion; ( g , i ) pie charts for controls; ( h , j ) pie charts for 2H7. Pie charts indicate different heterogeneous subsets; the coloured arcs correspond to the fraction of cells that express specific markers shown in the key. p = 0.2566 at 12 weeks and p = 0.1486 at 30 weeks (permutation test to compare the pie charts, performed by SPICE software). Graphical representations of heterogeneous subsets in pie slices are shown for 12 weeks ( k ) and 30 weeks ( l ) post depletion. Horizontal lines represent medians. Black circles, control IgG; white squares, 2H7. Cells were gated on CD8 − CD4 − CD19 + . B cell combinations where the frequency did not exceed 1% are not included in SPICE analysis. Data are representative of a minimum of four mice in each group, from at least two independent experiments. * p
Figure Legend Snippet: Islet-infiltrating B cells show no enrichment of regulatory cytokines. Pancreatic islets were isolated from mice aged 6–8 weeks ( a – c ) and 12–15 weeks ( d – f ) treated with control IgG (black lines/circles) or 2H7 anti-CD20 depleting antibody (grey lines/circles). ( a , d ) Number of CD19 + B cells. Data are expressed as mean ± SEM and numbers represent all islets recovered from individual pancreases. ( b , c , e , f ) Frequency of islet B cells stimulated with PMA/ionomycin expressing the intracellular cytokines IL-10 ( b , e ) and TGF-β ( c , f ). Horizontal lines represent medians. Black circles, control IgG; white squares, 2H7. ( g – l ) Multivariable analysis of B cells performed by SPICE software at 12 weeks ( g , h ) and 30 weeks ( i , j ) post depletion; ( g , i ) pie charts for controls; ( h , j ) pie charts for 2H7. Pie charts indicate different heterogeneous subsets; the coloured arcs correspond to the fraction of cells that express specific markers shown in the key. p = 0.2566 at 12 weeks and p = 0.1486 at 30 weeks (permutation test to compare the pie charts, performed by SPICE software). Graphical representations of heterogeneous subsets in pie slices are shown for 12 weeks ( k ) and 30 weeks ( l ) post depletion. Horizontal lines represent medians. Black circles, control IgG; white squares, 2H7. Cells were gated on CD8 − CD4 − CD19 + . B cell combinations where the frequency did not exceed 1% are not included in SPICE analysis. Data are representative of a minimum of four mice in each group, from at least two independent experiments. * p

Techniques Used: Isolation, Mouse Assay, Expressing, Software

2) Product Images from "EB1 binding restricts STIM1 translocation to ER–PM junctions and regulates store-operated Ca2+ entry"

Article Title: EB1 binding restricts STIM1 translocation to ER–PM junctions and regulates store-operated Ca2+ entry

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201711151

EB1 binding impedes STIM1 translocation to ER–PM junctions and Orai1 recruitment during ER Ca 2+ depletion. (A) Translocation of YFP-STIM1 and YFP-STIM1-TRNN to ER–PM junctions after 1 µM ionomycin treatment in HeLa cells, monitored by TIRF microscopy. Bar, 2 µm. (B) Relative translocation to ER–PM junctions of YFP-STIM1 and YFP-STIM1-TRNN as described in A. 14–15 cells from three independent experiments. Mean times to half-maximal translocation ( t 1/2 ) are indicated. (C) Relative translocation to ER–PM junctions of YFP-STIM1 subtypes and corresponding Orai1-mCherry after 1 µM TG treatment in HeLa cells, monitored by TIRF microscopy. Black, YFP-STIM1 coexpressed with Orai1-mCherry; red, STIM1-TRNN coexpressed with Orai1-mCherry. Mean traces are shown (15–23 cells from three to four independent experiments). (D) Time to the half-maximal translocation ( t 1/2 ) of YFP-STIM1 subtypes and Orai1-mCherry as described in C. Means ± SEM are shown. **, P
Figure Legend Snippet: EB1 binding impedes STIM1 translocation to ER–PM junctions and Orai1 recruitment during ER Ca 2+ depletion. (A) Translocation of YFP-STIM1 and YFP-STIM1-TRNN to ER–PM junctions after 1 µM ionomycin treatment in HeLa cells, monitored by TIRF microscopy. Bar, 2 µm. (B) Relative translocation to ER–PM junctions of YFP-STIM1 and YFP-STIM1-TRNN as described in A. 14–15 cells from three independent experiments. Mean times to half-maximal translocation ( t 1/2 ) are indicated. (C) Relative translocation to ER–PM junctions of YFP-STIM1 subtypes and corresponding Orai1-mCherry after 1 µM TG treatment in HeLa cells, monitored by TIRF microscopy. Black, YFP-STIM1 coexpressed with Orai1-mCherry; red, STIM1-TRNN coexpressed with Orai1-mCherry. Mean traces are shown (15–23 cells from three to four independent experiments). (D) Time to the half-maximal translocation ( t 1/2 ) of YFP-STIM1 subtypes and Orai1-mCherry as described in C. Means ± SEM are shown. **, P

Techniques Used: Binding Assay, Translocation Assay, Microscopy

Disruption of STIM1–EB1 interaction facilitated SOCE and resulted in ER Ca 2+ store overload. (A) Relative changes in cytosolic Ca 2+ concentration after 100 µM histamine and 1 µM TG treatment, monitored by Fura-2 ratio in HeLa cells transfected with siControl or siEB1. Four independent experiments. (B) SOCE triggered by 100 µM histamine and 1 µM TG treatment, monitored by Fura-2 ratio in HeLa cells transfected with siControl or siEB1. Three independent experiments. (C) Peak of SOCE in HeLa cells treated with siControl or siEB1 as described in B. Three independent experiments. (D) Slope of SOCE in HeLa cells treated with siControl or siEB1 as described in B. Three independent experiments. (E) SOCE triggered by 100 µM histamine and 1 µM TG treatment, monitored by Fura-2 ratio in HeLa cells transfected with YFP-STIM1 or YFP-STIM1-TRNN. Three independent experiments. (F) Peak of SOCE in HeLa cells transfected with YFP-STIM1 and YFP-STIM1-TRNN as described in E. Three independent experiments. (G) Slope of SOCE in HeLa cells transfected with YFP-STIM1 and YFP-STIM1-TRNN as described in E. Three independent experiments. (H) Relative changes in cytosolic Ca 2+ concentration in response to depletion and readdition of extracellular Ca 2+ , monitored by Fura-2 ratio in HeLa cells transfected with YFP-TM, YFP-STIM1-D76A, or YFP-STIM1-D76A-TRNN. Three to four independent experiments. (I) Peak ER Ca 2+ release by 1 µM ionomycin treatment in the absence of extracellular Ca 2+ , monitored by Fura-2 ratio in HeLa cells transfected with siControl or siEB1. Three independent experiments. (J) Relative ER Ca 2+ levels in the resting state (phase I), after 5 µM BHQ treatment (phase II), and after BHQ washout (phase III and IV), monitored by D1ER in HeLa cells transfected with mCherry-STIM1 or mCherry-STIM1-TRNN. 16–26 cells from three independent experiments. *, P
Figure Legend Snippet: Disruption of STIM1–EB1 interaction facilitated SOCE and resulted in ER Ca 2+ store overload. (A) Relative changes in cytosolic Ca 2+ concentration after 100 µM histamine and 1 µM TG treatment, monitored by Fura-2 ratio in HeLa cells transfected with siControl or siEB1. Four independent experiments. (B) SOCE triggered by 100 µM histamine and 1 µM TG treatment, monitored by Fura-2 ratio in HeLa cells transfected with siControl or siEB1. Three independent experiments. (C) Peak of SOCE in HeLa cells treated with siControl or siEB1 as described in B. Three independent experiments. (D) Slope of SOCE in HeLa cells treated with siControl or siEB1 as described in B. Three independent experiments. (E) SOCE triggered by 100 µM histamine and 1 µM TG treatment, monitored by Fura-2 ratio in HeLa cells transfected with YFP-STIM1 or YFP-STIM1-TRNN. Three independent experiments. (F) Peak of SOCE in HeLa cells transfected with YFP-STIM1 and YFP-STIM1-TRNN as described in E. Three independent experiments. (G) Slope of SOCE in HeLa cells transfected with YFP-STIM1 and YFP-STIM1-TRNN as described in E. Three independent experiments. (H) Relative changes in cytosolic Ca 2+ concentration in response to depletion and readdition of extracellular Ca 2+ , monitored by Fura-2 ratio in HeLa cells transfected with YFP-TM, YFP-STIM1-D76A, or YFP-STIM1-D76A-TRNN. Three to four independent experiments. (I) Peak ER Ca 2+ release by 1 µM ionomycin treatment in the absence of extracellular Ca 2+ , monitored by Fura-2 ratio in HeLa cells transfected with siControl or siEB1. Three independent experiments. (J) Relative ER Ca 2+ levels in the resting state (phase I), after 5 µM BHQ treatment (phase II), and after BHQ washout (phase III and IV), monitored by D1ER in HeLa cells transfected with mCherry-STIM1 or mCherry-STIM1-TRNN. 16–26 cells from three independent experiments. *, P

Techniques Used: Concentration Assay, Transfection

3) Product Images from "Interleukin-4 Receptor Alpha Expressing B Cells Are Essential to Down-Modulate Host Granulomatous Inflammation During Schistosomasis"

Article Title: Interleukin-4 Receptor Alpha Expressing B Cells Are Essential to Down-Modulate Host Granulomatous Inflammation During Schistosomasis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02928

Impaired Th2 immunity in mice lacking IL-4 producing B cells. Bone marrow chimeras were infected with 100 live S. mansoni cercariae and killed 7 weeks post-infection. Single cell suspensions were prepared from MLN and cells were restimulated with 20 μg/ml SEA or α-CD3 in vitro . (A,B) Cytokine production by restimulated total MLN cells was detected by ELISA. (C) Frequency of CD19 + B220 + B cells in the gut draining lymph node. (D,E) Detection of intracellular cytokines produced by CD19 + B cells after restimulation of total MLN cells with 50 ng/ml PMA and 250 ng/ml ionomycin. (F) Frequency of CD3 + CD4 + T cells in the MLN. (G,H) Intracellular cytokine production by CD3 + CD4 + T cells after stimulation of total MLN cells with 50 ng/ml PMA and 250 ng/ml ionomycin. (I) Frequency of CD3 + CD4 + T cells expressing Gata-3. (J) Dot plot showing gating on IL-4 producing CD4 + T cells by infected WT, B-IL-4 −/− and IL-4 −/− chimeras. Data are representative of two independent experiments. n = 4–6 mice.
Figure Legend Snippet: Impaired Th2 immunity in mice lacking IL-4 producing B cells. Bone marrow chimeras were infected with 100 live S. mansoni cercariae and killed 7 weeks post-infection. Single cell suspensions were prepared from MLN and cells were restimulated with 20 μg/ml SEA or α-CD3 in vitro . (A,B) Cytokine production by restimulated total MLN cells was detected by ELISA. (C) Frequency of CD19 + B220 + B cells in the gut draining lymph node. (D,E) Detection of intracellular cytokines produced by CD19 + B cells after restimulation of total MLN cells with 50 ng/ml PMA and 250 ng/ml ionomycin. (F) Frequency of CD3 + CD4 + T cells in the MLN. (G,H) Intracellular cytokine production by CD3 + CD4 + T cells after stimulation of total MLN cells with 50 ng/ml PMA and 250 ng/ml ionomycin. (I) Frequency of CD3 + CD4 + T cells expressing Gata-3. (J) Dot plot showing gating on IL-4 producing CD4 + T cells by infected WT, B-IL-4 −/− and IL-4 −/− chimeras. Data are representative of two independent experiments. n = 4–6 mice.

Techniques Used: Mouse Assay, Infection, In Vitro, Enzyme-linked Immunosorbent Assay, Produced, Expressing

Abrogated cellular immunity in mice lacking IL-4 producing B cells after synchronous S. mansoni eggs challenged. Single cell suspensions were prepared from mediastinal lymph nodes (MST) and cells were stained for flow cytometry. (A,B) Intracellular cytokine detection after stimulating total MST cells with 50 ng/ml PMA and 250 ng/ml ionomycin in vitro . (C) Total number of follicular B cells (FO, CD19 + CD23 hi CD21 hi ) cells recruited to the mediastinal lymph node (MST). (D,E) Total number of CD4 + T cells producing IL-4 and IL-10 in the MST. (F) Total number of CXCR5 + T follicular helper (T FH ) cells in the lung draining lymph nodes. Data are representative of two independent experiments. * p
Figure Legend Snippet: Abrogated cellular immunity in mice lacking IL-4 producing B cells after synchronous S. mansoni eggs challenged. Single cell suspensions were prepared from mediastinal lymph nodes (MST) and cells were stained for flow cytometry. (A,B) Intracellular cytokine detection after stimulating total MST cells with 50 ng/ml PMA and 250 ng/ml ionomycin in vitro . (C) Total number of follicular B cells (FO, CD19 + CD23 hi CD21 hi ) cells recruited to the mediastinal lymph node (MST). (D,E) Total number of CD4 + T cells producing IL-4 and IL-10 in the MST. (F) Total number of CXCR5 + T follicular helper (T FH ) cells in the lung draining lymph nodes. Data are representative of two independent experiments. * p

Techniques Used: Mouse Assay, Microscale Thermophoresis, Staining, Flow Cytometry, Cytometry, In Vitro

4) Product Images from "TFII-I Regulates Induction of Chromosomally Integrated Human Immunodeficiency Virus Type 1 Long Terminal Repeat in Cooperation with USF"

Article Title: TFII-I Regulates Induction of Chromosomally Integrated Human Immunodeficiency Virus Type 1 Long Terminal Repeat in Cooperation with USF

Journal: Journal of Virology

doi: 10.1128/JVI.79.7.4396-4406.2005

RBEIII is required for response of integrated HIV-1 LTR to T-cell activation signals. (A) Luciferase activities were measured from stable clones of Jurkat cells transfected with WT LTR-luciferase (W1, W2, W7, W14, W24, and W26) or RBEIII mutant LTR-luciferase (M1, M2, M3, M4, M10, and M20) or from pools of cells with stably transfected wild-type (WP) or RBEIII mutant (MP) reporters. Cells were untreated or were stimulated with PMA, TSA, and ionomycin. (B) Luciferase activities was measured in representative clones of stably transfected Jurkat cells bearing WT LTR-luciferase (W26, closed bars) or RBEIII mutant LTR-luciferase (M2, open bars) following treatment with TSA, TNF-α, PMA, PMA-TSA-ionomycin, T cell receptor cross-linking (TCR), or TNF-α--PMA. The results are presented as fold activities relative to untreated samples. (C) Stably transfected Jurkat clones bearing WT LTR-luciferase (W26, WT) or RBEIII mutant LTR-luciferase (M2, Mut.) were analyzed by FACS with anti-CD69 antibodies. Cells were unstimulated or were treated with PMA-TSA-ionomycin (PMA) or by cross-linking of the T-cell receptor (TCR).
Figure Legend Snippet: RBEIII is required for response of integrated HIV-1 LTR to T-cell activation signals. (A) Luciferase activities were measured from stable clones of Jurkat cells transfected with WT LTR-luciferase (W1, W2, W7, W14, W24, and W26) or RBEIII mutant LTR-luciferase (M1, M2, M3, M4, M10, and M20) or from pools of cells with stably transfected wild-type (WP) or RBEIII mutant (MP) reporters. Cells were untreated or were stimulated with PMA, TSA, and ionomycin. (B) Luciferase activities was measured in representative clones of stably transfected Jurkat cells bearing WT LTR-luciferase (W26, closed bars) or RBEIII mutant LTR-luciferase (M2, open bars) following treatment with TSA, TNF-α, PMA, PMA-TSA-ionomycin, T cell receptor cross-linking (TCR), or TNF-α--PMA. The results are presented as fold activities relative to untreated samples. (C) Stably transfected Jurkat clones bearing WT LTR-luciferase (W26, WT) or RBEIII mutant LTR-luciferase (M2, Mut.) were analyzed by FACS with anti-CD69 antibodies. Cells were unstimulated or were treated with PMA-TSA-ionomycin (PMA) or by cross-linking of the T-cell receptor (TCR).

Techniques Used: Activation Assay, Luciferase, Clone Assay, Transfection, Mutagenesis, Stable Transfection, FACS

USF1, USF2, and TFII-I are bound to RBEIII on the integrated WT HIV-1 LTR in unstimulated cells. (A) Chromatin immunoprecipitation was performed with formaldehyde cross-linked stable Jurkat clones bearing the RBEIII mutant LTR-luciferase (i and ii) or WT LTR-luciferase (iii and iv) reporter by the use of antibodies against USF1 (lane 3), USF2 (lane 4), TFII-I (lane 5), or NF-κB (lane 6). Samples were analyzed by PCR with oligonucleotides specific for the HIV-1 LTR flanking the RBEIII region (−220 to −1) and the β-globin promoter. A 100-bp ladder is shown in lane 1, and the input samples are shown in lane 2. Cells were untreated (i and iii) or stimulated with PMA-TSA-ionomycin (ii and iv).(B) The results from panel A were quantitated from a phosphorimager scan and normalized relative to the input sample and a β-globin internal control.
Figure Legend Snippet: USF1, USF2, and TFII-I are bound to RBEIII on the integrated WT HIV-1 LTR in unstimulated cells. (A) Chromatin immunoprecipitation was performed with formaldehyde cross-linked stable Jurkat clones bearing the RBEIII mutant LTR-luciferase (i and ii) or WT LTR-luciferase (iii and iv) reporter by the use of antibodies against USF1 (lane 3), USF2 (lane 4), TFII-I (lane 5), or NF-κB (lane 6). Samples were analyzed by PCR with oligonucleotides specific for the HIV-1 LTR flanking the RBEIII region (−220 to −1) and the β-globin promoter. A 100-bp ladder is shown in lane 1, and the input samples are shown in lane 2. Cells were untreated (i and iii) or stimulated with PMA-TSA-ionomycin (ii and iv).(B) The results from panel A were quantitated from a phosphorimager scan and normalized relative to the input sample and a β-globin internal control.

Techniques Used: Chromatin Immunoprecipitation, Clone Assay, Mutagenesis, Luciferase, Polymerase Chain Reaction

USF and TFII-I are required for induction of the integrated HIV-1 LTR. (A) Luciferase activities were measured from COS-1 cells cotransfected with minimal TK-luciferase reporter constructs bearing four upstream direct tandem repeats of the WT RBEIII oligonucleotide P3 (pTA-P3WT, closed bars) or the RBEIII mutant oligonucleotide P3M (pTA-P3mut, open bars) and plasmids expressing USF1, USF2, TFII-I, both USF1 and USF2, both USF1 and TFII-I, or a vector control (vector). (B) Jurkat clone W26 bearing the stably integrated WT LTR-luciferase reporter gene was cotransfected with a vector control or a plasmid expressing dominant negative USF (DN-USF), TFII-I (DN-TFII-I), or I-κb (DN-IκB) and a GFP expression plasmid at a 10:1 molar ratio. Transfected cells were enriched by sorting for GFP fluorescence, and luciferase activities were measured from unstimulated cells or following treatment with PMA-TSA-ionomycin. Activities are presented as fold stimulations relative to the unstimulated vector control.
Figure Legend Snippet: USF and TFII-I are required for induction of the integrated HIV-1 LTR. (A) Luciferase activities were measured from COS-1 cells cotransfected with minimal TK-luciferase reporter constructs bearing four upstream direct tandem repeats of the WT RBEIII oligonucleotide P3 (pTA-P3WT, closed bars) or the RBEIII mutant oligonucleotide P3M (pTA-P3mut, open bars) and plasmids expressing USF1, USF2, TFII-I, both USF1 and USF2, both USF1 and TFII-I, or a vector control (vector). (B) Jurkat clone W26 bearing the stably integrated WT LTR-luciferase reporter gene was cotransfected with a vector control or a plasmid expressing dominant negative USF (DN-USF), TFII-I (DN-TFII-I), or I-κb (DN-IκB) and a GFP expression plasmid at a 10:1 molar ratio. Transfected cells were enriched by sorting for GFP fluorescence, and luciferase activities were measured from unstimulated cells or following treatment with PMA-TSA-ionomycin. Activities are presented as fold stimulations relative to the unstimulated vector control.

