Structured Review

Valiant ionomycin
BSH effect on intracellular markers of systemic NK cells. Following NK cell enrichment, NK cells were stimulated with <t>PMA/Ionomycin</t> and blocked with Brefeldin A for 4hrs. The differences of day2 or day21 and day-1 are shown. Data are presented as mean±std.dev. N = 9–14. Data are shown as whiskers with 10–90 percentiles. *significantly different (p = 0.049), tested with two sample t test.
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Images

1) Product Images from "Effect of Broccoli Sprouts and Live Attenuated Influenza Virus on Peripheral Blood Natural Killer Cells: A Randomized, Double-Blind Study"

Article Title: Effect of Broccoli Sprouts and Live Attenuated Influenza Virus on Peripheral Blood Natural Killer Cells: A Randomized, Double-Blind Study

Journal: PLoS ONE

doi: 10.1371/journal.pone.0147742

BSH effect on intracellular markers of systemic NK cells. Following NK cell enrichment, NK cells were stimulated with PMA/Ionomycin and blocked with Brefeldin A for 4hrs. The differences of day2 or day21 and day-1 are shown. Data are presented as mean±std.dev. N = 9–14. Data are shown as whiskers with 10–90 percentiles. *significantly different (p = 0.049), tested with two sample t test.
Figure Legend Snippet: BSH effect on intracellular markers of systemic NK cells. Following NK cell enrichment, NK cells were stimulated with PMA/Ionomycin and blocked with Brefeldin A for 4hrs. The differences of day2 or day21 and day-1 are shown. Data are presented as mean±std.dev. N = 9–14. Data are shown as whiskers with 10–90 percentiles. *significantly different (p = 0.049), tested with two sample t test.

Techniques Used:

Overview of sample collection and processing. (A) Study design and sample collection. Details of the complete study have been published previously [ 1 ]. (B) Blood samples were stained for total leukocyte populations or used for NK cell enrichment. NK cells were analyzed for surface marker expression or cytokine production either naive or stimulated with PMA and ionomycin (Iono). Half of the peripheral blood mononuclear cells (PBMCs) were frozen and used later for the cytotoxicity assay.
Figure Legend Snippet: Overview of sample collection and processing. (A) Study design and sample collection. Details of the complete study have been published previously [ 1 ]. (B) Blood samples were stained for total leukocyte populations or used for NK cell enrichment. NK cells were analyzed for surface marker expression or cytokine production either naive or stimulated with PMA and ionomycin (Iono). Half of the peripheral blood mononuclear cells (PBMCs) were frozen and used later for the cytotoxicity assay.

Techniques Used: Staining, Marker, Expressing, Cytotoxicity Assay

2) Product Images from "Nuclear calcium is required for human T cell activation"

Article Title: Nuclear calcium is required for human T cell activation

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201602001

Nuclear calcium controls the expression of activation markers and cytokines in primary human T cells. T cells transfected with expression vectors for CaMBP4.mCherry (CaMBP4) or mCherry either were left unstimulated (ctr) or were stimulated with a combination of antibodies to CD3 and CD28 or with a combination of PMA and ionomycin. Expression of CD25 (A), CD69 (B), IL-2 (C), and IFNγ (D) in T cells was analyzed by flow cytometry with FITC-conjugated antibodies. Dot plots (left) are representative examples of three independent experiments; histograms (right) show the mean percentage of FITC-positive cells (blue dots) from three independent experiments. Error bars represent SEM. Statistically significant differences are indicated with asterisks (*, P
Figure Legend Snippet: Nuclear calcium controls the expression of activation markers and cytokines in primary human T cells. T cells transfected with expression vectors for CaMBP4.mCherry (CaMBP4) or mCherry either were left unstimulated (ctr) or were stimulated with a combination of antibodies to CD3 and CD28 or with a combination of PMA and ionomycin. Expression of CD25 (A), CD69 (B), IL-2 (C), and IFNγ (D) in T cells was analyzed by flow cytometry with FITC-conjugated antibodies. Dot plots (left) are representative examples of three independent experiments; histograms (right) show the mean percentage of FITC-positive cells (blue dots) from three independent experiments. Error bars represent SEM. Statistically significant differences are indicated with asterisks (*, P

Techniques Used: Expressing, Activation Assay, Transfection, Flow Cytometry, Cytometry