Techniques Used: Luciferase, Construct, Mutagenesis, Expressing, Plasmid Preparation, Stable Transfection, Dominant Negative Mutation, Transfection, Fluorescence

5) Product Images from "The Costimulatory Molecule ICOS Regulates Host Th1 and Follicular Th Cell Differentiation in Response to Plasmodium chabaudi chabaudi AS Infection"

Article Title: The Costimulatory Molecule ICOS Regulates Host Th1 and Follicular Th Cell Differentiation in Response to Plasmodium chabaudi chabaudi AS Infection

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1403206

Proinflammatory cytokine production is augmented in Icos −/− mice. ELISAs of serum ( A ) IL-12p40, ( B ) IL-12p70, and ( C ) IFN-γ (D6: p = 0.010]). ( D and E ) Cumulative data showing representative IFN-γ production at day 6 p.i., and percent and total number of IFN-γ + CD4 + or CD8 + T cells after ex vivo stimulation with PMA and ionomycin in the presence of brefeldin A. Vertical bold text indicates median fluorescence intensity of gated area. (D) FACS plots gated on live, CD4 + T cells (% D6: p = 0.016, D11: p = 0.031, total number D6: p = 0.031]. (E) FACS plots gated on live, CD8 + T cells (% D6: p = 0.016; total number D6: p = 0.016, D11: p = 0.029). Data are pooled from three independent experiments: (A and C) n = 8–12 mice/group, (B) n = 4–10 mice/group. Data are representative of three independent experiments (D and E); n = 3–5 mice/group. Symbols represent individual mice. Error bars represent SEM. * p
Figure Legend Snippet: Proinflammatory cytokine production is augmented in Icos −/− mice. ELISAs of serum ( A ) IL-12p40, ( B ) IL-12p70, and ( C ) IFN-γ (D6: p = 0.010]). ( D and E ) Cumulative data showing representative IFN-γ production at day 6 p.i., and percent and total number of IFN-γ + CD4 + or CD8 + T cells after ex vivo stimulation with PMA and ionomycin in the presence of brefeldin A. Vertical bold text indicates median fluorescence intensity of gated area. (D) FACS plots gated on live, CD4 + T cells (% D6: p = 0.016, D11: p = 0.031, total number D6: p = 0.031]. (E) FACS plots gated on live, CD8 + T cells (% D6: p = 0.016; total number D6: p = 0.016, D11: p = 0.029). Data are pooled from three independent experiments: (A and C) n = 8–12 mice/group, (B) n = 4–10 mice/group. Data are representative of three independent experiments (D and E); n = 3–5 mice/group. Symbols represent individual mice. Error bars represent SEM. * p

Techniques Used: Mouse Assay, Ex Vivo, Fluorescence, FACS

Icos −/− T FH s fail to expand past day 6 p.i. ( A ) Cumulative data showing representative PD-1 and CXCR5 expression. FACS plots gated on live, CD4 + T cells. ( B ) Bcl6 expression of gated area in (A). ( C ) Cumulative data showing representative IL-21 production at day 6 p.i., and percent IL-21 + CD4 + T cells after ex vivo stimulation with PMA and ionomycin in the presence of brefeldin A. FACS plots gated on live, CD4 + T cells (D21: p = 0.0006). ( D ) Total number of CD4 + PD-1 + Bcl6 + CXCR5 + cells per spleen (D11: p = 0.032, D21: p = 0.0006). ( E ) Frequency of Ki-67 + CD4 + T cells of gated area in (A) (D21: p = 0.018). ( F ) Cumulative data showing representative PI staining and Annexin V expression and percent PI + Annexin V + CD4 + PD-1 + CXCR5 + T FH s at day 11 p.i. (D11 p = 0.002). Data are representative of three independent experiments (A–D); n = 4–5 mice/group. Data are pooled from two independent experiments (E and F); n = 4–8 mice/group. Symbols (C–F) represent individual mice. Error bars represent SEM. * p
Figure Legend Snippet: Icos −/− T FH s fail to expand past day 6 p.i. ( A ) Cumulative data showing representative PD-1 and CXCR5 expression. FACS plots gated on live, CD4 + T cells. ( B ) Bcl6 expression of gated area in (A). ( C ) Cumulative data showing representative IL-21 production at day 6 p.i., and percent IL-21 + CD4 + T cells after ex vivo stimulation with PMA and ionomycin in the presence of brefeldin A. FACS plots gated on live, CD4 + T cells (D21: p = 0.0006). ( D ) Total number of CD4 + PD-1 + Bcl6 + CXCR5 + cells per spleen (D11: p = 0.032, D21: p = 0.0006). ( E ) Frequency of Ki-67 + CD4 + T cells of gated area in (A) (D21: p = 0.018). ( F ) Cumulative data showing representative PI staining and Annexin V expression and percent PI + Annexin V + CD4 + PD-1 + CXCR5 + T FH s at day 11 p.i. (D11 p = 0.002). Data are representative of three independent experiments (A–D); n = 4–5 mice/group. Data are pooled from two independent experiments (E and F); n = 4–8 mice/group. Symbols (C–F) represent individual mice. Error bars represent SEM. * p

Techniques Used: Expressing, FACS, Ex Vivo, Staining, Mouse Assay

6) Product Images from "Enhanced interleukin-4 production in CD4+ T cells and elevated immunoglobulin E levels in antigen-primed mice by bisphenol A and nonylphenol, endocrine disruptors: involvement of nuclear factor-AT and Ca2+"

Article Title: Enhanced interleukin-4 production in CD4+ T cells and elevated immunoglobulin E levels in antigen-primed mice by bisphenol A and nonylphenol, endocrine disruptors: involvement of nuclear factor-AT and Ca2+

Journal: Immunology

doi: 10.1046/j.1365-2567.2003.01631.x

BPA or NP enhances the PMA/ionomycin-induced binding of nuclear proteins to the NF-AT site, but not the AP-1 site. Nuclear extracts were prepared from resting and PMA/ionomycin-activated EL4 cells treated for 24 hr with varying concentrations of BPA or NP, and then analysed by EMSA using a radiolabelled oligonucleotide encompassing the NF-AT (a) or AP-1 (b), respectively. S and NS indicate the presence of an unlabelled, specific oligonucleotide (NF-AT or AP-1) and non-specific oligonucleotide (CRE or NF-κB), respectively. The specific NF-AT or AP-1 complexes are indicated. Similar results were obtained in two additional experiments.
Figure Legend Snippet: BPA or NP enhances the PMA/ionomycin-induced binding of nuclear proteins to the NF-AT site, but not the AP-1 site. Nuclear extracts were prepared from resting and PMA/ionomycin-activated EL4 cells treated for 24 hr with varying concentrations of BPA or NP, and then analysed by EMSA using a radiolabelled oligonucleotide encompassing the NF-AT (a) or AP-1 (b), respectively. S and NS indicate the presence of an unlabelled, specific oligonucleotide (NF-AT or AP-1) and non-specific oligonucleotide (CRE or NF-κB), respectively. The specific NF-AT or AP-1 complexes are indicated. Similar results were obtained in two additional experiments.

Techniques Used: Binding Assay

Effect of BPA or NP on IL-4 gene promoter activity stimulated by PMA/ionomycin. (a) Schematic representation of murine IL-4 promoter constructs containing P0–P4 NF-AT elements. (b) EL4 cells were transiently transfected with the IL-4 promoter constructs, followed by stimulation for 24 hr with PMA/ionomycin in the presence of BPA or NP. Afterward, the cells were lysed and reporter gene expression was analysed by luminometry. The results are represented as induction fold over the value obtained with the unstimulated EL4 cells transfected with each of promoter constructs, given as an arbitrary value of 1. The data are representative of three independent experiments.
Figure Legend Snippet: Effect of BPA or NP on IL-4 gene promoter activity stimulated by PMA/ionomycin. (a) Schematic representation of murine IL-4 promoter constructs containing P0–P4 NF-AT elements. (b) EL4 cells were transiently transfected with the IL-4 promoter constructs, followed by stimulation for 24 hr with PMA/ionomycin in the presence of BPA or NP. Afterward, the cells were lysed and reporter gene expression was analysed by luminometry. The results are represented as induction fold over the value obtained with the unstimulated EL4 cells transfected with each of promoter constructs, given as an arbitrary value of 1. The data are representative of three independent experiments.

Techniques Used: Activity Assay, Construct, Transfection, Expressing

7) Product Images from "Plasma membrane calcium ATPase regulates bone mass by fine-tuning osteoclast differentiation and survival"

Article Title: Plasma membrane calcium ATPase regulates bone mass by fine-tuning osteoclast differentiation and survival

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201204067

PMCA reduced the RANKL-dependent Ca 2+ oscillations and subsequent NFATc1 nuclear localization in preosteoclasts. (A–C) BMMs on glass coverslips were transfected with scrambled control siRNA or PMCA siRNA and further incubated for 2 d with 30 ng/ml M-CSF and 100 ng/ml RANKL. (A) Cells were loaded with Fura-2/AM for the recording of Ca 2+ oscillations (presented as a ratio of maximal fluorescence) in individual preosteoclast (BMMs treated with RANKL for 2 d). Each colored line represents Ca 2+ oscillations in a single cell. 10 µM ionomycin was added at the end of the experiment (arrow) to determine the maximal Ca 2+ -induced fluorescence. (B and C) Cells were stained with NFATc1 antibody (FITC labeled) and lamin B antibody (Cy3 labeled) to detect the nuclear localization of NFATc1 (arrows). Treatment of 1 µM cyclosporineA (CyA) for 3 h induced exclusive translocation of NFATc1 to the cytosol (arrowheads). Bars, 50 µm. (D) NFATc1 localization was examined biochemically by subjecting nuclear fractions from the cells treated as in B to Western blotting. (E and F) PMCA1 (pcDNA-rPMCA1) or PMCA4 (pMX-PMCA4) were overexpressed in BMMs. (E) Ca 2+ oscillations were monitored in BMMs after RANKL treatment for 2 d (top). TRAP staining was performed after culturing the cells for 4 d (bottom). Bars, 200 µm. (F) PMCA1 or PMCA4 expression levels were examined by Western blotting after culturing the cells for 2 d. (G) The number of TRAP-positive multinucleated cells was counted from E. Data are means ± SD, representative of more than three experiments performed in triplicate (**, P
Figure Legend Snippet: PMCA reduced the RANKL-dependent Ca 2+ oscillations and subsequent NFATc1 nuclear localization in preosteoclasts. (A–C) BMMs on glass coverslips were transfected with scrambled control siRNA or PMCA siRNA and further incubated for 2 d with 30 ng/ml M-CSF and 100 ng/ml RANKL. (A) Cells were loaded with Fura-2/AM for the recording of Ca 2+ oscillations (presented as a ratio of maximal fluorescence) in individual preosteoclast (BMMs treated with RANKL for 2 d). Each colored line represents Ca 2+ oscillations in a single cell. 10 µM ionomycin was added at the end of the experiment (arrow) to determine the maximal Ca 2+ -induced fluorescence. (B and C) Cells were stained with NFATc1 antibody (FITC labeled) and lamin B antibody (Cy3 labeled) to detect the nuclear localization of NFATc1 (arrows). Treatment of 1 µM cyclosporineA (CyA) for 3 h induced exclusive translocation of NFATc1 to the cytosol (arrowheads). Bars, 50 µm. (D) NFATc1 localization was examined biochemically by subjecting nuclear fractions from the cells treated as in B to Western blotting. (E and F) PMCA1 (pcDNA-rPMCA1) or PMCA4 (pMX-PMCA4) were overexpressed in BMMs. (E) Ca 2+ oscillations were monitored in BMMs after RANKL treatment for 2 d (top). TRAP staining was performed after culturing the cells for 4 d (bottom). Bars, 200 µm. (F) PMCA1 or PMCA4 expression levels were examined by Western blotting after culturing the cells for 2 d. (G) The number of TRAP-positive multinucleated cells was counted from E. Data are means ± SD, representative of more than three experiments performed in triplicate (**, P

Techniques Used: Transfection, Incubation, Fluorescence, Staining, Labeling, Translocation Assay, Western Blot, Expressing

8) Product Images from "ChIP-on-chip analysis identifies IL-22 as direct target gene of ectopically expressed FOXP3 transcription factor in human T cells"

Article Title: ChIP-on-chip analysis identifies IL-22 as direct target gene of ectopically expressed FOXP3 transcription factor in human T cells

Journal: BMC Genomics

doi: 10.1186/1471-2164-13-705

Validation of FOXP3-dependent gene repression. Expression of IL-22, IL-26 and TGF-β2 in resting and PMA/ionomycin-stimulated J-FOXP3 T cells by quantitative real-time RT PCR. Bars represent mean ± SD of duplicate measurements and are representative for two independent experiments.
Figure Legend Snippet: Validation of FOXP3-dependent gene repression. Expression of IL-22, IL-26 and TGF-β2 in resting and PMA/ionomycin-stimulated J-FOXP3 T cells by quantitative real-time RT PCR. Bars represent mean ± SD of duplicate measurements and are representative for two independent experiments.

Techniques Used: Expressing, Quantitative RT-PCR

Expression of FOXP3 and IL-22 in ex vivo isolated human T cells. Human CD4 + CD25 - (T naiv ) and CD4 + CD25 + (T reg ) cells were isolated from healthy donors (n = 6-8) using AutoMACS technology and were either stimulated with PMA and ionomycin for 4 h or left untreated. Cells were subsequently analyzed by quantitative real-time RT-PCR ( A ) Expression of FOXP3 normalized to median expression of resting T naiv . ( B ) Fold induction of IL-22 comparing stimulated vs. resting cells. The median and individual measurements are shown. Asterisk indicates p
Figure Legend Snippet: Expression of FOXP3 and IL-22 in ex vivo isolated human T cells. Human CD4 + CD25 - (T naiv ) and CD4 + CD25 + (T reg ) cells were isolated from healthy donors (n = 6-8) using AutoMACS technology and were either stimulated with PMA and ionomycin for 4 h or left untreated. Cells were subsequently analyzed by quantitative real-time RT-PCR ( A ) Expression of FOXP3 normalized to median expression of resting T naiv . ( B ) Fold induction of IL-22 comparing stimulated vs. resting cells. The median and individual measurements are shown. Asterisk indicates p

Techniques Used: Expressing, Ex Vivo, Isolation, Quantitative RT-PCR

IL-2 expression in resting and PMA/ionomycin–stimulated Jurkat cell lines. ( A ) Quantitative real-time RT-PCR for IL-2 expression. Bars represent mean ± SD of duplicate measurements. ( B ) FACS analysis of intracellular IL-2 staining. All data are representative of at least two independent experiments.
Figure Legend Snippet: IL-2 expression in resting and PMA/ionomycin–stimulated Jurkat cell lines. ( A ) Quantitative real-time RT-PCR for IL-2 expression. Bars represent mean ± SD of duplicate measurements. ( B ) FACS analysis of intracellular IL-2 staining. All data are representative of at least two independent experiments.

Techniques Used: Expressing, Quantitative RT-PCR, FACS, Staining

Cluster analysis of FOXP3-dependent expressional changes in resting and PMA/ionomycin–stimulated J-FOXP3 and J-GFP cells. Color coding represents z -scores of normalized expression signal intensities. Black boxes indicate the presence of at least one FOXP3 ChIP region within the transcript’s promoter region in the range of -10 kb to +2.45 kb around the TSS as determined by ChIP-on-chip analysis and GPAT annotation of resting and stimulated J-FOXP3 cells.
Figure Legend Snippet: Cluster analysis of FOXP3-dependent expressional changes in resting and PMA/ionomycin–stimulated J-FOXP3 and J-GFP cells. Color coding represents z -scores of normalized expression signal intensities. Black boxes indicate the presence of at least one FOXP3 ChIP region within the transcript’s promoter region in the range of -10 kb to +2.45 kb around the TSS as determined by ChIP-on-chip analysis and GPAT annotation of resting and stimulated J-FOXP3 cells.

Techniques Used: Expressing, Chromatin Immunoprecipitation

IL-22 gene locus on chromosome 12 in more detail. Generated ChIP-on-Chip data within the tiling array’s coverage range of the IL22 promoter alongside with RefSeq annotation. ( A ) ChIP peaks represent -10 x log 10 of p-values derived from the local comparison of probe signal intensities from tiling microarrays of FOXP3-specific and isotype control IPs from two independent experiments using resting and PMA/ionomycin–stimulated J-FOXP3 cells. Positional overlap of ChIP regions above the p-value threshold (95th percentile) in the two replicate ChIP experiments is summarized in red below. ( B ) Sequence of summarized ChIP region chr12.119 identified in A and aligned with FOXP3 consensus sequence [ 8 ]. Chromosomal coordinates refer to hg18 genomic reference assembly. ( C ) Fold enrichment of ChIP region chr12.119 in FOXP3-precipitated J-FOXP3 material as determined by genomic quantitative real-time PCR.
Figure Legend Snippet: IL-22 gene locus on chromosome 12 in more detail. Generated ChIP-on-Chip data within the tiling array’s coverage range of the IL22 promoter alongside with RefSeq annotation. ( A ) ChIP peaks represent -10 x log 10 of p-values derived from the local comparison of probe signal intensities from tiling microarrays of FOXP3-specific and isotype control IPs from two independent experiments using resting and PMA/ionomycin–stimulated J-FOXP3 cells. Positional overlap of ChIP regions above the p-value threshold (95th percentile) in the two replicate ChIP experiments is summarized in red below. ( B ) Sequence of summarized ChIP region chr12.119 identified in A and aligned with FOXP3 consensus sequence [ 8 ]. Chromosomal coordinates refer to hg18 genomic reference assembly. ( C ) Fold enrichment of ChIP region chr12.119 in FOXP3-precipitated J-FOXP3 material as determined by genomic quantitative real-time PCR.

Techniques Used: Generated, Chromatin Immunoprecipitation, Derivative Assay, Sequencing, Real-time Polymerase Chain Reaction

9) Product Images from "Microgravity Inhibits Resting T Cell Immunity in an Exposure Time-Dependent Manner"

Article Title: Microgravity Inhibits Resting T Cell Immunity in an Exposure Time-Dependent Manner

Journal: International Journal of Medical Sciences

doi: 10.7150/ijms.7651

The cytokine production of T cell subsets and the IL-2 effect on their proliferation in response to ConA after MMg pre-exposure. After 16h-MMg pre-exposure, the splenocytes were activated with ConA at a static conditions or PMA plus Ionomycin, and then IL-2 and IFN-γ productions were investigated. A) IL-2 and IFN-γ mRNA expressions of splenocytes were evaluated after 24h-ConA or 6h-PMA/Ionomycin stimulation determined by Real-time PCR assays. B) The FACS profile analysis of IL-2 and IFN-γ intracellular staining gated on CD4 + and CD8 + T cells was shown. C) The percentages of T cell subsets positive for IL-2 and IFN-γ after 6h-PMA/Ionomycin stimulation were summarized. The FACS profile analysis (D) and the proportional changes of proliferation of CD4 + and CD8 + T cells (E) after a 72h-ConA stimulation with or without IL-2 supplying were shown. Data were represented as means±SD. *, p
Figure Legend Snippet: The cytokine production of T cell subsets and the IL-2 effect on their proliferation in response to ConA after MMg pre-exposure. After 16h-MMg pre-exposure, the splenocytes were activated with ConA at a static conditions or PMA plus Ionomycin, and then IL-2 and IFN-γ productions were investigated. A) IL-2 and IFN-γ mRNA expressions of splenocytes were evaluated after 24h-ConA or 6h-PMA/Ionomycin stimulation determined by Real-time PCR assays. B) The FACS profile analysis of IL-2 and IFN-γ intracellular staining gated on CD4 + and CD8 + T cells was shown. C) The percentages of T cell subsets positive for IL-2 and IFN-γ after 6h-PMA/Ionomycin stimulation were summarized. The FACS profile analysis (D) and the proportional changes of proliferation of CD4 + and CD8 + T cells (E) after a 72h-ConA stimulation with or without IL-2 supplying were shown. Data were represented as means±SD. *, p

Techniques Used: Real-time Polymerase Chain Reaction, FACS, Staining

10) Product Images from "The Acute Environment, Rather than T Cell Subset Pre-Commitment, Regulates Expression of the Human T Cell Cytokine Amphiregulin"

Article Title: The Acute Environment, Rather than T Cell Subset Pre-Commitment, Regulates Expression of the Human T Cell Cytokine Amphiregulin

Journal: PLoS ONE

doi: 10.1371/journal.pone.0039072

Several human CD4 T cell subsets can produce AR. (A) Allogeneic Th1 and Th2 cell lines from three subjects were stimulated with PMA + ionomycin for 6 hours. The percentage of cells expressing IFNγ, IL-4, and AR was analyzed by ICS. (B) The expression of AR and other cytokines was measured in SEB-stimulated PBMC from four subjects by ICS, calculating the frequencies of single cytokine producers, and all possible combinations of double-producers, among the CD154+ CD4+ T cells. The figure shows the ratio between the observed frequencies of double-producing T cells for each cytokine pair, and the expected frequencies (calculated as the product of the individual frequencies for each cytokine). Values represent the ratios for the double-producer combination defined by the row and column labels. Ratios above or below 1 are indicated by solid or open symbols, respectively. (C) IL-4, IFNγ and IL-2 mRNA levels were measured by RT-PCR in the sorted populations described in Figure 4C . (D) PBMC were treated with influenza H1N1 peptides or tetanus (five subjects each), or the allergens Fel d1 (solid symbols) or Der p1 (open symbols)(three subjects each). The numbers of memory CD4 T cells expressing AR and other cytokines were measured by ICS. The backgrounds (no antigen) have been subtracted. Each symbol represents one individual and the filled bar is the mean of all tested subjects. (E) CD69+ CD4+ T cells (Control_CD69+) were sorted from PBMC incubated in medium alone. CD69+IFNγ+ and CD69+IFNγ- CD4 T cells were sorted from influenza peptide-treated PBMC using the cytokine secretion assay. The mRNA levels of IFNγ and AR were measured by RT-PCR. Results in (A-C) are representative of at least three experiments, (D) represents two experiments using a total of 5 independent subjects, and (E) represents two experiments.
Figure Legend Snippet: Several human CD4 T cell subsets can produce AR. (A) Allogeneic Th1 and Th2 cell lines from three subjects were stimulated with PMA + ionomycin for 6 hours. The percentage of cells expressing IFNγ, IL-4, and AR was analyzed by ICS. (B) The expression of AR and other cytokines was measured in SEB-stimulated PBMC from four subjects by ICS, calculating the frequencies of single cytokine producers, and all possible combinations of double-producers, among the CD154+ CD4+ T cells. The figure shows the ratio between the observed frequencies of double-producing T cells for each cytokine pair, and the expected frequencies (calculated as the product of the individual frequencies for each cytokine). Values represent the ratios for the double-producer combination defined by the row and column labels. Ratios above or below 1 are indicated by solid or open symbols, respectively. (C) IL-4, IFNγ and IL-2 mRNA levels were measured by RT-PCR in the sorted populations described in Figure 4C . (D) PBMC were treated with influenza H1N1 peptides or tetanus (five subjects each), or the allergens Fel d1 (solid symbols) or Der p1 (open symbols)(three subjects each). The numbers of memory CD4 T cells expressing AR and other cytokines were measured by ICS. The backgrounds (no antigen) have been subtracted. Each symbol represents one individual and the filled bar is the mean of all tested subjects. (E) CD69+ CD4+ T cells (Control_CD69+) were sorted from PBMC incubated in medium alone. CD69+IFNγ+ and CD69+IFNγ- CD4 T cells were sorted from influenza peptide-treated PBMC using the cytokine secretion assay. The mRNA levels of IFNγ and AR were measured by RT-PCR. Results in (A-C) are representative of at least three experiments, (D) represents two experiments using a total of 5 independent subjects, and (E) represents two experiments.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation

11) Product Images from "Dual molecular imaging for targeting metalloproteinase activity and apoptosis in atherosclerosis: molecular imaging facilitates understanding of pathogenesis"

Article Title: Dual molecular imaging for targeting metalloproteinase activity and apoptosis in atherosclerosis: molecular imaging facilitates understanding of pathogenesis

Journal: Journal of Nuclear Cardiology

doi: 10.1007/s12350-009-9107-8

Effect of caspase inhibitors on the expression of MMP-9 in CASP1-transfected THP-1 cells. A Zymogram: Z-VAD-FMK, a cell-permeable broad-spectrum caspase inhibitor and YVAD-CHO, a CASP1 specific inhibitor, show marked inhibition of MMP-9 release from THP-1 cells. DEVD-CHO, a caspase-3 specific inhibitor, had no effect on MMP-9 expression and release. B , C . MMP-9 enzymatic activity in CASP1 expressing THP-1 cell lysate is significantly reduced by a CASP1 specific inhibitor YVAD-CHO. D Effect of ER stress on the expression of MMP-9 in THP-1 cells, as shown by oxysterols, 7-ketocholesterol, and cholesterol-5β induce MMP-9 release from THP-1 cells. SERCA inhibitor thapsigargin also induces MMP-9 expression while it remains unchanged in ionophore ionomycin-treated cells. E Cells treated with LPS release MMP-9 into the medium. IFNγ has no effect on MMP-9 expression. MMP-2 level remains unchanged in both groups
Figure Legend Snippet: Effect of caspase inhibitors on the expression of MMP-9 in CASP1-transfected THP-1 cells. A Zymogram: Z-VAD-FMK, a cell-permeable broad-spectrum caspase inhibitor and YVAD-CHO, a CASP1 specific inhibitor, show marked inhibition of MMP-9 release from THP-1 cells. DEVD-CHO, a caspase-3 specific inhibitor, had no effect on MMP-9 expression and release. B , C . MMP-9 enzymatic activity in CASP1 expressing THP-1 cell lysate is significantly reduced by a CASP1 specific inhibitor YVAD-CHO. D Effect of ER stress on the expression of MMP-9 in THP-1 cells, as shown by oxysterols, 7-ketocholesterol, and cholesterol-5β induce MMP-9 release from THP-1 cells. SERCA inhibitor thapsigargin also induces MMP-9 expression while it remains unchanged in ionophore ionomycin-treated cells. E Cells treated with LPS release MMP-9 into the medium. IFNγ has no effect on MMP-9 expression. MMP-2 level remains unchanged in both groups

Techniques Used: Expressing, Transfection, Inhibition, Activity Assay

12) Product Images from "Astrocyte Stellation, a Process Dependent on Rac1 Is Sustained by the Regulated Exocytosis of Enlargeosomes"

Article Title: Astrocyte Stellation, a Process Dependent on Rac1 Is Sustained by the Regulated Exocytosis of Enlargeosomes

Journal: Glia

doi: 10.1002/glia.22280

Enlargeosome exocytosis induced by Y27632 depends on Rac1. ( A ) A resting flat astrocyte surface-negative for the enlargeosome marker, Ahnak. ( B ) Ahnak surface-labeling in astrocyte stimulated with ionomycin (1μM, 10 min). ( C ) Surface Ahnak labeling in astrocytes stellated by Y27632 (25 μM, 1 h). ( D ) Quantitative immuno-labeling of surface Ahnak labelling in groups of at least 25 cells, treated with Y27632 (25 μM) for the indicated times. ( E ) Ahnak surface labelling in astrocytes transfected (see Fig. 3 ) with the scrambled (left) or the Rac1-specific (right) shRNA, and then treated with Y27632 (25 μM, 1 h). Bars in (A), valid for (B, C) left panel; (C) right panel and (E) are 10 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Figure Legend Snippet: Enlargeosome exocytosis induced by Y27632 depends on Rac1. ( A ) A resting flat astrocyte surface-negative for the enlargeosome marker, Ahnak. ( B ) Ahnak surface-labeling in astrocyte stimulated with ionomycin (1μM, 10 min). ( C ) Surface Ahnak labeling in astrocytes stellated by Y27632 (25 μM, 1 h). ( D ) Quantitative immuno-labeling of surface Ahnak labelling in groups of at least 25 cells, treated with Y27632 (25 μM) for the indicated times. ( E ) Ahnak surface labelling in astrocytes transfected (see Fig. 3 ) with the scrambled (left) or the Rac1-specific (right) shRNA, and then treated with Y27632 (25 μM, 1 h). Bars in (A), valid for (B, C) left panel; (C) right panel and (E) are 10 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Techniques Used: Marker, Labeling, Immunolabeling, Transfection, shRNA

13) Product Images from "Regulation of in vitro human T cell development through interleukin-7 deprivation and anti-CD3 stimulation"

Article Title: Regulation of in vitro human T cell development through interleukin-7 deprivation and anti-CD3 stimulation

Journal: BMC Immunology

doi: 10.1186/1471-2172-13-46

In vitro maturation and functional analysis of CD4 SP T cells upon IL-7 deprivation and anti-CD3/CD28 co-stimulation in the absence of stromal cell environment. A , Analysis of T cell development kinetics upon IL-7 removal. Diagram of key events and markers for precursor T cell development is illustrated at top. Adult HPCs were co-cultured with stromal cells for 21 days and continued on with IL-7 (IL-7 present, i and iii) or transferred to culture without IL-7 (IL-7 deprived, ii and iv) for an additional 17 days. The cells were then transferred to 96 well U bottom plates and stimulated with anti-CD3/CD28 beads for an additional 14 days. Representative results of analysis of T cell surface markers two weeks after stimulation are shown with percentage of cells indicated in the flow graph quadrons. The percentages of CD4 + CD3 + TCRαβ + cell population of four independent T cell development experiments are shown. B , Analysis of effector functions of the in vitro -derived CD4 T cells. PBMCs of healthy donors and the in vitro -derived CD4 T cells were stimulated using anti-CD3/CD28 beads in the presence of IL-2, IL-7 and IL-15 for two weeks. Expression of intracellular effector cytokine (IFNγ) and T helper functional markers (IL-4, IL-17) was detected after Brefeldin A treatment; unstimulated (left panel) or PMA and ionomycin stimulated (right panel) cells were analyzed by antibody staining and flow cytometry. Note that the small percentage of in vitro -derived CD8 + cells were not CD3 + or viable propagating cells.
Figure Legend Snippet: In vitro maturation and functional analysis of CD4 SP T cells upon IL-7 deprivation and anti-CD3/CD28 co-stimulation in the absence of stromal cell environment. A , Analysis of T cell development kinetics upon IL-7 removal. Diagram of key events and markers for precursor T cell development is illustrated at top. Adult HPCs were co-cultured with stromal cells for 21 days and continued on with IL-7 (IL-7 present, i and iii) or transferred to culture without IL-7 (IL-7 deprived, ii and iv) for an additional 17 days. The cells were then transferred to 96 well U bottom plates and stimulated with anti-CD3/CD28 beads for an additional 14 days. Representative results of analysis of T cell surface markers two weeks after stimulation are shown with percentage of cells indicated in the flow graph quadrons. The percentages of CD4 + CD3 + TCRαβ + cell population of four independent T cell development experiments are shown. B , Analysis of effector functions of the in vitro -derived CD4 T cells. PBMCs of healthy donors and the in vitro -derived CD4 T cells were stimulated using anti-CD3/CD28 beads in the presence of IL-2, IL-7 and IL-15 for two weeks. Expression of intracellular effector cytokine (IFNγ) and T helper functional markers (IL-4, IL-17) was detected after Brefeldin A treatment; unstimulated (left panel) or PMA and ionomycin stimulated (right panel) cells were analyzed by antibody staining and flow cytometry. Note that the small percentage of in vitro -derived CD8 + cells were not CD3 + or viable propagating cells.

Techniques Used: In Vitro, Functional Assay, Cell Culture, Flow Cytometry, Derivative Assay, Expressing, Staining, Cytometry

14) Product Images from "Regulation of Anti-DNA B Cells in Recombination-activating Gene-deficient Mice "

Article Title: Regulation of Anti-DNA B Cells in Recombination-activating Gene-deficient Mice

Journal: The Journal of Experimental Medicine

doi:

Binding of 3H9Vk4 antibody to apoptotic cells. ( a ) DNA fragmentation of apoptotic cells. DNA from freshly isolated splenocytes (lane 1 ) and cells cultured overnight in the presence of ionomycin (lane 2 ) was electrophoresed in 1% agar gel. Ionomycin-treated cells show DNA-fragmentation typical of apoptotic cells. ( b ) Binding of 3H9Vk4 antibody to apoptotic cells. Ionomycin-treated ( bold line ) and fresh ( broken line ) spleen cells were incubated with FITC-labeled 3H9Vk4 antibody ( left ) and the isotype-matched control antibody MOPC141 ( right ) and analyzed by flow cytometry. ( c ) Binding of 3H9Vk4 antibody to bone marrow cells. Bone marrow cells of RAG-2 −/− ( bold line ) and control RAG-2 +/− ( broken line ) were isolated, stained with FITC-3H9Vk4 antibody ( left ) and MOPC 141 ( right ), and analyzed by flow cytometry.
Figure Legend Snippet: Binding of 3H9Vk4 antibody to apoptotic cells. ( a ) DNA fragmentation of apoptotic cells. DNA from freshly isolated splenocytes (lane 1 ) and cells cultured overnight in the presence of ionomycin (lane 2 ) was electrophoresed in 1% agar gel. Ionomycin-treated cells show DNA-fragmentation typical of apoptotic cells. ( b ) Binding of 3H9Vk4 antibody to apoptotic cells. Ionomycin-treated ( bold line ) and fresh ( broken line ) spleen cells were incubated with FITC-labeled 3H9Vk4 antibody ( left ) and the isotype-matched control antibody MOPC141 ( right ) and analyzed by flow cytometry. ( c ) Binding of 3H9Vk4 antibody to bone marrow cells. Bone marrow cells of RAG-2 −/− ( bold line ) and control RAG-2 +/− ( broken line ) were isolated, stained with FITC-3H9Vk4 antibody ( left ) and MOPC 141 ( right ), and analyzed by flow cytometry.

Techniques Used: Binding Assay, Isolation, Cell Culture, Incubation, Labeling, Flow Cytometry, Cytometry, Staining

15) Product Images from "Epithelial-intrinsic IKKα expression regulates group 3 innate lymphoid cell responses and antibacterial immunity"

Article Title: Epithelial-intrinsic IKKα expression regulates group 3 innate lymphoid cell responses and antibacterial immunity

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20141831

Neutralization of TSLP partially restores immunity to C. rodentium infection in IKKα ΔIEC mice. IKKα ΔIEC mice were infected with C. rodentium and injected i.p. with either 0.5 mg rat IgG control or anti-TSLP mAb every 3 d (littermate control IKKα F/F mice received rat IgG only). (a and c) C. rodentium CFU in the feces on day 6 p.i. (a) and liver on day 11 p.i. (b). (c) Percentage of initial body weight. (d) H E staining of colon tissue sections of day 11 C. rodentium –infected mice. Bar, 50 µm. (e) IL-22 expression by splenic ILCs at day 4 p.i., after ex vivo PMA and ionomycin stimulation. (f) IL-22 protein expression within colon homogenate tissue from day 4 infected mice. Data for a–d are representative of three experiments (IKKα F/F + rat IgG, n = 13; IKKα ΔIEC + rat IgG, n = 10; IKKα ΔIEC + anti-TSLP, n = 9), and data for e and f are representative of two experiments (IKKα F/F + rat IgG, n = 8; IKKα ΔIEC + rat IgG, n = 6; IKKα ΔIEC + anti-TSLP, n = 6). Data are expressed as mean ± SEM. *, P
Figure Legend Snippet: Neutralization of TSLP partially restores immunity to C. rodentium infection in IKKα ΔIEC mice. IKKα ΔIEC mice were infected with C. rodentium and injected i.p. with either 0.5 mg rat IgG control or anti-TSLP mAb every 3 d (littermate control IKKα F/F mice received rat IgG only). (a and c) C. rodentium CFU in the feces on day 6 p.i. (a) and liver on day 11 p.i. (b). (c) Percentage of initial body weight. (d) H E staining of colon tissue sections of day 11 C. rodentium –infected mice. Bar, 50 µm. (e) IL-22 expression by splenic ILCs at day 4 p.i., after ex vivo PMA and ionomycin stimulation. (f) IL-22 protein expression within colon homogenate tissue from day 4 infected mice. Data for a–d are representative of three experiments (IKKα F/F + rat IgG, n = 13; IKKα ΔIEC + rat IgG, n = 10; IKKα ΔIEC + anti-TSLP, n = 9), and data for e and f are representative of two experiments (IKKα F/F + rat IgG, n = 8; IKKα ΔIEC + rat IgG, n = 6; IKKα ΔIEC + anti-TSLP, n = 6). Data are expressed as mean ± SEM. *, P

Techniques Used: Neutralization, Infection, Mouse Assay, Injection, Staining, Expressing, Ex Vivo

C. rodentium infection–induced ILC-dependent IL-22 responses are diminished in IKKα ΔIEC mice. IKKα F/F mice were infected (INF) with C. rodentium and sacrificed at day 4 p.i. (a) Representative plots displaying frequencies of IL-22 + cells in the mLNs. (b) Expression of T cell (CD3 and CD5) and NK cell (NK1.1) markers among IL-22 + cells. (c) Expression of ILC surface markers in gated CD3 − CD5 − NK1.1 − IL-22 + cells (black, open histograms) compared with CD19 + B cells (gray, closed histograms). (d) Representative plots displaying frequencies of CD3 − CD5 − CD19 − CD11c − NK1.1 − , RORγt + CD25 + ILCs in the mLNs of naive and C. rodentium –infected littermate control IKKα F/F and IKKα ΔIEC mice. (e) Total mLN ILC3s. (f) Frequencies of IL-22–expressing RORγt + ILCs in the mLN after ex vivo PMA and ionomycin stimulation. (g) Frequencies of IL-22–expressing RORγt + ILCs in the mLNs after stimulation with rIL-23, PMA, and ionomycin. (h) Frequencies of CD3 + CD4 + T cells in the mLNs expressing IL-22 after ex vivo PMA and ionomycin stimulation. Data for a–f and h are representative of five experiments (IKKα F/F , total n = 21; IKKα ΔIEC , n = 17 + 1 naive mouse of each genotype per experiment), and data for g are representative of three experiments (IKKα F/F , total n = 13; IKKα ΔIEC , n = 11 + 1 naive mouse of each genotype per experiment). Data are expressed as mean ± SEM. *, P
Figure Legend Snippet: C. rodentium infection–induced ILC-dependent IL-22 responses are diminished in IKKα ΔIEC mice. IKKα F/F mice were infected (INF) with C. rodentium and sacrificed at day 4 p.i. (a) Representative plots displaying frequencies of IL-22 + cells in the mLNs. (b) Expression of T cell (CD3 and CD5) and NK cell (NK1.1) markers among IL-22 + cells. (c) Expression of ILC surface markers in gated CD3 − CD5 − NK1.1 − IL-22 + cells (black, open histograms) compared with CD19 + B cells (gray, closed histograms). (d) Representative plots displaying frequencies of CD3 − CD5 − CD19 − CD11c − NK1.1 − , RORγt + CD25 + ILCs in the mLNs of naive and C. rodentium –infected littermate control IKKα F/F and IKKα ΔIEC mice. (e) Total mLN ILC3s. (f) Frequencies of IL-22–expressing RORγt + ILCs in the mLN after ex vivo PMA and ionomycin stimulation. (g) Frequencies of IL-22–expressing RORγt + ILCs in the mLNs after stimulation with rIL-23, PMA, and ionomycin. (h) Frequencies of CD3 + CD4 + T cells in the mLNs expressing IL-22 after ex vivo PMA and ionomycin stimulation. Data for a–f and h are representative of five experiments (IKKα F/F , total n = 21; IKKα ΔIEC , n = 17 + 1 naive mouse of each genotype per experiment), and data for g are representative of three experiments (IKKα F/F , total n = 13; IKKα ΔIEC , n = 11 + 1 naive mouse of each genotype per experiment). Data are expressed as mean ± SEM. *, P