Analysis of nuclear calcium–regulated human T cell adaptogenomics. Representation of gene profiling data of human T cells transfected with expression vectors for CaMBP4.GFP (CaMBP4) or GFP and stimulated with a combination of PMA and ionomycin for 2, 6, and 16 h. (A) Heatmap with cluster dendrogram of 939 genes differentially expressed (log2 fold change) in at least one condition. Maximum value is set to 2.0. Fold ratio values greater than or equal to 2.0 are depicted with saturated red; values less than or equal to −2.0 are depicted with saturated green. (B and C) Number of genes induced by nuclear calcium signaling (i.e., down-regulated by CaMBP4.GFP; B) and repressed by nuclear calcium signaling (up-regulated by CaMBP4; C) at different time points on the basis of Illumina Human Sentrix-12 BeadChip arrays. (D–G) Validation of gene regulatory events by QRT-PCR. Gene expression was analyzed in T cells transfected with expression vectors for CaMBP4.GFP (CaMBP4) or GFP. Cells were stimulated with a combination of PMA and ionomycin for 2 h ( Egr2 and Egr3 ) and 6 h ( DUSP2 and Ikzf5 ) or were left unstimulated (ctr). mRNA levels are expressed relative to those in GFP-expressing T cells stimulated with PMA and ionomycin, which was set to 100% ( n = 3). Statistically significant differences are indicated with an asterisk (*, P
Figure Legend Snippet: Analysis of nuclear calcium–regulated human T cell adaptogenomics. Representation of gene profiling data of human T cells transfected with expression vectors for CaMBP4.GFP (CaMBP4) or GFP and stimulated with a combination of PMA and ionomycin for 2, 6, and 16 h. (A) Heatmap with cluster dendrogram of 939 genes differentially expressed (log2 fold change) in at least one condition. Maximum value is set to 2.0. Fold ratio values greater than or equal to 2.0 are depicted with saturated red; values less than or equal to −2.0 are depicted with saturated green. (B and C) Number of genes induced by nuclear calcium signaling (i.e., down-regulated by CaMBP4.GFP; B) and repressed by nuclear calcium signaling (up-regulated by CaMBP4; C) at different time points on the basis of Illumina Human Sentrix-12 BeadChip arrays. (D–G) Validation of gene regulatory events by QRT-PCR. Gene expression was analyzed in T cells transfected with expression vectors for CaMBP4.GFP (CaMBP4) or GFP. Cells were stimulated with a combination of PMA and ionomycin for 2 h ( Egr2 and Egr3 ) and 6 h ( DUSP2 and Ikzf5 ) or were left unstimulated (ctr). mRNA levels are expressed relative to those in GFP-expressing T cells stimulated with PMA and ionomycin, which was set to 100% ( n = 3). Statistically significant differences are indicated with an asterisk (*, P

Techniques Used: Transfection, Expressing, Quantitative RT-PCR

Nuclear calcium signaling does not regulate nuclear translocation of NFATc1 in primary human T cells. (A) T cells transfected with expression vectors for CaMBP4.mCherry (CaMBP4) or mCherry were left unstimulated (ctr), were stimulated with a combination of antibodies to CD3 and CD28 in the presence or absence of cyclosporine A (CsA) and FK506, or were stimulated with ionomycin (iono) for 30 min. Cells were stained with antibodies against NFATc1. Nuclei were stained with Hoechst. Bar, 5 µm. (B) Quantitative analysis of NFATc1 nuclear localization ( n = 3). Error bars represent SEM.
Figure Legend Snippet: Nuclear calcium signaling does not regulate nuclear translocation of NFATc1 in primary human T cells. (A) T cells transfected with expression vectors for CaMBP4.mCherry (CaMBP4) or mCherry were left unstimulated (ctr), were stimulated with a combination of antibodies to CD3 and CD28 in the presence or absence of cyclosporine A (CsA) and FK506, or were stimulated with ionomycin (iono) for 30 min. Cells were stained with antibodies against NFATc1. Nuclei were stained with Hoechst. Bar, 5 µm. (B) Quantitative analysis of NFATc1 nuclear localization ( n = 3). Error bars represent SEM.

Techniques Used: Translocation Assay, Transfection, Expressing, Staining

Nuclear calcium signaling differentially regulates CREB activation and CREM expression in primary human T cells. (A, left) Immunoblot analysis of CREB and the serine 133–phosphorylated form of CREB (pCREB) in T cells transfected with expression vectors for CaMBP4.GFP (CaMBP4) or GFP and stimulated with a combination of PMA and ionomycin for 30 or 120 min. A representative example is shown. (right) Quantitative analysis of three experiments; fold induction is relative to the pCREB/CREB ratio in unstimulated GFP-expressing T cells. (B) QRT-PCR analysis of Crem mRNA in T cells transfected with expression vectors for CaMBP4.GFP (CaMBP4) or GFP. Cells were stimulated with a combination of PMA and ionomycin for 6 h or were left unstimulated (ctr). mRNA levels are expressed relative to the mRNA level in GFP-expressing T cells stimulated with a combination of PMA and ionomycin, which was set to 100%. n = 3. Statistically significant differences are indicated with an asterisk (*, P
Figure Legend Snippet: Nuclear calcium signaling differentially regulates CREB activation and CREM expression in primary human T cells. (A, left) Immunoblot analysis of CREB and the serine 133–phosphorylated form of CREB (pCREB) in T cells transfected with expression vectors for CaMBP4.GFP (CaMBP4) or GFP and stimulated with a combination of PMA and ionomycin for 30 or 120 min. A representative example is shown. (right) Quantitative analysis of three experiments; fold induction is relative to the pCREB/CREB ratio in unstimulated GFP-expressing T cells. (B) QRT-PCR analysis of Crem mRNA in T cells transfected with expression vectors for CaMBP4.GFP (CaMBP4) or GFP. Cells were stimulated with a combination of PMA and ionomycin for 6 h or were left unstimulated (ctr). mRNA levels are expressed relative to the mRNA level in GFP-expressing T cells stimulated with a combination of PMA and ionomycin, which was set to 100%. n = 3. Statistically significant differences are indicated with an asterisk (*, P