Techniques Used: Infection, Mouse Assay, Expressing, Ex Vivo

16) Product Images from "A2B adenosine receptor activation switches differentiation of bone marrow cells to a CD11c+Gr‐1+ dendritic cell subset that promotes the Th17 response"

Article Title: A2B adenosine receptor activation switches differentiation of bone marrow cells to a CD11c+Gr‐1+ dendritic cell subset that promotes the Th17 response

Journal: Immunity, Inflammation and Disease

doi: 10.1002/iid3.74

Differentiation of bone marrow cells into BMDCs in the presence of the non‐specific AR agonist NECA results in increased Th17‐stimulating ability. A: Stimulatory effect of mouse BMDCs under non‐polarizing conditions. Bone marrow cells were cultured for 5 days in medium continuing GM‐CSF (10 ng/mL) in the absence or presence of NECA (100 nM), then were detached, washed, and seeded (5 × 10 4 /well) into 24‐well plates Responder CD3 T cells, isolated from immunized B6 mice (10 6 cells/well) were added to the plates (T cell/DC ratio 20:1), then the cells were incubated in the presence of graded doses of the immunizing peptide for 48 h and T cell proliferation was assessed by 3 H‐thymidine incorporation. The results shown are the mean ± SD for one study doing in triplicated wells and the experiment was repeated 3 times with similar results. B: Stimulatory effect of mouse BMDCs on Th1 and Th17 autoreactive T cells under polarizing conditions. Responder T cells were co‐cultured for 48 h with each of the two DC preparations (generated in the absence or presence of NECA) and the immunizing peptide under polarizing conditions favoring Th1 cell proliferation (culture medium containing IL‐12) or Th17 cell proliferation (culture medium containing IL‐23), then the culture supernatants were assayed for IL‐17 (top panel) or IFN‐γ (bottom panel). C: Intracellular staining of the proliferating T cells for IL‐17 or IFN‐γ expression. The activated T cells generated in (B) using DCs generated in the absence (top panels) or presence of NECA (bottom panels) were separated on day 2 and cultured for 3 days, then the separated, activated T cells were treated for 4 h with 50 ng/mL of phorbol myristic acetate, 1 μg/mL of ionomycin, and 1 μg/mL of brefeldin A, fixed, permeabilized overnight with Cytofix/Cytoperm buffer, and stained intracellularly with antibodies against IL‐17 (left panels) or IFN‐γ (right panels) and analyzed on a FACScalibur.
Figure Legend Snippet: Differentiation of bone marrow cells into BMDCs in the presence of the non‐specific AR agonist NECA results in increased Th17‐stimulating ability. A: Stimulatory effect of mouse BMDCs under non‐polarizing conditions. Bone marrow cells were cultured for 5 days in medium continuing GM‐CSF (10 ng/mL) in the absence or presence of NECA (100 nM), then were detached, washed, and seeded (5 × 10 4 /well) into 24‐well plates Responder CD3 T cells, isolated from immunized B6 mice (10 6 cells/well) were added to the plates (T cell/DC ratio 20:1), then the cells were incubated in the presence of graded doses of the immunizing peptide for 48 h and T cell proliferation was assessed by 3 H‐thymidine incorporation. The results shown are the mean ± SD for one study doing in triplicated wells and the experiment was repeated 3 times with similar results. B: Stimulatory effect of mouse BMDCs on Th1 and Th17 autoreactive T cells under polarizing conditions. Responder T cells were co‐cultured for 48 h with each of the two DC preparations (generated in the absence or presence of NECA) and the immunizing peptide under polarizing conditions favoring Th1 cell proliferation (culture medium containing IL‐12) or Th17 cell proliferation (culture medium containing IL‐23), then the culture supernatants were assayed for IL‐17 (top panel) or IFN‐γ (bottom panel). C: Intracellular staining of the proliferating T cells for IL‐17 or IFN‐γ expression. The activated T cells generated in (B) using DCs generated in the absence (top panels) or presence of NECA (bottom panels) were separated on day 2 and cultured for 3 days, then the separated, activated T cells were treated for 4 h with 50 ng/mL of phorbol myristic acetate, 1 μg/mL of ionomycin, and 1 μg/mL of brefeldin A, fixed, permeabilized overnight with Cytofix/Cytoperm buffer, and stained intracellularly with antibodies against IL‐17 (left panels) or IFN‐γ (right panels) and analyzed on a FACScalibur.

Techniques Used: Cell Culture, Isolation, Mouse Assay, Incubation, Generated, Staining, Expressing

17) Product Images from "Roles of Calcium/Calmodulin-Dependent Kinase II in Long-Term Memory Formation in Crickets"

Article Title: Roles of Calcium/Calmodulin-Dependent Kinase II in Long-Term Memory Formation in Crickets

Journal: PLoS ONE

doi: 10.1371/journal.pone.0107442

Effects of co-injection of a CaMKII inhibitor and ionomycin on LTM formation. Four groups of animals were each injected with 3 µl of saline or saline containing 20 µM ionomycin, 200 µM ionomycin, or 200 µM ionomycin and 2 mM KN-62 20 min prior to 1-trial conditioning. The saline contained 1% DMSO. Relative preference between the rewarded odor and control odor was tested before and at 1 day after training. PIs for the rewarded odor before (white bars) and after (grey bars) training are shown as box and whisker diagrams. Odor preferences before and after training are compared by WCX test. Odor preferences after training of different groups were compared by the M-W test. The results of statistical comparisons are shown by asterisks (** P
Figure Legend Snippet: Effects of co-injection of a CaMKII inhibitor and ionomycin on LTM formation. Four groups of animals were each injected with 3 µl of saline or saline containing 20 µM ionomycin, 200 µM ionomycin, or 200 µM ionomycin and 2 mM KN-62 20 min prior to 1-trial conditioning. The saline contained 1% DMSO. Relative preference between the rewarded odor and control odor was tested before and at 1 day after training. PIs for the rewarded odor before (white bars) and after (grey bars) training are shown as box and whisker diagrams. Odor preferences before and after training are compared by WCX test. Odor preferences after training of different groups were compared by the M-W test. The results of statistical comparisons are shown by asterisks (** P

Techniques Used: Injection, Whisker Assay

18) Product Images from "Citrullination of Histone H3 Interferes with HP1-Mediated Transcriptional Repression"

Article Title: Citrullination of Histone H3 Interferes with HP1-Mediated Transcriptional Repression

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1002934

PADI activity facilitates activation of TNFα and IL8 transcription in T cells. (A) ChIP with anti-HP1α antibodies was carried out with chromatin from Jurkat cells either untreated or treated with PMA and ionophore (ionomycin). The relative enrichments of HP1α on the indicated promoters were measured by qPCR. Data are presented relative to non-immune IgG. Changes in enrichment are presented relative to the un-induced control (set to 1). Values are means ± SEM from two PCR measures of two independent ChIP experiments. (B) Total RNA was isolated from Jurkat cells either un-stimulated or treated with ionomycin and PMA for 2 hour. Changes in mRNA levels for the indicated genes were quantified by RT-qPCR. The data are presented as the means ± SEM of triplicate experiments. (C) ChIP with anti-H3cit8K9me3 antibodies was carried out as in A. Data are presented as a percentage of histone H3. Changes in enrichment are presented relative to the un-induced control (set to 1). Values are means ± SEM from two PCR measures of two independent ChIP experiments. (D) Total RNA was isolated from Jurkat cells treated as in B minus or plus PADI-inhibitor cl-amidine as indicated. Changes in mRNA levels for the indicated genes were quantified by RT-qPCR. The data are presented as the means ± SEM of duplicate experiments.
Figure Legend Snippet: PADI activity facilitates activation of TNFα and IL8 transcription in T cells. (A) ChIP with anti-HP1α antibodies was carried out with chromatin from Jurkat cells either untreated or treated with PMA and ionophore (ionomycin). The relative enrichments of HP1α on the indicated promoters were measured by qPCR. Data are presented relative to non-immune IgG. Changes in enrichment are presented relative to the un-induced control (set to 1). Values are means ± SEM from two PCR measures of two independent ChIP experiments. (B) Total RNA was isolated from Jurkat cells either un-stimulated or treated with ionomycin and PMA for 2 hour. Changes in mRNA levels for the indicated genes were quantified by RT-qPCR. The data are presented as the means ± SEM of triplicate experiments. (C) ChIP with anti-H3cit8K9me3 antibodies was carried out as in A. Data are presented as a percentage of histone H3. Changes in enrichment are presented relative to the un-induced control (set to 1). Values are means ± SEM from two PCR measures of two independent ChIP experiments. (D) Total RNA was isolated from Jurkat cells treated as in B minus or plus PADI-inhibitor cl-amidine as indicated. Changes in mRNA levels for the indicated genes were quantified by RT-qPCR. The data are presented as the means ± SEM of duplicate experiments.

Techniques Used: Activity Assay, Activation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Isolation, Quantitative RT-PCR

19) Product Images from "A genetically-encoded reporter of synaptic activity in vivo"

Article Title: A genetically-encoded reporter of synaptic activity in vivo

Journal: Nature methods

doi: 10.1038/nmeth.1399

The dynamic range of SyGCaMP2 responses ( a ) Average SyGCaMP2 responses to trains of 1, 2, 3, 5, 10, 20 and 40 APs at 20 Hz. Each trace represents 500 synapses from 6 different cover slips. Error bars show s.e.m. ( b ) Peak amplitude of the SyGCaMP2 response (squares) taken from a as a function of the number of APs delivered. GCaMP2 responses (circles) are also plotted (Error bars, s.e.m.; n=450 synapses from 5 different cover slips). When using GCaMP2, boutons were identified by co-expressing mRFP-VAMP2. The response of SyGCaMP2 was linear up to ~8 APs, with a proportionality constant of 7 ± 0.3 % per AP for SyGCaMP2 and 5 ± 0.4 % for GCaMP2. ( c ) Comparison of experimental SyGCaMP2 measurements (black traces) and their simulations (red). The model accurately predicts the response to 1 AP as well as the saturating response to 20 APs at 20 Hz. The model does not account for the slower recovery of the SyGCaMP2 signal after the introduction of larger calcium loads. ( d ) SyGCaMP2 response to 40 APs delivered at 20 Hz (70% increase). Neurons were then perfused with ionomycin (5 μM), 0 Ca 2+ , and 10 mM EGTA. The minimum fluorescence (ΔF/F 0min ) was −0.55 relative to rest. The external [Ca 2+ ] was then increased to 2.5 mM to saturate SyGCaMP2. The peak signal (ΔF/F 0max ) was 2.1. Assuming a Hill coefficient of 4 for the binding of Ca 2+ to GCaMP2 and a K d of 150 nM, the resting free [Ca 2+ ] is estimated to be ~2 nM.
Figure Legend Snippet: The dynamic range of SyGCaMP2 responses ( a ) Average SyGCaMP2 responses to trains of 1, 2, 3, 5, 10, 20 and 40 APs at 20 Hz. Each trace represents 500 synapses from 6 different cover slips. Error bars show s.e.m. ( b ) Peak amplitude of the SyGCaMP2 response (squares) taken from a as a function of the number of APs delivered. GCaMP2 responses (circles) are also plotted (Error bars, s.e.m.; n=450 synapses from 5 different cover slips). When using GCaMP2, boutons were identified by co-expressing mRFP-VAMP2. The response of SyGCaMP2 was linear up to ~8 APs, with a proportionality constant of 7 ± 0.3 % per AP for SyGCaMP2 and 5 ± 0.4 % for GCaMP2. ( c ) Comparison of experimental SyGCaMP2 measurements (black traces) and their simulations (red). The model accurately predicts the response to 1 AP as well as the saturating response to 20 APs at 20 Hz. The model does not account for the slower recovery of the SyGCaMP2 signal after the introduction of larger calcium loads. ( d ) SyGCaMP2 response to 40 APs delivered at 20 Hz (70% increase). Neurons were then perfused with ionomycin (5 μM), 0 Ca 2+ , and 10 mM EGTA. The minimum fluorescence (ΔF/F 0min ) was −0.55 relative to rest. The external [Ca 2+ ] was then increased to 2.5 mM to saturate SyGCaMP2. The peak signal (ΔF/F 0max ) was 2.1. Assuming a Hill coefficient of 4 for the binding of Ca 2+ to GCaMP2 and a K d of 150 nM, the resting free [Ca 2+ ] is estimated to be ~2 nM.

Techniques Used: Expressing, Fluorescence, Binding Assay

20) Product Images from "IL-33 Induces IL-9 Production in Human CD4+ T Cells and Basophils"

Article Title: IL-33 Induces IL-9 Production in Human CD4+ T Cells and Basophils

Journal: PLoS ONE

doi: 10.1371/journal.pone.0021695

TGF-β induces IL-9 expression in Th1 and Th2 cells. Naïve CD4+ T cells were activated with fibroblast-bound anti-CD3/CD28 under classical Th1 and Th2 conditions, with IL-12 and anti-IL-4 or IL-4, anti-IFN-γ, and anti-IL-12 respectively for 5 days, restimulated at day 5 and split in cultures with and without TGF-β or anti-TGF-β for 5 more days of stimulation. (A, B, C, D, E and F) Percentage positive LIVE + CD4+ cells for, respectively IL-9, PU.1, IL-13, IFN-γ, GATA-3, and T-bet at day 10 after restimulation with PMA and ionomycin for 6 h in the presence of Bref A for the last 4 h. Each donor is represented by a symbol and connected with a line. Data are from four independent experiments, each with two donors.* p
Figure Legend Snippet: TGF-β induces IL-9 expression in Th1 and Th2 cells. Naïve CD4+ T cells were activated with fibroblast-bound anti-CD3/CD28 under classical Th1 and Th2 conditions, with IL-12 and anti-IL-4 or IL-4, anti-IFN-γ, and anti-IL-12 respectively for 5 days, restimulated at day 5 and split in cultures with and without TGF-β or anti-TGF-β for 5 more days of stimulation. (A, B, C, D, E and F) Percentage positive LIVE + CD4+ cells for, respectively IL-9, PU.1, IL-13, IFN-γ, GATA-3, and T-bet at day 10 after restimulation with PMA and ionomycin for 6 h in the presence of Bref A for the last 4 h. Each donor is represented by a symbol and connected with a line. Data are from four independent experiments, each with two donors.* p

Techniques Used: Expressing

A and B. IL-33 and TGF-β induce IL-9 secretion in Th cells. Naïve CD4+ T cells were activated with fibroblast-bound anti-CD3/CD28 for five 5 days in the presence of blocking antibodies against IFN-γ and IL-12 (Th0) or these antibodies plus IL-4 (Th2). At day 5, these cultures were restimulated and split in cultures with TGF-β or anti-TGF-βplus IL-33 for an additional 5 days of stimulation. (A) Supernatant multiplex analysis of IL-9, at day 10, after restimulation with PMA and ionomycin for 6 h in the presence of Bref A for the last 4 h. (B) qRT-PCR gene expression analysis of the relative expression of IL9 at day 10 related to the Th0 culture. Data are from four independent experiments, each with two donors. Vertical lines represent means (SEM). All tested cultures are significantly different p
Figure Legend Snippet: A and B. IL-33 and TGF-β induce IL-9 secretion in Th cells. Naïve CD4+ T cells were activated with fibroblast-bound anti-CD3/CD28 for five 5 days in the presence of blocking antibodies against IFN-γ and IL-12 (Th0) or these antibodies plus IL-4 (Th2). At day 5, these cultures were restimulated and split in cultures with TGF-β or anti-TGF-βplus IL-33 for an additional 5 days of stimulation. (A) Supernatant multiplex analysis of IL-9, at day 10, after restimulation with PMA and ionomycin for 6 h in the presence of Bref A for the last 4 h. (B) qRT-PCR gene expression analysis of the relative expression of IL9 at day 10 related to the Th0 culture. Data are from four independent experiments, each with two donors. Vertical lines represent means (SEM). All tested cultures are significantly different p

Techniques Used: Blocking Assay, Multiplex Assay, Quantitative RT-PCR, Expressing

TGF-β induces IL-9 secretion in Th1 and Th2 cells. Naïve CD4+ T cells were activated with fibroblast-bound anti-CD3/CD28 under classical Th1 and Th2 conditions, with IL-12 and anti-IL-4 or IL-4, anti-IFN-γ, and anti-IL-12 respectively for 5 days, restimulated at day 5 and split in cultures with and without TGF-β or anti-TGF-β for 5 more days of stimulation. (A, B and C) Supernatant concentrations of IL-9, IL-5 and IFN-γ at day 10 after restimulation with PMA and ionomycin for 6 h in the presence of Bref A for the last 4 h. Each donor is represented by a specific symbol and connected with a line. Data are from four independent experiments, each with two donors. * p
Figure Legend Snippet: TGF-β induces IL-9 secretion in Th1 and Th2 cells. Naïve CD4+ T cells were activated with fibroblast-bound anti-CD3/CD28 under classical Th1 and Th2 conditions, with IL-12 and anti-IL-4 or IL-4, anti-IFN-γ, and anti-IL-12 respectively for 5 days, restimulated at day 5 and split in cultures with and without TGF-β or anti-TGF-β for 5 more days of stimulation. (A, B and C) Supernatant concentrations of IL-9, IL-5 and IFN-γ at day 10 after restimulation with PMA and ionomycin for 6 h in the presence of Bref A for the last 4 h. Each donor is represented by a specific symbol and connected with a line. Data are from four independent experiments, each with two donors. * p

Techniques Used:

21) Product Images from "Calcium Regulation of EGF-Induced ERK5 Activation: Role of Lad1-MEKK2 Interaction"

Article Title: Calcium Regulation of EGF-Induced ERK5 Activation: Role of Lad1-MEKK2 Interaction

Journal: PLoS ONE

doi: 10.1371/journal.pone.0012627

EGF-induced ERK5 phosphorylation is mediated by c-Src and Lad1 but not by WNK1. ( A ) HeLa cells were transfected with GFP-Lad1 or vector control followed by treatment with EGF (20 ng/ml) for the indicated times. ERK5 phosphorylation was determined using upshift of ERK5 in blot. ( B ) Lad1 shRNA (Sh1 – 2 µg or Sh2 – 6 µg) or empty vector (con) were transfected into HeLa cells. The cells were stimulated by EGF and ERK5 phosphorylation was determined by Western blot as in (A). The levels of endogenous Lad1 and MEKK2 were determined by their corresponding Abs as indicated. ( C ) HeLa cells were transfected with GFP-Lad1 and the activation of ERK5 in response to EGF in the presence of ionomycin or BAPTA-AM was detected by band shift. All the experiments in this figure were reproduced at least three times.
Figure Legend Snippet: EGF-induced ERK5 phosphorylation is mediated by c-Src and Lad1 but not by WNK1. ( A ) HeLa cells were transfected with GFP-Lad1 or vector control followed by treatment with EGF (20 ng/ml) for the indicated times. ERK5 phosphorylation was determined using upshift of ERK5 in blot. ( B ) Lad1 shRNA (Sh1 – 2 µg or Sh2 – 6 µg) or empty vector (con) were transfected into HeLa cells. The cells were stimulated by EGF and ERK5 phosphorylation was determined by Western blot as in (A). The levels of endogenous Lad1 and MEKK2 were determined by their corresponding Abs as indicated. ( C ) HeLa cells were transfected with GFP-Lad1 and the activation of ERK5 in response to EGF in the presence of ionomycin or BAPTA-AM was detected by band shift. All the experiments in this figure were reproduced at least three times.

Techniques Used: Transfection, Plasmid Preparation, shRNA, Western Blot, Activation Assay, Electrophoretic Mobility Shift Assay

Calcium modulates Tyr phosphorylation of MEKK2 but not of Lad1. HeLa cells transfected with GFP-Lad1 were subject to different treatments as indicated (EGF 20 ng/ml; ionomycin (1 µM) or BAPTA-AM (15 µM) for 15 min; PP2 3 µM). GFP-Lad1 was immunoprecipitated with anti GFP Ab ( A, B ). Endogenous MEKK2 was immunoprecipitated by anti-MEKK2 Ab ( C, D ). Their phsophorylation on Tyr residues was detected by pY99 Ab. The experiments in this figure were reproduced 3 times.
Figure Legend Snippet: Calcium modulates Tyr phosphorylation of MEKK2 but not of Lad1. HeLa cells transfected with GFP-Lad1 were subject to different treatments as indicated (EGF 20 ng/ml; ionomycin (1 µM) or BAPTA-AM (15 µM) for 15 min; PP2 3 µM). GFP-Lad1 was immunoprecipitated with anti GFP Ab ( A, B ). Endogenous MEKK2 was immunoprecipitated by anti-MEKK2 Ab ( C, D ). Their phsophorylation on Tyr residues was detected by pY99 Ab. The experiments in this figure were reproduced 3 times.

Techniques Used: Transfection, Immunoprecipitation

Calcium modifiers affect either MEKK2 or its upstream components, without involvement of CaMKII. ( A ) MEK5-HA transfected HeLa cells were either stimulated with EGF or left untreated. MEK5-HA was immunoprecipitated and its in vitro kinase activity towards K/A ERK5 (1 – 397)-GST was measured in the presence of the indicated calcium concentrations. ( B ) Exogenously expressed MEK5-HA was immunoprecipitated from HeLa cells, which were either stimulated with EGF (20 ng/ml, two upper panels) or were left without treatment (two lower panels). The MEK5 was than subjected to in vitro binding assay with K/A ERK5(1 – 397)-GST, in the presence of indicated calcium concentrations or calcium chelator EGTA. ( C ) MEKK2 was immunoprecipitated from the cells treated with vehicle, BAPTA-AM or ionomycin in combination with or without EGF (20 ng/ml). Its activity was measured by the incorporation of radioactive phosphate to recombinant MEK5-GST. ( D ) HeLa cells were pretreated with 30 µM of KN-93 for 30 min and then stimulated with EGF (20 ng/ml) for 10 min. ERK5 activation was measured by band up-shift of endogenous ERK5 detected by anti general ERK5 Ab, CREB phosphorylation was detected by anti pCREB Ab, and anti tubulin Ab was used as a loading control. ( E ) ERK5-HA transfected HeLa cells were pretreated with KN-62 in different concentrations for 1 h. After EGF stimulation, ERK5-HA activation was measured with anti pERK5 Ab in Western blot analysis. All the experiments in this figure were reproduced at least three times.
Figure Legend Snippet: Calcium modifiers affect either MEKK2 or its upstream components, without involvement of CaMKII. ( A ) MEK5-HA transfected HeLa cells were either stimulated with EGF or left untreated. MEK5-HA was immunoprecipitated and its in vitro kinase activity towards K/A ERK5 (1 – 397)-GST was measured in the presence of the indicated calcium concentrations. ( B ) Exogenously expressed MEK5-HA was immunoprecipitated from HeLa cells, which were either stimulated with EGF (20 ng/ml, two upper panels) or were left without treatment (two lower panels). The MEK5 was than subjected to in vitro binding assay with K/A ERK5(1 – 397)-GST, in the presence of indicated calcium concentrations or calcium chelator EGTA. ( C ) MEKK2 was immunoprecipitated from the cells treated with vehicle, BAPTA-AM or ionomycin in combination with or without EGF (20 ng/ml). Its activity was measured by the incorporation of radioactive phosphate to recombinant MEK5-GST. ( D ) HeLa cells were pretreated with 30 µM of KN-93 for 30 min and then stimulated with EGF (20 ng/ml) for 10 min. ERK5 activation was measured by band up-shift of endogenous ERK5 detected by anti general ERK5 Ab, CREB phosphorylation was detected by anti pCREB Ab, and anti tubulin Ab was used as a loading control. ( E ) ERK5-HA transfected HeLa cells were pretreated with KN-62 in different concentrations for 1 h. After EGF stimulation, ERK5-HA activation was measured with anti pERK5 Ab in Western blot analysis. All the experiments in this figure were reproduced at least three times.

Techniques Used: Transfection, Immunoprecipitation, In Vitro, Activity Assay, Binding Assay, Recombinant, Activation Assay, Western Blot

Changes in calcium concentrations do not affect Lad1 localization but inhibit nuclear MEKK2 accumulation. Serum starved HeLa cells were pretreated with vehicle, ionomycin (1 µM) or BAPTA-AM (15 µM) for 15 min and then stimulated with EGF (20 ng/ml) for 10 min. The cells were stained with anti Lad1 or anti MEKK2 Abs and visualized with fluorescent microscopy. This experiment was reproduced 3 times.
Figure Legend Snippet: Changes in calcium concentrations do not affect Lad1 localization but inhibit nuclear MEKK2 accumulation. Serum starved HeLa cells were pretreated with vehicle, ionomycin (1 µM) or BAPTA-AM (15 µM) for 15 min and then stimulated with EGF (20 ng/ml) for 10 min. The cells were stained with anti Lad1 or anti MEKK2 Abs and visualized with fluorescent microscopy. This experiment was reproduced 3 times.

Techniques Used: Staining, Microscopy

Intracellular calcium levels affect ERK5 activation. ( A ) ERK5-HA was transfected into HeLa cells. After starvation, the cells were treated with vehicle, ionomycin (1 µM) or BAPTA-AM (15 µM) for 15 min and then with EGF (20 ng/ml) for 15 min. ERK5 phosphorylation was detected with anti pERK5 Ab. The experiment was reproduced five times. ( B ) Activation of endogenous ERK5 from cells after different treatments was detected by up-shift of ERK5 detected by anti general ERK5 Ab. The experiment was reproduced five times. ( C–E ) The effects of ionomycin or BAPTA-AM were observed at different time points. Quantification of three independent experiments is presented in (E). The error bars represent standard deviation. P values (*: P
Figure Legend Snippet: Intracellular calcium levels affect ERK5 activation. ( A ) ERK5-HA was transfected into HeLa cells. After starvation, the cells were treated with vehicle, ionomycin (1 µM) or BAPTA-AM (15 µM) for 15 min and then with EGF (20 ng/ml) for 15 min. ERK5 phosphorylation was detected with anti pERK5 Ab. The experiment was reproduced five times. ( B ) Activation of endogenous ERK5 from cells after different treatments was detected by up-shift of ERK5 detected by anti general ERK5 Ab. The experiment was reproduced five times. ( C–E ) The effects of ionomycin or BAPTA-AM were observed at different time points. Quantification of three independent experiments is presented in (E). The error bars represent standard deviation. P values (*: P

Techniques Used: Activation Assay, Transfection, Standard Deviation

22) Product Images from "Response to Treatment with TNFα Inhibitors in Rheumatoid Arthritis Is Associated with High Levels of GM-CSF and GM-CSF+ T Lymphocytes"

Article Title: Response to Treatment with TNFα Inhibitors in Rheumatoid Arthritis Is Associated with High Levels of GM-CSF and GM-CSF+ T Lymphocytes

Journal: Clinical Reviews in Allergy & Immunology

doi: 10.1007/s12016-017-8610-y

GM-CSF + and IL-17 + T cells segregate into two different subsets. Enriched T cells were stimulated with PMA/Ionomycin for 16 h and stained for membrane proteins and intracellular cytokines as indicated. a Contour plot showing the percentage of CCR6 + and CCR6 + IL-17 + in blood CD4 + cells and b TNFα + and TNF + GM-CSF + T cells in the blood of RA patients. CCR6 + IL-17 + in a were gated and the frequency of T cells expressing intracellular GM-CSF, TNFα and IFNγ + and membrane CD161 determined by FACS. b PMA/Ionomycin-stimulated T cells were stained for intracellular TNFα and GM-CSF and the frequency in responder and non-responder patients determined. The frequency of TNFα + GM-CSF + T cells that co-expressed INFγ and IL-17 membrane CD161 was determined by multicolour FACS. The data represent the mean ± SEM values from seven responder and three non-responder patients. c Contour plots showing characteristics of the RA patients GM-CSF + T cells (expression of CD45RA, CD45RO and CD161) pre-treatment. Percentages of differentially stained cell subsets are given inside the quadrants. d Linear regression showing the correlation between the level of IL-17 and GM-CSF produced by patients’ T cells activated with anti-CD3/anti-CD28 mAbs
Figure Legend Snippet: GM-CSF + and IL-17 + T cells segregate into two different subsets. Enriched T cells were stimulated with PMA/Ionomycin for 16 h and stained for membrane proteins and intracellular cytokines as indicated. a Contour plot showing the percentage of CCR6 + and CCR6 + IL-17 + in blood CD4 + cells and b TNFα + and TNF + GM-CSF + T cells in the blood of RA patients. CCR6 + IL-17 + in a were gated and the frequency of T cells expressing intracellular GM-CSF, TNFα and IFNγ + and membrane CD161 determined by FACS. b PMA/Ionomycin-stimulated T cells were stained for intracellular TNFα and GM-CSF and the frequency in responder and non-responder patients determined. The frequency of TNFα + GM-CSF + T cells that co-expressed INFγ and IL-17 membrane CD161 was determined by multicolour FACS. The data represent the mean ± SEM values from seven responder and three non-responder patients. c Contour plots showing characteristics of the RA patients GM-CSF + T cells (expression of CD45RA, CD45RO and CD161) pre-treatment. Percentages of differentially stained cell subsets are given inside the quadrants. d Linear regression showing the correlation between the level of IL-17 and GM-CSF produced by patients’ T cells activated with anti-CD3/anti-CD28 mAbs

Techniques Used: Staining, Expressing, FACS, Produced

Cytokine production by T cells, B cells and monocytes in rheumatoid arthritis patients prior to and after treatment with anti-TNFα. T cells, B cells and monocytes were enriched by negative selection from the blood of RA patients immediately before treatment with anti-TNFα agents and then after 1 and 3 months. The cells were stimulated for 48 h and the level of cytokines produced determined using MSD multiplex kits. Levels of 16 cytokines were determined but only results of cytokines with notable differences are presented: a, b Data on TNFα; c IL-1β and d GM-CSF. a TNFα produced by T cells stimulated with anti-CD3/anti-CD28 mAbs, B cells stimulated with anti-IgM/anti-CD40 and monocytes stimulated with LPS. b Mean ± standard error of the mean (SEM) for TNFα produced by T cells and monocytes from 3 healthy controls and stimulated for 48 h with PMA and ionomycin. c , d Levels of IL-1β and GM-CSF produced by T and B cells and monocytes activated and cultured as described in a. e Data on GM-CSF production by T cells as described for a before treatment (0 months) and after 1 and then 3 months. f Data on the frequency of GM-CSF + T cells before treatment and after 1 and 3 months in responder and non-responder RA patients. Enriched T cells were stimulated with PMA/ionomycin for 16 h and stained for intracellular GM-CSF. g IL-6 production by T cells and h CCL2 production by monocytes. The data before and after treatment are for 67 patients. Differences between responders and non-responders were assessed using two-tailed Mann Whitney U test. Difference at different time points in the same group was assessed using Wilcoxon matched-pairs signed rank test. Single asterisk indicates P
Figure Legend Snippet: Cytokine production by T cells, B cells and monocytes in rheumatoid arthritis patients prior to and after treatment with anti-TNFα. T cells, B cells and monocytes were enriched by negative selection from the blood of RA patients immediately before treatment with anti-TNFα agents and then after 1 and 3 months. The cells were stimulated for 48 h and the level of cytokines produced determined using MSD multiplex kits. Levels of 16 cytokines were determined but only results of cytokines with notable differences are presented: a, b Data on TNFα; c IL-1β and d GM-CSF. a TNFα produced by T cells stimulated with anti-CD3/anti-CD28 mAbs, B cells stimulated with anti-IgM/anti-CD40 and monocytes stimulated with LPS. b Mean ± standard error of the mean (SEM) for TNFα produced by T cells and monocytes from 3 healthy controls and stimulated for 48 h with PMA and ionomycin. c , d Levels of IL-1β and GM-CSF produced by T and B cells and monocytes activated and cultured as described in a. e Data on GM-CSF production by T cells as described for a before treatment (0 months) and after 1 and then 3 months. f Data on the frequency of GM-CSF + T cells before treatment and after 1 and 3 months in responder and non-responder RA patients. Enriched T cells were stimulated with PMA/ionomycin for 16 h and stained for intracellular GM-CSF. g IL-6 production by T cells and h CCL2 production by monocytes. The data before and after treatment are for 67 patients. Differences between responders and non-responders were assessed using two-tailed Mann Whitney U test. Difference at different time points in the same group was assessed using Wilcoxon matched-pairs signed rank test. Single asterisk indicates P

Techniques Used: Selection, Produced, Multiplex Assay, Cell Culture, Staining, Two Tailed Test, MANN-WHITNEY

23) Product Images from "Impedance-Based Living Cell Analysis for Clinical Diagnosis of Type I Allergy"

Article Title: Impedance-Based Living Cell Analysis for Clinical Diagnosis of Type I Allergy

Journal: Sensors (Basel, Switzerland)

doi: 10.3390/s17112503

( a ) Effects of activators (DNP-HSA 50 ng/mL, PMA 50 nM, Ionomycin 1 μM) and inhibitors (Genistein 100 μM, Cytochalasin D 1 μM, Nocodazole 10 μM) on CI changes (ΔCI) of RBL-2H3 cells. The gray bar indicates the presence of each inhibitor, and the black bar indicates the presence of antigen, activator, or Triton. ( b ) Effects of activators and inhibitors on degranulation of RBL-2H3 cells. The graph is representative of three experiments. Data were obtained from quadruplicate measurements. ( c ) Effects of Cytochalasin D on cell spreading in response to antigen. The white bar shows ca. 10 μm. ( d ) Relative cell area of RBL-2H3 cells before and after stimulation by DNP-HSA with or without Cytochalasin D. The area of cell adhesion was measured by using Image-Pro Plus 6.3J. Differences between the areas in each group were analyzed using one-way analysis of variance followed by Tukey’s test (* p
Figure Legend Snippet: ( a ) Effects of activators (DNP-HSA 50 ng/mL, PMA 50 nM, Ionomycin 1 μM) and inhibitors (Genistein 100 μM, Cytochalasin D 1 μM, Nocodazole 10 μM) on CI changes (ΔCI) of RBL-2H3 cells. The gray bar indicates the presence of each inhibitor, and the black bar indicates the presence of antigen, activator, or Triton. ( b ) Effects of activators and inhibitors on degranulation of RBL-2H3 cells. The graph is representative of three experiments. Data were obtained from quadruplicate measurements. ( c ) Effects of Cytochalasin D on cell spreading in response to antigen. The white bar shows ca. 10 μm. ( d ) Relative cell area of RBL-2H3 cells before and after stimulation by DNP-HSA with or without Cytochalasin D. The area of cell adhesion was measured by using Image-Pro Plus 6.3J. Differences between the areas in each group were analyzed using one-way analysis of variance followed by Tukey’s test (* p

Techniques Used:

24) Product Images from "The Probiotic Compound VSL#3 Modulates Mucosal, Peripheral, and Systemic Immunity Following Murine Broad-Spectrum Antibiotic Treatment"

Article Title: The Probiotic Compound VSL#3 Modulates Mucosal, Peripheral, and Systemic Immunity Following Murine Broad-Spectrum Antibiotic Treatment

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2017.00167

IFN-γ and IL-10 producing CD4+ cells in intestinal and systemic compartments of secondary abiotic mice following recolonization with VSL#3 or complex murine microbiota . Lymphocytes were isolated from small intestinal and colonic lamina propria, MLN, and spleen and stimulated with PMA/ionomycin in presence of brefeldin A and subsequently analyzed by flow cytometry. The percentages of IFN-γ (left panel A,C,E,G ) and IL-10 (right panel B,D,F,H ) producing CD4+ cells in the small intestine (A,B) , colon (C,D) , MLN (E,F) , and spleen (G,H) in naive conventional mice (N), by antibiotic treatment generated secondary abiotic mice (ABx), and mice subjected to VSL#3 recolonization or fecal microbiota transplantation (FMT) were determined on day 28 following peroral reassociation. Box plots represent the 75th and 25th percentiles of the medians (black bar inside the boxes). Total range and significance levels ( p -values) determined with one-way ANOVA test followed by Tukey post-correction test for multiple comparisons are indicated. Data shown were pooled from two independent experiments ( n = 10–15/group).
Figure Legend Snippet: IFN-γ and IL-10 producing CD4+ cells in intestinal and systemic compartments of secondary abiotic mice following recolonization with VSL#3 or complex murine microbiota . Lymphocytes were isolated from small intestinal and colonic lamina propria, MLN, and spleen and stimulated with PMA/ionomycin in presence of brefeldin A and subsequently analyzed by flow cytometry. The percentages of IFN-γ (left panel A,C,E,G ) and IL-10 (right panel B,D,F,H ) producing CD4+ cells in the small intestine (A,B) , colon (C,D) , MLN (E,F) , and spleen (G,H) in naive conventional mice (N), by antibiotic treatment generated secondary abiotic mice (ABx), and mice subjected to VSL#3 recolonization or fecal microbiota transplantation (FMT) were determined on day 28 following peroral reassociation. Box plots represent the 75th and 25th percentiles of the medians (black bar inside the boxes). Total range and significance levels ( p -values) determined with one-way ANOVA test followed by Tukey post-correction test for multiple comparisons are indicated. Data shown were pooled from two independent experiments ( n = 10–15/group).

Techniques Used: Mouse Assay, Isolation, Flow Cytometry, Cytometry, Generated, Transplantation Assay

IL-17 and IL-22 producing CD4+ cells in intestinal and systemic compartments of secondary abiotic mice following recolonization with VSL#3 or complex murine microbiota . Lymphocytes were isolated from small intestinal and colonic lamina propria, MLN, and spleen and stimulated with PMA/ionomycin in presence of brefeldin A and subsequently analyzed by flow cytometry. The percentages of IL-17 (left panel A,C,E,G ) and IL-22 (right panel B,D,F,H ) producing CD4+ cells in the small intestine (A,B) , colon (C,D) , MLN (E,F) , and spleen (G,H) in naive conventional mice (N), by antibiotic treatment generated secondary abiotic mice (ABx), and mice subjected to VSL#3 recolonization or fecal microbiota transplantation (FMT) were determined on day 28 following peroral reassociation. Box plots represent the 75th and 25th percentiles of the medians (black bar inside the boxes). Total range and significance levels ( p -values) determined with one-way ANOVA test followed by Tukey post-correction test for multiple comparisons are indicated. Data shown were pooled from two independent experiments ( n = 10–15/group).
Figure Legend Snippet: IL-17 and IL-22 producing CD4+ cells in intestinal and systemic compartments of secondary abiotic mice following recolonization with VSL#3 or complex murine microbiota . Lymphocytes were isolated from small intestinal and colonic lamina propria, MLN, and spleen and stimulated with PMA/ionomycin in presence of brefeldin A and subsequently analyzed by flow cytometry. The percentages of IL-17 (left panel A,C,E,G ) and IL-22 (right panel B,D,F,H ) producing CD4+ cells in the small intestine (A,B) , colon (C,D) , MLN (E,F) , and spleen (G,H) in naive conventional mice (N), by antibiotic treatment generated secondary abiotic mice (ABx), and mice subjected to VSL#3 recolonization or fecal microbiota transplantation (FMT) were determined on day 28 following peroral reassociation. Box plots represent the 75th and 25th percentiles of the medians (black bar inside the boxes). Total range and significance levels ( p -values) determined with one-way ANOVA test followed by Tukey post-correction test for multiple comparisons are indicated. Data shown were pooled from two independent experiments ( n = 10–15/group).

Techniques Used: Mouse Assay, Isolation, Flow Cytometry, Cytometry, Generated, Transplantation Assay

25) Product Images from "Schistosome egg antigens, including the glycoprotein IPSE/alpha-1, trigger the development of regulatory B cells"

Article Title: Schistosome egg antigens, including the glycoprotein IPSE/alpha-1, trigger the development of regulatory B cells

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006539

Breg cells can be generated in vitro by stimulation with schistosome egg antigens which involves lysosomal processing. (A-E) Splenic B cells from naïve mice were cultured for 3 days with 20 μg/ml SEA or medium as control. Cytokine concentration in culture supernatants of (A) total B cells (6 experiments) or (B) sorted B cell subsets (2 experiments with N = 6). (C) Intracellular IL-10 expression of total B cells after addition of PMA, ionomycin and Brefeldin A to the last 4 hours of the culture (4 experiments). (D) Mean fluorescence intensity of the activation markers CD40 and CD86 (4 experiments). (E) Splenic B cells were stimulated in vitro as above, and subsequently co-cultured with CD4 + CD25 - sorted splenic T cells. After 4 days, the frequency of CD25 + Foxp3 + Treg cells within the CD4 T cell population was determined by flow cytometry. Summary of 4 experiments. (F, G) Splenic B cells from naïve mice were cultured in vitro with SEA (20 μg/ml), CpG (5 μg/ml) or Pam3Cys (10 μg/ml) for 2 days. Every day, chloroquine (5 μM) was added to the culture. Intracellular IL-10 expression after addition of PMA, ionomycin and Brefeldin A during the last 4 hours of the culture (F) , and cytokine concentrations in culture supernatants (G) . Summary of 2 experiments with N = 3–4. Significant differences are indicated with * p
Figure Legend Snippet: Breg cells can be generated in vitro by stimulation with schistosome egg antigens which involves lysosomal processing. (A-E) Splenic B cells from naïve mice were cultured for 3 days with 20 μg/ml SEA or medium as control. Cytokine concentration in culture supernatants of (A) total B cells (6 experiments) or (B) sorted B cell subsets (2 experiments with N = 6). (C) Intracellular IL-10 expression of total B cells after addition of PMA, ionomycin and Brefeldin A to the last 4 hours of the culture (4 experiments). (D) Mean fluorescence intensity of the activation markers CD40 and CD86 (4 experiments). (E) Splenic B cells were stimulated in vitro as above, and subsequently co-cultured with CD4 + CD25 - sorted splenic T cells. After 4 days, the frequency of CD25 + Foxp3 + Treg cells within the CD4 T cell population was determined by flow cytometry. Summary of 4 experiments. (F, G) Splenic B cells from naïve mice were cultured in vitro with SEA (20 μg/ml), CpG (5 μg/ml) or Pam3Cys (10 μg/ml) for 2 days. Every day, chloroquine (5 μM) was added to the culture. Intracellular IL-10 expression after addition of PMA, ionomycin and Brefeldin A during the last 4 hours of the culture (F) , and cytokine concentrations in culture supernatants (G) . Summary of 2 experiments with N = 3–4. Significant differences are indicated with * p

Techniques Used: Generated, In Vitro, Mouse Assay, Cell Culture, Concentration Assay, Expressing, Fluorescence, Activation Assay, Flow Cytometry, Cytometry

26) Product Images from "Gαq Regulates the Development of Rheumatoid Arthritis by Modulating Th1 Differentiation"

Article Title: Gαq Regulates the Development of Rheumatoid Arthritis by Modulating Th1 Differentiation

Journal: Mediators of Inflammation

doi: 10.1155/2017/4639081

The percentage of Th1 cells is increased in Gnaq−/− BM chimeras suffering from inflammatory arthritis. (a) Single cell suspension was deprived from spleen of Gnaq−/− BM chimeras suffering from inflammatory arthritis and WT controls, stimulated with PMA, ionomycin, and monensin for 4 hours. After culture, cells were stained with PE-conjugated anti-CD4, followed by intracellular staining with FITC-conjugated anti-IFN- γ , and analyzed by flow cytometry. Gated on CD4 + cells. (b) The percentage of Th1 cells is presented as mean ± SE, ∗∗ P
Figure Legend Snippet: The percentage of Th1 cells is increased in Gnaq−/− BM chimeras suffering from inflammatory arthritis. (a) Single cell suspension was deprived from spleen of Gnaq−/− BM chimeras suffering from inflammatory arthritis and WT controls, stimulated with PMA, ionomycin, and monensin for 4 hours. After culture, cells were stained with PE-conjugated anti-CD4, followed by intracellular staining with FITC-conjugated anti-IFN- γ , and analyzed by flow cytometry. Gated on CD4 + cells. (b) The percentage of Th1 cells is presented as mean ± SE, ∗∗ P

Techniques Used: Staining, Flow Cytometry, Cytometry

Loss of G α q enhances the differentiation of Th1 cells. Purified naïve CD4 + T cells from WT and Gnaq−/− mice were stimulated with anti-CD3/CD28 (3 μ g/mL), in the presence of mouse IL-12 (20 ng/mL), mouse IL-2 (20 ng/mL), and anti-IL-4 (10 μ g/mL) for five days. Cells were harvested and analyzed. (a) WT and Gnaq−/− CD4 + T cells were stimulated with PMA, ionomycin, and monensin, fixed, permeabilized, and stained with FITC-conjugated anti-IFN- γ , followed by flow cytometry. (b) The percentage of IFN- γ + cells was calculated. (c) IFN- γ secretion was detected by ELISA. Cultured CD4 + T cells were harvested, adjusted to same concentration, and stimulated by anti-CD3/CD28 (1 μ g/mL) for 24 hours. Supernatants were collected for ELISA assay. All data are presented as mean ± SD; ∗ P
Figure Legend Snippet: Loss of G α q enhances the differentiation of Th1 cells. Purified naïve CD4 + T cells from WT and Gnaq−/− mice were stimulated with anti-CD3/CD28 (3 μ g/mL), in the presence of mouse IL-12 (20 ng/mL), mouse IL-2 (20 ng/mL), and anti-IL-4 (10 μ g/mL) for five days. Cells were harvested and analyzed. (a) WT and Gnaq−/− CD4 + T cells were stimulated with PMA, ionomycin, and monensin, fixed, permeabilized, and stained with FITC-conjugated anti-IFN- γ , followed by flow cytometry. (b) The percentage of IFN- γ + cells was calculated. (c) IFN- γ secretion was detected by ELISA. Cultured CD4 + T cells were harvested, adjusted to same concentration, and stimulated by anti-CD3/CD28 (1 μ g/mL) for 24 hours. Supernatants were collected for ELISA assay. All data are presented as mean ± SD; ∗ P

Techniques Used: Purification, Mouse Assay, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Concentration Assay

27) Product Images from "Interleukin 2 gene transcription is regulated by Ikaros-induced changes in histone acetylation in anergic T cells"

Article Title: Interleukin 2 gene transcription is regulated by Ikaros-induced changes in histone acetylation in anergic T cells

Journal:

doi: 10.1182/blood-2006-07-037754

Anergizing stimuli induce H4 deacetylation of the il2 promoter in anergic Th1 cells . (A) Th1 cells were treated with 1 μM ionomycin (Iono) for 16 hours and analyzed by intracellular IL-2 staining following stimulation with anti-CD3 and anti-CD28.
Figure Legend Snippet: Anergizing stimuli induce H4 deacetylation of the il2 promoter in anergic Th1 cells . (A) Th1 cells were treated with 1 μM ionomycin (Iono) for 16 hours and analyzed by intracellular IL-2 staining following stimulation with anti-CD3 and anti-CD28.

Techniques Used: Staining

HDAC inhibition interferes with IL-2 expression blockade in anergic Th1 cells . (A) Th1 cells were treated with ionomycin in the presence or absence of TSA. Untreated and TSA-treated cells were used as controls. After a 16-hour treatment, cells were allowed
Figure Legend Snippet: HDAC inhibition interferes with IL-2 expression blockade in anergic Th1 cells . (A) Th1 cells were treated with ionomycin in the presence or absence of TSA. Untreated and TSA-treated cells were used as controls. After a 16-hour treatment, cells were allowed

Techniques Used: Inhibition, Expressing

Calcium signaling induces increased expression and binding of Ikaros to the il2 promoter in anergic Th1 cells . (A) Th1 cells were treated with ionomycin (Iono) for 6 hours and the expression of Ikaros mRNA was measured by qPCR compared with resting cells
Figure Legend Snippet: Calcium signaling induces increased expression and binding of Ikaros to the il2 promoter in anergic Th1 cells . (A) Th1 cells were treated with ionomycin (Iono) for 6 hours and the expression of Ikaros mRNA was measured by qPCR compared with resting cells

Techniques Used: Expressing, Binding Assay, Real-time Polymerase Chain Reaction

28) Product Images from "Modulation of ATP-induced Calcium Signaling by progesterone in T47D-Y Breast Cancer Cells"

Article Title: Modulation of ATP-induced Calcium Signaling by progesterone in T47D-Y Breast Cancer Cells

Journal: Molecular and cellular endocrinology

doi: 10.1016/j.mce.2010.01.004

Progesterone pretreatment does not affect mobilization of intracellular stores by Ionomycin
Figure Legend Snippet: Progesterone pretreatment does not affect mobilization of intracellular stores by Ionomycin

Techniques Used:

29) Product Images from "Hydroquinone, a reactive metabolite of benzene, enhances interleukin-4 production in CD4+ T cells and increases immunoglobulin E levels in antigen-primed mice"

Article Title: Hydroquinone, a reactive metabolite of benzene, enhances interleukin-4 production in CD4+ T cells and increases immunoglobulin E levels in antigen-primed mice

Journal: Immunology

doi: 10.1046/j.1365-2567.2002.01451.x

HQ-mediated the enhancement of IL-4 promoter activated by PMA/ionomycin. EL-4 cells were transiently transfected with the IL-4 promoter constructs followed by stimulation with PMA/ionomycin in the presence of HQ. The results are represented as induction fold over the value obtained with unstimulated EL-4 cells transfected with each of promoter constructs, given as an arbitrary value of 1. The data are representative of three independent experiments.
Figure Legend Snippet: HQ-mediated the enhancement of IL-4 promoter activated by PMA/ionomycin. EL-4 cells were transiently transfected with the IL-4 promoter constructs followed by stimulation with PMA/ionomycin in the presence of HQ. The results are represented as induction fold over the value obtained with unstimulated EL-4 cells transfected with each of promoter constructs, given as an arbitrary value of 1. The data are representative of three independent experiments.

Techniques Used: Transfection, Construct

30) Product Images from "Three-Color Flow Cytometry Detection of Intracellular Cytokines in Peripheral Blood Mononuclear Cells: Comparative Analysis of Phorbol Myristate Acetate-Ionomycin and Phytohemagglutinin Stimulation"

Article Title: Three-Color Flow Cytometry Detection of Intracellular Cytokines in Peripheral Blood Mononuclear Cells: Comparative Analysis of Phorbol Myristate Acetate-Ionomycin and Phytohemagglutinin Stimulation

Journal: Clinical and Diagnostic Laboratory Immunology

doi: 10.1128/CDLI.8.2.303-313.2001

Quantification of TNF-β- and IL-10-producing CD3 + cells by flow cytometry. PBMCs cultured for 48 h in the medium (left set of panels under “control”) or stimulated with PMA-ionomycin (middle set of panels) or PHA (right set of panels) in the presence of monensin were labeled for surface CD3 (PE/Cy5) and intracellular TNF-β or IL-10 (PE) expression. Cells were gated according to CD3 expression and SSC (region R1). Histogram overlays show FL2 (orange fluorescence) intensity corresponding to a given cytokine (solid line) compared to the intensity for the isotype-specific control (dotted line).
Figure Legend Snippet: Quantification of TNF-β- and IL-10-producing CD3 + cells by flow cytometry. PBMCs cultured for 48 h in the medium (left set of panels under “control”) or stimulated with PMA-ionomycin (middle set of panels) or PHA (right set of panels) in the presence of monensin were labeled for surface CD3 (PE/Cy5) and intracellular TNF-β or IL-10 (PE) expression. Cells were gated according to CD3 expression and SSC (region R1). Histogram overlays show FL2 (orange fluorescence) intensity corresponding to a given cytokine (solid line) compared to the intensity for the isotype-specific control (dotted line).

Techniques Used: Flow Cytometry, Cytometry, Cell Culture, Labeling, Expressing, Fluorescence

Flow cytometry analysis of intracellular cytokine expression in control PBMC (left set of panels) and following either PMA-ionomycin activation (middle set of panels) or PHA activation (right set of panels). PBMCs were cultured in medium alone or were stimulated with PMA-ionomycin or PHA in the presence of monensin for 6 h and stained with PE-labeled anti-cytokine MAbs. Histogram overlays show the FL2 (orange fluorescence) intensity corresponding to a given cytokine (solid line) compared to the intensity for the isotype-specific control (dotted line). The numbers indicate the percentages of positive cells and the mean fluorescence intensity (mfi). The results from one representative experiment of five experiments performed are shown.
Figure Legend Snippet: Flow cytometry analysis of intracellular cytokine expression in control PBMC (left set of panels) and following either PMA-ionomycin activation (middle set of panels) or PHA activation (right set of panels). PBMCs were cultured in medium alone or were stimulated with PMA-ionomycin or PHA in the presence of monensin for 6 h and stained with PE-labeled anti-cytokine MAbs. Histogram overlays show the FL2 (orange fluorescence) intensity corresponding to a given cytokine (solid line) compared to the intensity for the isotype-specific control (dotted line). The numbers indicate the percentages of positive cells and the mean fluorescence intensity (mfi). The results from one representative experiment of five experiments performed are shown.

Techniques Used: Flow Cytometry, Cytometry, Expressing, Activation Assay, Cell Culture, Staining, Labeling, Fluorescence

Alterations of cell size (FSC) and cell granularity (SSC) of PBMCs after activation with PMA-ionomycin or PHA. PBMCs were cultured for 6 h in the medium (left) or were stimulated with PMA-ionomycin (middle) or PHA (right) in the presence of monensin. Dot plots (FSC versus SSC) show the changes in cellular morphology after cell activation. Regions R1 and R2, defined in a control, correspond to lymphocytes and monocytes, respectively.
Figure Legend Snippet: Alterations of cell size (FSC) and cell granularity (SSC) of PBMCs after activation with PMA-ionomycin or PHA. PBMCs were cultured for 6 h in the medium (left) or were stimulated with PMA-ionomycin (middle) or PHA (right) in the presence of monensin. Dot plots (FSC versus SSC) show the changes in cellular morphology after cell activation. Regions R1 and R2, defined in a control, correspond to lymphocytes and monocytes, respectively.

Techniques Used: Activation Assay, Cell Culture

Surface CD4 and CD14 and intracellular TNF-α expression in PBMCs. PBMCs were cultured for 6 h in medium (left panels) or were stimulated with PMA-ionomycin (middle panels) or PHA (right panels) in the presence of monensin. Dot plots of CD4-FITC (FL1 [green fluorescence]) (A) or CD14-FITC (B) versus TNF-α–PE (FL2 [orange fluorescence]) expression in the whole PBMC population are shown. Numbers show percentages of positive cells. Data from one representative experiment of three experiments performed are shown.
Figure Legend Snippet: Surface CD4 and CD14 and intracellular TNF-α expression in PBMCs. PBMCs were cultured for 6 h in medium (left panels) or were stimulated with PMA-ionomycin (middle panels) or PHA (right panels) in the presence of monensin. Dot plots of CD4-FITC (FL1 [green fluorescence]) (A) or CD14-FITC (B) versus TNF-α–PE (FL2 [orange fluorescence]) expression in the whole PBMC population are shown. Numbers show percentages of positive cells. Data from one representative experiment of three experiments performed are shown.

Techniques Used: Expressing, Cell Culture, Fluorescence

Intracellular cytokine expression in gated CD3 + T cells by three-color flow cytometry. PBMCs cultured for 6 h in medium (left set of panels) or stimulated with PMA-ionomycin (middle set of panels) or PHA (right set of panels) in the presence of monensin were labeled for the surface expression of CD3 (PE/Cy5) and CD4 (FITC) and for the intracellular presence of different cytokines (PE). Dot plots of CD4-FITC (FL1 [green fluorescence]) versus relevant cytokine-PE (FL2 [orange fluorescence]) after T-cell gating according to CD3-PE/Cy5 (FL3 [red fluorescence]) expression are shown. Numbers show the percentages of CD4 − and CD4 + T cells producing cytokines set according to the isotype-matched control. Data from one representative experiment of five experiments performed are shown.
Figure Legend Snippet: Intracellular cytokine expression in gated CD3 + T cells by three-color flow cytometry. PBMCs cultured for 6 h in medium (left set of panels) or stimulated with PMA-ionomycin (middle set of panels) or PHA (right set of panels) in the presence of monensin were labeled for the surface expression of CD3 (PE/Cy5) and CD4 (FITC) and for the intracellular presence of different cytokines (PE). Dot plots of CD4-FITC (FL1 [green fluorescence]) versus relevant cytokine-PE (FL2 [orange fluorescence]) after T-cell gating according to CD3-PE/Cy5 (FL3 [red fluorescence]) expression are shown. Numbers show the percentages of CD4 − and CD4 + T cells producing cytokines set according to the isotype-matched control. Data from one representative experiment of five experiments performed are shown.

Techniques Used: Expressing, Flow Cytometry, Cytometry, Cell Culture, Labeling, Fluorescence

Flow cytometry analysis of TNF-β- and IL-10-producing CD33 + cells. PBMCs cultured for 48 h in medium (left set of panels under “control”) or stimulated with PMA-ionomycin (middle set of panels) or PHA (right set of panels) in the presence of monensin were labeled for surface CD33 (PE/Cy5) and intracellular TNF-β or IL-10. Cells were gated according to CD33 expression and SSC (region R1). Histogram overlays show FL2 (orange fluorescence) intensity corresponding to a given cytokine (solid line) compared to the intensity for the isotype-specific control (dotted line). Results from one representative experiment of five experiments performed are shown.
Figure Legend Snippet: Flow cytometry analysis of TNF-β- and IL-10-producing CD33 + cells. PBMCs cultured for 48 h in medium (left set of panels under “control”) or stimulated with PMA-ionomycin (middle set of panels) or PHA (right set of panels) in the presence of monensin were labeled for surface CD33 (PE/Cy5) and intracellular TNF-β or IL-10. Cells were gated according to CD33 expression and SSC (region R1). Histogram overlays show FL2 (orange fluorescence) intensity corresponding to a given cytokine (solid line) compared to the intensity for the isotype-specific control (dotted line). Results from one representative experiment of five experiments performed are shown.

Techniques Used: Flow Cytometry, Cytometry, Cell Culture, Labeling, Expressing, Fluorescence

31) Product Images from "Opposing Action of Casein Kinase 1 and Calcineurin in Nucleo-cytoplasmic Shuttling of Mammalian Translation Initiation Factor eIF6 *"

Article Title: Opposing Action of Casein Kinase 1 and Calcineurin in Nucleo-cytoplasmic Shuttling of Mammalian Translation Initiation Factor eIF6 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.188565

Nuclear import of eIF6 from the cytoplasm is sensitive to calcineurin activator and inhibitor in vivo . A , COS-7 cells were treated either with 2 μ m calcium ionophore ionomycin to activate calcineurin, or with 5 μ m CsA, a specific calcineurin
Figure Legend Snippet: Nuclear import of eIF6 from the cytoplasm is sensitive to calcineurin activator and inhibitor in vivo . A , COS-7 cells were treated either with 2 μ m calcium ionophore ionomycin to activate calcineurin, or with 5 μ m CsA, a specific calcineurin

Techniques Used: In Vivo

32) Product Images from "Preclinical testing of anti-human CD28 Fab’ antibody in a novel nonhuman primate (NHP) small animal rodent model of xenogenic graft-versus-host disease (GVHD)"

Article Title: Preclinical testing of anti-human CD28 Fab’ antibody in a novel nonhuman primate (NHP) small animal rodent model of xenogenic graft-versus-host disease (GVHD)

Journal: Transplantation

doi: 10.1097/TP.0000000000001465

CD28 blockade with FR104 inhibits T cell expansion and effector differentiation To assess the effects of FR104 on NHP T cell expansion and differentiation in secondary lymphoid organs, spleens were harvested on day 10 from mice receiving PBMNC + control IgG or FR104. Splenocytes were isolated, stimulated with phorbol PMA/Ionomycin, and cytokine expressing cells determined by intracellular cytokine staining. (A) Average number of CD4+, CD8+ and CD4+8+ T cells (±SEM) present in spleen. Percentage and average number of CD4+, CD8+ and CD4+8+ T cells producing IFNγ (B), IL-17 (C) or GzmB (D). n=4 mice per group for elective sac on day 10. p-values as indicated.
Figure Legend Snippet: CD28 blockade with FR104 inhibits T cell expansion and effector differentiation To assess the effects of FR104 on NHP T cell expansion and differentiation in secondary lymphoid organs, spleens were harvested on day 10 from mice receiving PBMNC + control IgG or FR104. Splenocytes were isolated, stimulated with phorbol PMA/Ionomycin, and cytokine expressing cells determined by intracellular cytokine staining. (A) Average number of CD4+, CD8+ and CD4+8+ T cells (±SEM) present in spleen. Percentage and average number of CD4+, CD8+ and CD4+8+ T cells producing IFNγ (B), IL-17 (C) or GzmB (D). n=4 mice per group for elective sac on day 10. p-values as indicated.

Techniques Used: Mouse Assay, Isolation, Expressing, Staining

33) Product Images from "Counterregulation of clathrin-mediated endocytosis by the actin and microtubular cytoskeleton in human neutrophils"

Article Title: Counterregulation of clathrin-mediated endocytosis by the actin and microtubular cytoskeleton in human neutrophils

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00454.2008

Actin disruption induces Alexa fluor-488-conjugated BSA and transferrin uptake after ionomycin (Iono) stimulation. Neutrophils (4 × 10 6 cells/ml) were incubated for 30 min in the presence or absence of 225 mM sucrose, 40 μM chlorpromazine,
Figure Legend Snippet: Actin disruption induces Alexa fluor-488-conjugated BSA and transferrin uptake after ionomycin (Iono) stimulation. Neutrophils (4 × 10 6 cells/ml) were incubated for 30 min in the presence or absence of 225 mM sucrose, 40 μM chlorpromazine,

Techniques Used: Incubation

34) Product Images from "Genetic disruption of p38α Tyr323 phosphorylation prevents T-cell receptor–mediated p38α activation and impairs interferon-γ production"

Article Title: Genetic disruption of p38α Tyr323 phosphorylation prevents T-cell receptor–mediated p38α activation and impairs interferon-γ production

Journal: Blood

doi: 10.1182/blood-2008-04-153304

Onset of proliferation is delayed in p38α Y323F T cells stimulated through TCR/CD28. (A) Wild-type (■; WT) and p38 α Y323F (□; Y323F) T cells were stimulated with plate-bound anti-CD3/CD28 antibodies or with PMA plus ionomycin
Figure Legend Snippet: Onset of proliferation is delayed in p38α Y323F T cells stimulated through TCR/CD28. (A) Wild-type (■; WT) and p38 α Y323F (□; Y323F) T cells were stimulated with plate-bound anti-CD3/CD28 antibodies or with PMA plus ionomycin

Techniques Used:

35) Product Images from "A presenilin-1/?-secretase cleavage releases the E-cadherin intracellular domain and regulates disassembly of adherens junctions"

Article Title: A presenilin-1/?-secretase cleavage releases the E-cadherin intracellular domain and regulates disassembly of adherens junctions

Journal: The EMBO Journal

doi: 10.1093/emboj/21.8.1948

Fig. 5. A PS1/γ - secretase cleavage promotes disassembly of the E-cadherin–catenin adhesion complex. ( A and B ) A431 cell cultures were pre-incubated for 30 min in the absence (–) or presence (+) of L-685,458 and then treated for the indicated times with ionomycin (10 µM). Cell extracts were fractionated, and the Triton X-100-insoluble and cytosolic soluble fractions were analyzed on western blots with antibodies against cytoplasmic E-cadherin C36 (A) or β- and α-catenins (B). The immunoblots are representative of three independent experiments. ( C ) Signals from cytosolic β-catenin (upper graph) or α-catenin (lower graph) obtained from ionomycin-treated cultures in the presence (+L-685,458) or absence (–L-685,458) of γ-secretase inhibitor L-685,458 were quantified by densitometric analysis. The graphs show the averaged immunoreactivities observed in three independent experiments.
Figure Legend Snippet: Fig. 5. A PS1/γ - secretase cleavage promotes disassembly of the E-cadherin–catenin adhesion complex. ( A and B ) A431 cell cultures were pre-incubated for 30 min in the absence (–) or presence (+) of L-685,458 and then treated for the indicated times with ionomycin (10 µM). Cell extracts were fractionated, and the Triton X-100-insoluble and cytosolic soluble fractions were analyzed on western blots with antibodies against cytoplasmic E-cadherin C36 (A) or β- and α-catenins (B). The immunoblots are representative of three independent experiments. ( C ) Signals from cytosolic β-catenin (upper graph) or α-catenin (lower graph) obtained from ionomycin-treated cultures in the presence (+L-685,458) or absence (–L-685,458) of γ-secretase inhibitor L-685,458 were quantified by densitometric analysis. The graphs show the averaged immunoreactivities observed in three independent experiments.

Techniques Used: Incubation, Western Blot

Fig. 6. Immunostaining and LSCM analysis of ionomycin- and L-685,458-treated A431 cultures. A431 cells were pre-incubated for 30 min in the presence or absence of L-685,458 and then treated for 45 min with ionomycin. Control cells were not treated. Following the ionomycin-induced cell–cell dissociation, the distribution of PS1, E-cadherin, β-catenin and α-catenin in all cultures was analyzed by LSCM using the constant detector setting. Cells were double labeled either with anti-PS1/NTF antibody R222 ( A – C ) and anti-cytoplasmic E-cadherin antibody C36 ( D – F ), or with anti-ectodomain E-cadherin antibody H108 ( G – I ) and anti-β-catenin antibody ( J – L ). Cells were also labeled for α-catenin ( M – O ). Arrows indicate a cell population showing β-catenin immunoreactivity at the cell surface without ectodomain E-cadherin labeling (I and L). Scale bar = 30 µm.
Figure Legend Snippet: Fig. 6. Immunostaining and LSCM analysis of ionomycin- and L-685,458-treated A431 cultures. A431 cells were pre-incubated for 30 min in the presence or absence of L-685,458 and then treated for 45 min with ionomycin. Control cells were not treated. Following the ionomycin-induced cell–cell dissociation, the distribution of PS1, E-cadherin, β-catenin and α-catenin in all cultures was analyzed by LSCM using the constant detector setting. Cells were double labeled either with anti-PS1/NTF antibody R222 ( A – C ) and anti-cytoplasmic E-cadherin antibody C36 ( D – F ), or with anti-ectodomain E-cadherin antibody H108 ( G – I ) and anti-β-catenin antibody ( J – L ). Cells were also labeled for α-catenin ( M – O ). Arrows indicate a cell population showing β-catenin immunoreactivity at the cell surface without ectodomain E-cadherin labeling (I and L). Scale bar = 30 µm.

Techniques Used: Immunostaining, Incubation, Labeling

Fig. 7. E-cadherin mutation GGG759-761AAA prevents binding to PS1 and inhibits γ-secretase cleavage of E-cadherin and cytosolic release of catenins. ( A ) Right panel: extracts from A431D cells stably transfected with vector, wild-type E-cadherin (WT E-Cad) or E-cadherin mutant GGG759-761AAA (761AAA) were immunoprecipitated with anti-PS1/NTF antibody R222 (PS1 IP) and the IPs were probed with either anti-E-cadherin antibody C36 (upper panel) or anti-PS1/CTF antibody 33B10 (lower panel). The left panel shows relative E-cadherin levels in transfectants. ( B ) A431D cells transfected with either wild-type E-cadherin or E-cadherin mutant 761AAA were incubated in the absence (–) or presence (+) of ionomycin for 45 min, and RIPA extracts were probed on western blots with C36 antibody (upper panel). Cytosolic fractions of the above cultures were probed on western blots with antibodies against E-cadherin (C36, second panel), β-catenin (third panel) or α-catenin (lower panel). ( C ) Schematic representation of the PS1/γ-secretase-mediated disassembly of CAJs. An MMP-mediated proteolytic activity cleaves the extracellular domain of cytoskeletal E-cadherin and releases E-Cad/NTF1 to the extracellular medium (a). Fragment E-Cad/CTF1 containing the transmembrane and cytoplasmic sequence of E-cadherin remains bound to PS1, β-catenin, α-catenin and the actin cytoskeleton. E-Cad/CTF1 is then cleaved by a PS1/γ-secretase activity at the membrane–cytosol interface to produce E-Cad/CTF2, which dissociates from both PS1 and F-actin and is released to the cytosol in a complex with β-catenin (b). Full-length E-cadherin bound to the cytoskeleton can also be cleaved by the PS1/γ-secretase activity (c). No E-Cad/CTF2–α-catenin complex was detected, suggesting that α-catenin dissociates from E-Cad/CTF2 (unpublished observations). α, α-catenin; β, β-catenin.
Figure Legend Snippet: Fig. 7. E-cadherin mutation GGG759-761AAA prevents binding to PS1 and inhibits γ-secretase cleavage of E-cadherin and cytosolic release of catenins. ( A ) Right panel: extracts from A431D cells stably transfected with vector, wild-type E-cadherin (WT E-Cad) or E-cadherin mutant GGG759-761AAA (761AAA) were immunoprecipitated with anti-PS1/NTF antibody R222 (PS1 IP) and the IPs were probed with either anti-E-cadherin antibody C36 (upper panel) or anti-PS1/CTF antibody 33B10 (lower panel). The left panel shows relative E-cadherin levels in transfectants. ( B ) A431D cells transfected with either wild-type E-cadherin or E-cadherin mutant 761AAA were incubated in the absence (–) or presence (+) of ionomycin for 45 min, and RIPA extracts were probed on western blots with C36 antibody (upper panel). Cytosolic fractions of the above cultures were probed on western blots with antibodies against E-cadherin (C36, second panel), β-catenin (third panel) or α-catenin (lower panel). ( C ) Schematic representation of the PS1/γ-secretase-mediated disassembly of CAJs. An MMP-mediated proteolytic activity cleaves the extracellular domain of cytoskeletal E-cadherin and releases E-Cad/NTF1 to the extracellular medium (a). Fragment E-Cad/CTF1 containing the transmembrane and cytoplasmic sequence of E-cadherin remains bound to PS1, β-catenin, α-catenin and the actin cytoskeleton. E-Cad/CTF1 is then cleaved by a PS1/γ-secretase activity at the membrane–cytosol interface to produce E-Cad/CTF2, which dissociates from both PS1 and F-actin and is released to the cytosol in a complex with β-catenin (b). Full-length E-cadherin bound to the cytoskeleton can also be cleaved by the PS1/γ-secretase activity (c). No E-Cad/CTF2–α-catenin complex was detected, suggesting that α-catenin dissociates from E-Cad/CTF2 (unpublished observations). α, α-catenin; β, β-catenin.

Techniques Used: Mutagenesis, Binding Assay, Stable Transfection, Transfection, Plasmid Preparation, Immunoprecipitation, Incubation, Western Blot, Activity Assay, Sequencing

36) Product Images from "Id1 expression promotes T regulatory cell differentiation by facilitating TCR costimulation"

Article Title: Id1 expression promotes T regulatory cell differentiation by facilitating TCR costimulation

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

doi: 10.4049/jimmunol.1302554

Attenuated EAE development in Id1 transgenic mice is associated with increased Treg counts and reduced encephalitogenic effector T cells (A) EAE was induced in 7-week-old WT and TG female littermates using MOG peptide and pertussis toxin. Disease progression was scored daily for 36 days using the criteria described in Materials and Methods. Results are presented as mean with SD. (B) The numbers of mononuclear cells harvested from the spinal cord after proteolytic digestion of the tissue are shown as mean with SD. (C) Expression of FoxP3 in draining lymph nodes and spinal cord was measured on day 14 after immunization and representative dot plots and average percentages of the indicated subsets are shown. (D) Examination of CD4 + effector T cells in draining lymph nodes and spinal cord. Cells were stimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) for 4 hours. IL-17A and IFNγ expression was detected using intracellular staining. Average percentages of the indicated subsets are shown.
Figure Legend Snippet: Attenuated EAE development in Id1 transgenic mice is associated with increased Treg counts and reduced encephalitogenic effector T cells (A) EAE was induced in 7-week-old WT and TG female littermates using MOG peptide and pertussis toxin. Disease progression was scored daily for 36 days using the criteria described in Materials and Methods. Results are presented as mean with SD. (B) The numbers of mononuclear cells harvested from the spinal cord after proteolytic digestion of the tissue are shown as mean with SD. (C) Expression of FoxP3 in draining lymph nodes and spinal cord was measured on day 14 after immunization and representative dot plots and average percentages of the indicated subsets are shown. (D) Examination of CD4 + effector T cells in draining lymph nodes and spinal cord. Cells were stimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) for 4 hours. IL-17A and IFNγ expression was detected using intracellular staining. Average percentages of the indicated subsets are shown.

Techniques Used: Transgenic Assay, Mouse Assay, Expressing, Staining

37) Product Images from "Identification of Novel Nuclear Factor of Activated T Cell (NFAT)-associated Proteins in T Cells *"

Article Title: Identification of Novel Nuclear Factor of Activated T Cell (NFAT)-associated Proteins in T Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.739326

Validation of the association of NFATc2 with identified partners. A, NFATc2-BT-containing protein complexes were isolated from nuclear extracts ( nucl. extr. ) of the indicated transgenic Jurkat cells after stimulation (PMA/ionomycin for 2 h) using streptavidin-coupled agarose beads. Proteins were eluted from the beads by TEV protease cleavage and analyzed by immunoblotting. Cells overexpressing wild-type NFATc2 (without tag) and BirA-ligase served as control cells. Images are representative for two independent experiments. IP, immunoprecipitation. B, proximity ligation assay pictures for Ikaros and NFATc2 in primary human CD4 + T cells show nuclear staining (DAPI) in blue and PLA signals in red . For control experiments, one of the primary antibodies was exchanged for an isotype-matched control antibody. C, statistical analysis of B ; n > 170 for all samples. D, confirmation of CRISPR/Cas9 induced knock-out of IKZF1 and NFATc2 in Jurkat cells. Jurkat IKZF1 −/− and NFATC2 −/− cells show no signal for the respective protein in immunoblot staining. Asterisk marks an unspecific band of the used NFATc2 antibody. E, PLA in stimulated Jurkat WT, IKZF1 −/− , or NFATC2 −/− cells; n > 1000 cells for each sample. p values were obtained by Kruskal-Wallis test. Size bars shown are 30 μm. All data are representative of two independent experiments. P/I, PMA/ionomycin; CsA, cyclosporine A.
Figure Legend Snippet: Validation of the association of NFATc2 with identified partners. A, NFATc2-BT-containing protein complexes were isolated from nuclear extracts ( nucl. extr. ) of the indicated transgenic Jurkat cells after stimulation (PMA/ionomycin for 2 h) using streptavidin-coupled agarose beads. Proteins were eluted from the beads by TEV protease cleavage and analyzed by immunoblotting. Cells overexpressing wild-type NFATc2 (without tag) and BirA-ligase served as control cells. Images are representative for two independent experiments. IP, immunoprecipitation. B, proximity ligation assay pictures for Ikaros and NFATc2 in primary human CD4 + T cells show nuclear staining (DAPI) in blue and PLA signals in red . For control experiments, one of the primary antibodies was exchanged for an isotype-matched control antibody. C, statistical analysis of B ; n > 170 for all samples. D, confirmation of CRISPR/Cas9 induced knock-out of IKZF1 and NFATc2 in Jurkat cells. Jurkat IKZF1 −/− and NFATC2 −/− cells show no signal for the respective protein in immunoblot staining. Asterisk marks an unspecific band of the used NFATc2 antibody. E, PLA in stimulated Jurkat WT, IKZF1 −/− , or NFATC2 −/− cells; n > 1000 cells for each sample. p values were obtained by Kruskal-Wallis test. Size bars shown are 30 μm. All data are representative of two independent experiments. P/I, PMA/ionomycin; CsA, cyclosporine A.

Techniques Used: Isolation, Transgenic Assay, Immunoprecipitation, Proximity Ligation Assay, Staining, CRISPR, Knock-Out

Dependence of NFATc2 protein associations on DNA binding. A, observed associations between NFAT and other proteins ( green circle ) can occur dependently or independently of DNA binding. In the first case, the proteins can either associate in complexes that are mediated/stabilized by DNA binding or can bind to the same DNA molecule independent from each other. The different modi can be distinguished by performing CoIP in the presence of either DNase I or GelRed. B–F, NFATc2-BT- or BT-GFP-containing complexes were isolated from nuclear extracts ( nucl. extr. ) of the indicated transgenic Jurkat cells after stimulation (PMA/ionomycin for 2 h) using streptavidin-coupled agarose beads, either in the presence or absence of the DNA intercalating agent GelRed ( B and C ) or DNase I treatment ( D–F ). DNA digestion was controlled by agarose gel electrophoresis of purified DNA from treated and untreated extracts ( D ). Proteins were eluted from the beads by boiling in 2× Laemmli buffer and analyzed by immunoblotting. Images are representative for three independent experiments. C and F show relative quantification of three independent experiments. Bars represent mean; underlying values are marked by x .
Figure Legend Snippet: Dependence of NFATc2 protein associations on DNA binding. A, observed associations between NFAT and other proteins ( green circle ) can occur dependently or independently of DNA binding. In the first case, the proteins can either associate in complexes that are mediated/stabilized by DNA binding or can bind to the same DNA molecule independent from each other. The different modi can be distinguished by performing CoIP in the presence of either DNase I or GelRed. B–F, NFATc2-BT- or BT-GFP-containing complexes were isolated from nuclear extracts ( nucl. extr. ) of the indicated transgenic Jurkat cells after stimulation (PMA/ionomycin for 2 h) using streptavidin-coupled agarose beads, either in the presence or absence of the DNA intercalating agent GelRed ( B and C ) or DNase I treatment ( D–F ). DNA digestion was controlled by agarose gel electrophoresis of purified DNA from treated and untreated extracts ( D ). Proteins were eluted from the beads by boiling in 2× Laemmli buffer and analyzed by immunoblotting. Images are representative for three independent experiments. C and F show relative quantification of three independent experiments. Bars represent mean; underlying values are marked by x .

Techniques Used: Binding Assay, Co-Immunoprecipitation Assay, Isolation, Transgenic Assay, Agarose Gel Electrophoresis, Purification

Co-purification of associated proteins with different NFAT isoforms. A , protein complexes containing biotinylated proteins were isolated from nuclear extracts of the indicated transgenic Jurkat cells after stimulation (PMA/ionomycin for 2 h) using streptavidin-coupled agarose beads. Proteins were eluted from the beads by boiling in 2× Laemmli buffer and analyzed by immunoblotting. Images are representative for three independent experiments. B , signals from two (NFATc1/αA, p300) or three experiments were quantified, normalized to the signal of the respective purified bait proteins, and depicted in relation to the NFATc2/CoIP-signals. Bars represent mean; underlying values are marked by x .
Figure Legend Snippet: Co-purification of associated proteins with different NFAT isoforms. A , protein complexes containing biotinylated proteins were isolated from nuclear extracts of the indicated transgenic Jurkat cells after stimulation (PMA/ionomycin for 2 h) using streptavidin-coupled agarose beads. Proteins were eluted from the beads by boiling in 2× Laemmli buffer and analyzed by immunoblotting. Images are representative for three independent experiments. B , signals from two (NFATc1/αA, p300) or three experiments were quantified, normalized to the signal of the respective purified bait proteins, and depicted in relation to the NFATc2/CoIP-signals. Bars represent mean; underlying values are marked by x .

Techniques Used: Copurification, Isolation, Transgenic Assay, Purification, Co-Immunoprecipitation Assay

38) Product Images from "The NEMO Mutation Creating the Most-Upstream Premature Stop Codon Is Hypomorphic Because of a Reinitiation of Translation"

Article Title: The NEMO Mutation Creating the Most-Upstream Premature Stop Codon Is Hypomorphic Because of a Reinitiation of Translation

Journal: American Journal of Human Genetics

doi:

Whole blood cell activation. A, IL-10 production. B, IL-6 production. C, TNF-α production. D–E, IFN-γ and IL-12p40 production, respectively, in the culture supernatants of whole blood cells from a control and from the patient after 48 h of activation with TNF-α, IL-1β, LPS, heat-killed S. aureus, and PMA/ionomycin; after 24 h of stimulation with agonists of TLR1-9 and whole bacteria ( E. coli and M. tb ); and after 24 or 48 h with BCG, BCG plus IL-12, or BCG plus IFNγ. Cytokine production was normalized for 10 6 peripheral blood mononuclear cells (PBMC). F–G, B-cell proliferation and B-cell immunoglobulin class switching of PBMC from a control and the patient on IL-4, soluble recombinant CD154, and CD154 plus IL-4. When indicated, means and SDs have been calculated from three to five different controls, and the patient has been tested two to four times.
Figure Legend Snippet: Whole blood cell activation. A, IL-10 production. B, IL-6 production. C, TNF-α production. D–E, IFN-γ and IL-12p40 production, respectively, in the culture supernatants of whole blood cells from a control and from the patient after 48 h of activation with TNF-α, IL-1β, LPS, heat-killed S. aureus, and PMA/ionomycin; after 24 h of stimulation with agonists of TLR1-9 and whole bacteria ( E. coli and M. tb ); and after 24 or 48 h with BCG, BCG plus IL-12, or BCG plus IFNγ. Cytokine production was normalized for 10 6 peripheral blood mononuclear cells (PBMC). F–G, B-cell proliferation and B-cell immunoglobulin class switching of PBMC from a control and the patient on IL-4, soluble recombinant CD154, and CD154 plus IL-4. When indicated, means and SDs have been calculated from three to five different controls, and the patient has been tested two to four times.

Techniques Used: Activation Assay, Recombinant

NF-κB signaling in SV40-transformed fibroblasts. A, Time-course analysis of IκBα protein degradation and resynthesis detected by western blot after stimulation with IL-1β for 20, 40, 60, and 120 min; the antibody against p38 served as a control for protein loading. B, NF-κB DNA-binding activity, measured by EMSA after stimulation for 20 and 40 min with IL-1β. γ-Activating–factor DNA-binding activity after 20 min of stimulation with IFN-γ and competition with unlabeled NF-κB–specific probe (*) served as internal activation control and specificity control, respectively. C, IL-6 production after 18 h of exposure to IL-1β, TNF-α, and PMA/ionomycin, in fibroblasts from a healthy control, from the patient, from a patient with XR-OL-EDA-ID (X420W), and from a patient with XD-IP ( ex4_10del ). Results shown are representative of two to three independent experiments.
Figure Legend Snippet: NF-κB signaling in SV40-transformed fibroblasts. A, Time-course analysis of IκBα protein degradation and resynthesis detected by western blot after stimulation with IL-1β for 20, 40, 60, and 120 min; the antibody against p38 served as a control for protein loading. B, NF-κB DNA-binding activity, measured by EMSA after stimulation for 20 and 40 min with IL-1β. γ-Activating–factor DNA-binding activity after 20 min of stimulation with IFN-γ and competition with unlabeled NF-κB–specific probe (*) served as internal activation control and specificity control, respectively. C, IL-6 production after 18 h of exposure to IL-1β, TNF-α, and PMA/ionomycin, in fibroblasts from a healthy control, from the patient, from a patient with XR-OL-EDA-ID (X420W), and from a patient with XD-IP ( ex4_10del ). Results shown are representative of two to three independent experiments.

Techniques Used: Transformation Assay, Western Blot, Binding Assay, Activity Assay, Activation Assay

39) Product Images from "Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis"

Article Title: Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis

Journal: Blood

doi: 10.1182/blood-2009-10-246116

Perforin increases clathrin-mediated endocytosis . (A) Within 7 minutes of treatment, sublytic rPFN and SLO activate uptake of A488-GzmB, whereas ionomycin, even at the highest lytic concentration, does not. Mean fluorescence intensity (mean ±
Figure Legend Snippet: Perforin increases clathrin-mediated endocytosis . (A) Within 7 minutes of treatment, sublytic rPFN and SLO activate uptake of A488-GzmB, whereas ionomycin, even at the highest lytic concentration, does not. Mean fluorescence intensity (mean ±

Techniques Used: Concentration Assay, Fluorescence

40) Product Images from "A novel form of integrin dysfunction involving ?1, ?2, and ?3 integrins"

Article Title: A novel form of integrin dysfunction involving ?1, ?2, and ?3 integrins

Journal: Journal of Clinical Investigation

doi: 10.1172/JCI200314076

Adhesion of patient and control T cells to LFA-1 ligands ICAM-1 and ICAM-3, to α4β1 ligand VCAM-1, and to α4β1/α5β1 ligand fibronectin. The binding of control (light gray bars) and patient (black bars) T cells to plates coated with ( a ) ICAM-1, ( b ) ICAM-3, ( c ) VCAM-1, and ( d ) fibronectin when stimulated with 50 nM PdBu, 1 μM ionomycin, 10 μg/ml UCHT-1, 10 μg/ml KIM 185 or TS2/16, 5 mM MgCl 2 /1 mM EGTA, or 0.5 mM MnCl 2 . The presence of αL mAb 38 at 10 μg/ml in a and b , α4 mAb HP2/1 at 10 μg/ml in c , and α4 mAb HP2/1 plus α5 mAb SAM-1 both at 10 μg/ml in d inhibits adhesion of control (white bars) and patient (dark gray bars) cells. Data (mean of triplicates ± SD) from one representative experiment ( n = 3) are shown. Unstim, unstimulated.
Figure Legend Snippet: Adhesion of patient and control T cells to LFA-1 ligands ICAM-1 and ICAM-3, to α4β1 ligand VCAM-1, and to α4β1/α5β1 ligand fibronectin. The binding of control (light gray bars) and patient (black bars) T cells to plates coated with ( a ) ICAM-1, ( b ) ICAM-3, ( c ) VCAM-1, and ( d ) fibronectin when stimulated with 50 nM PdBu, 1 μM ionomycin, 10 μg/ml UCHT-1, 10 μg/ml KIM 185 or TS2/16, 5 mM MgCl 2 /1 mM EGTA, or 0.5 mM MnCl 2 . The presence of αL mAb 38 at 10 μg/ml in a and b , α4 mAb HP2/1 at 10 μg/ml in c , and α4 mAb HP2/1 plus α5 mAb SAM-1 both at 10 μg/ml in d inhibits adhesion of control (white bars) and patient (dark gray bars) cells. Data (mean of triplicates ± SD) from one representative experiment ( n = 3) are shown. Unstim, unstimulated.

Techniques Used: Binding Assay

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Cell Culture:

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Mouse Assay:

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Concentration Assay:

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Incubation:

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Blocking Assay:

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Staining:

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    The FFA induced Ca 2+ activation is store-independent in GEnC but is store-dependent in ciPod. GEnC and ciPods loaded with Fura-2AM were pre-incubated with TG to deplete stores, then stimulated with FFA. (A) An example of an entire experiment in ciGEnC showing response to vehicle, TG then FFA, <t>ionomycin</t> (IM) then quench (MnCl). (B) Representative trace of the response to FFA in GEnC after store depletion, taken from (A). (C) Representative trace of the response to FFA in ciPod after store depletion. (D) Summary of data represented in (B) and (C) presented as area under the curve above 1, unpaired t -test, n = 6 and 5 respectively * p
    Ionomycin Im, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 88 stars, based on 3 article reviews
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    ionomycin im - by Bioz Stars, 2020-09
    88/100 stars
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    97
    Millipore ionomycin
    ( a ) Effects of activators (DNP-HSA 50 ng/mL, PMA 50 nM, <t>Ionomycin</t> 1 μM) and inhibitors (Genistein 100 μM, Cytochalasin D 1 μM, Nocodazole 10 μM) on CI changes (ΔCI) of RBL-2H3 cells. The gray bar indicates the presence of each inhibitor, and the black bar indicates the presence of antigen, activator, or Triton. ( b ) Effects of activators and inhibitors on degranulation of RBL-2H3 cells. The graph is representative of three experiments. Data were obtained from quadruplicate measurements. ( c ) Effects of Cytochalasin D on cell spreading in response to antigen. The white bar shows ca. 10 μm. ( d ) Relative cell area of RBL-2H3 cells before and after stimulation by DNP-HSA with or without Cytochalasin D. The area of cell adhesion was measured by using Image-Pro Plus 6.3J. Differences between the areas in each group were analyzed using one-way analysis of variance followed by Tukey’s test (* p
    Ionomycin, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 5718 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ionomycin/product/Millipore
    Average 97 stars, based on 5718 article reviews
    Price from $9.99 to $1999.99
    ionomycin - by Bioz Stars, 2020-09
    97/100 stars
      Buy from Supplier

    Image Search Results


    The FFA induced Ca 2+ activation is store-independent in GEnC but is store-dependent in ciPod. GEnC and ciPods loaded with Fura-2AM were pre-incubated with TG to deplete stores, then stimulated with FFA. (A) An example of an entire experiment in ciGEnC showing response to vehicle, TG then FFA, ionomycin (IM) then quench (MnCl). (B) Representative trace of the response to FFA in GEnC after store depletion, taken from (A). (C) Representative trace of the response to FFA in ciPod after store depletion. (D) Summary of data represented in (B) and (C) presented as area under the curve above 1, unpaired t -test, n = 6 and 5 respectively * p

    Journal: Cell Calcium

    Article Title: Functional distinctions in cytosolic calcium regulation between cells of the glomerular filtration barrier

    doi: 10.1016/j.ceca.2010.06.005

    Figure Lengend Snippet: The FFA induced Ca 2+ activation is store-independent in GEnC but is store-dependent in ciPod. GEnC and ciPods loaded with Fura-2AM were pre-incubated with TG to deplete stores, then stimulated with FFA. (A) An example of an entire experiment in ciGEnC showing response to vehicle, TG then FFA, ionomycin (IM) then quench (MnCl). (B) Representative trace of the response to FFA in GEnC after store depletion, taken from (A). (C) Representative trace of the response to FFA in ciPod after store depletion. (D) Summary of data represented in (B) and (C) presented as area under the curve above 1, unpaired t -test, n = 6 and 5 respectively * p

    Article Snippet: Where minimal Ca2+ was used, 10 μM ionomycin (IM) only induced a minimal increase in [Ca2+ ]i following stimulation with 200 nM thapsigargin (TG, an inhibitor of sarco-endoplasmic Ca2+ ATPases, Calbiochem, San Diego, CA), demonstrating that the external Ca2+ concentration was successfully reduced (data not shown).

    Techniques: Activation Assay, Incubation

    ( a ) Effects of activators (DNP-HSA 50 ng/mL, PMA 50 nM, Ionomycin 1 μM) and inhibitors (Genistein 100 μM, Cytochalasin D 1 μM, Nocodazole 10 μM) on CI changes (ΔCI) of RBL-2H3 cells. The gray bar indicates the presence of each inhibitor, and the black bar indicates the presence of antigen, activator, or Triton. ( b ) Effects of activators and inhibitors on degranulation of RBL-2H3 cells. The graph is representative of three experiments. Data were obtained from quadruplicate measurements. ( c ) Effects of Cytochalasin D on cell spreading in response to antigen. The white bar shows ca. 10 μm. ( d ) Relative cell area of RBL-2H3 cells before and after stimulation by DNP-HSA with or without Cytochalasin D. The area of cell adhesion was measured by using Image-Pro Plus 6.3J. Differences between the areas in each group were analyzed using one-way analysis of variance followed by Tukey’s test (* p

    Journal: Sensors (Basel, Switzerland)

    Article Title: Impedance-Based Living Cell Analysis for Clinical Diagnosis of Type I Allergy

    doi: 10.3390/s17112503

    Figure Lengend Snippet: ( a ) Effects of activators (DNP-HSA 50 ng/mL, PMA 50 nM, Ionomycin 1 μM) and inhibitors (Genistein 100 μM, Cytochalasin D 1 μM, Nocodazole 10 μM) on CI changes (ΔCI) of RBL-2H3 cells. The gray bar indicates the presence of each inhibitor, and the black bar indicates the presence of antigen, activator, or Triton. ( b ) Effects of activators and inhibitors on degranulation of RBL-2H3 cells. The graph is representative of three experiments. Data were obtained from quadruplicate measurements. ( c ) Effects of Cytochalasin D on cell spreading in response to antigen. The white bar shows ca. 10 μm. ( d ) Relative cell area of RBL-2H3 cells before and after stimulation by DNP-HSA with or without Cytochalasin D. The area of cell adhesion was measured by using Image-Pro Plus 6.3J. Differences between the areas in each group were analyzed using one-way analysis of variance followed by Tukey’s test (* p

    Article Snippet: Reagents Chemicals were obtained from the following sources: bovine serum albumin (BSA), dinitro-phenol-conjugated human serum albumin (DNP-HSA), DNP-specific rat monoclonal IgE and Phalloidin-TRITC from Sigma-Aldrich Japan (Tokyo, Japan); anti-IgE from BETYL (Montgomery, TX, USA); fetal calf serum (FCS) from Biowest (Nuaillé, France); penicillin/streptomycin, trypsin, and G418 from Life Technologies (Carlsbad, CA, USA); phorbol 12-myristate 13-acetate (PMA), ionomycin, genistein, cytochalasin D and nocodazole from Calbiochem (San Diego, CA, USA); epidermal growth factor (EGF) from R & D Systems (Minneapolis, MN, USA); DAPI from Dojindo (Kumamoto, Japan).

    Techniques:

    Anergizing stimuli induce H4 deacetylation of the il2 promoter in anergic Th1 cells . (A) Th1 cells were treated with 1 μM ionomycin (Iono) for 16 hours and analyzed by intracellular IL-2 staining following stimulation with anti-CD3 and anti-CD28.

    Journal:

    Article Title: Interleukin 2 gene transcription is regulated by Ikaros-induced changes in histone acetylation in anergic T cells

    doi: 10.1182/blood-2006-07-037754

    Figure Lengend Snippet: Anergizing stimuli induce H4 deacetylation of the il2 promoter in anergic Th1 cells . (A) Th1 cells were treated with 1 μM ionomycin (Iono) for 16 hours and analyzed by intracellular IL-2 staining following stimulation with anti-CD3 and anti-CD28.

    Article Snippet: Twenty-four hours after transfection, cells were stimulated with 500 nM ionomycin and 20 nM phorbol-12-myristate-13-acetate (PMA; Calbiochem) or plate-bound anti–human CD3 and CD28 (BD) at 1 μg/mL.

    Techniques: Staining

    HDAC inhibition interferes with IL-2 expression blockade in anergic Th1 cells . (A) Th1 cells were treated with ionomycin in the presence or absence of TSA. Untreated and TSA-treated cells were used as controls. After a 16-hour treatment, cells were allowed

    Journal:

    Article Title: Interleukin 2 gene transcription is regulated by Ikaros-induced changes in histone acetylation in anergic T cells

    doi: 10.1182/blood-2006-07-037754

    Figure Lengend Snippet: HDAC inhibition interferes with IL-2 expression blockade in anergic Th1 cells . (A) Th1 cells were treated with ionomycin in the presence or absence of TSA. Untreated and TSA-treated cells were used as controls. After a 16-hour treatment, cells were allowed

    Article Snippet: Twenty-four hours after transfection, cells were stimulated with 500 nM ionomycin and 20 nM phorbol-12-myristate-13-acetate (PMA; Calbiochem) or plate-bound anti–human CD3 and CD28 (BD) at 1 μg/mL.

    Techniques: Inhibition, Expressing

    Calcium signaling induces increased expression and binding of Ikaros to the il2 promoter in anergic Th1 cells . (A) Th1 cells were treated with ionomycin (Iono) for 6 hours and the expression of Ikaros mRNA was measured by qPCR compared with resting cells

    Journal:

    Article Title: Interleukin 2 gene transcription is regulated by Ikaros-induced changes in histone acetylation in anergic T cells

    doi: 10.1182/blood-2006-07-037754

    Figure Lengend Snippet: Calcium signaling induces increased expression and binding of Ikaros to the il2 promoter in anergic Th1 cells . (A) Th1 cells were treated with ionomycin (Iono) for 6 hours and the expression of Ikaros mRNA was measured by qPCR compared with resting cells

    Article Snippet: Twenty-four hours after transfection, cells were stimulated with 500 nM ionomycin and 20 nM phorbol-12-myristate-13-acetate (PMA; Calbiochem) or plate-bound anti–human CD3 and CD28 (BD) at 1 μg/mL.

    Techniques: Expressing, Binding Assay, Real-time Polymerase Chain Reaction

    Nuclear import of eIF6 from the cytoplasm is sensitive to calcineurin activator and inhibitor in vivo . A , COS-7 cells were treated either with 2 μ m calcium ionophore ionomycin to activate calcineurin, or with 5 μ m CsA, a specific calcineurin

    Journal: The Journal of Biological Chemistry

    Article Title: Opposing Action of Casein Kinase 1 and Calcineurin in Nucleo-cytoplasmic Shuttling of Mammalian Translation Initiation Factor eIF6 *

    doi: 10.1074/jbc.M110.188565

    Figure Lengend Snippet: Nuclear import of eIF6 from the cytoplasm is sensitive to calcineurin activator and inhibitor in vivo . A , COS-7 cells were treated either with 2 μ m calcium ionophore ionomycin to activate calcineurin, or with 5 μ m CsA, a specific calcineurin

    Article Snippet: The other reagents used in this work: cyclosporin A (CsA), FK-520 (analog of FK-506), ionomycin, D4476, and leptomycin B were purchased from Calbiochem; Hoechst 33258 was purchased from Molecular Probes Inc.; Protease inhibitors were purchased from Roche Diagnostics Corporation.

    Techniques: In Vivo