Techniques Used: Activation Assay, Expressing, Transfection, Quantitative RT-PCR

TCR activation induces calcium transients in the cytoplasm and the nucleus in primary human T cells. (A) Confocal images showing the subcellular localization of the genetically encoded calcium indicators GCaMP3 (cytoplasm and nucleus), GCaMP3.NLS (nucleus), and GCaMP3.NES (cytoplasm). Bar, 5 µm. (B–D) Representative experiments showing calcium transients in human T cells transfected with the indicated calcium sensors and stimulated with CD3 antibodies. Anti–mouse IgG (black arrows) was added for antibody cross-linking, and calcium signals were measured for 9 min. Ionomycin (red arrows) and EGTA (blue arrows) were added to obtain the maximum ( F max ) and minimum ( F min ) signal, respectively. Calcium signals were analyzed by monitoring changes in GFP mean fluorescence as a function of time. The dot plots (left) are representative examples of at least four independent experiments (a.u., arbitrary units). Histograms (right) show mean GFP fluorescence (Δ F / F 0 ) from seven (GCaMP3), four (GCaMP3.NLS), and four (GCaMP3.NES) independent experiments. Error bars represent SEM.
Figure Legend Snippet: TCR activation induces calcium transients in the cytoplasm and the nucleus in primary human T cells. (A) Confocal images showing the subcellular localization of the genetically encoded calcium indicators GCaMP3 (cytoplasm and nucleus), GCaMP3.NLS (nucleus), and GCaMP3.NES (cytoplasm). Bar, 5 µm. (B–D) Representative experiments showing calcium transients in human T cells transfected with the indicated calcium sensors and stimulated with CD3 antibodies. Anti–mouse IgG (black arrows) was added for antibody cross-linking, and calcium signals were measured for 9 min. Ionomycin (red arrows) and EGTA (blue arrows) were added to obtain the maximum ( F max ) and minimum ( F min ) signal, respectively. Calcium signals were analyzed by monitoring changes in GFP mean fluorescence as a function of time. The dot plots (left) are representative examples of at least four independent experiments (a.u., arbitrary units). Histograms (right) show mean GFP fluorescence (Δ F / F 0 ) from seven (GCaMP3), four (GCaMP3.NLS), and four (GCaMP3.NES) independent experiments. Error bars represent SEM.

Techniques Used: Activation Assay, Transfection, Fluorescence

3) Product Images from "From the Cover: Interplay Between IFN-γ and IL-6 Impacts the Inflammatory Response and Expression of Interferon-Regulated Genes in Environmental-Induced Autoimmunity"

Article Title: From the Cover: Interplay Between IFN-γ and IL-6 Impacts the Inflammatory Response and Expression of Interferon-Regulated Genes in Environmental-Induced Autoimmunity

Journal: Toxicological Sciences

doi: 10.1093/toxsci/kfx083

IFN-γ expression in the skin of mercury-exposed mice. B10.S were injected subcutaneously with PBS or 40 μg HgCl 2 in PBS twice/week for up to 28 days. Skin and spleen cells were isolated at the indicated times and treated for 8 h with PMA, ionomycin and Golgi plug. IFN-γ production in both skin and spleen cells was assessed by intracellular FACS staining. Top: Representative FACS profile (left panel) for intracellular expression of IFN-γ in the skin after 7 days of PBS (shaded histogram) or mercury (bold histogram) treatment. The average of viable IFN-γ positive cells in the skin of PBS or mercury-treated animals for each indicated times are summarized (right panel). Bottom: Representative FACS profile (left panel) for intracellular expression of IFN-γ in the spleen after 7 days of PBS (shaded histogram) or mercury (bold histogram) treatment. The average of viable IFN-γ positive cells in the spleen of PBS or mercury-treated animals for each indicated times are summarized ( right ). Data (mean ± SEM) are from 4 mice in each group. Statistical significance was assigned based on un-paired two tailed Mann–Whitney U test comparing PBS- and mercury-treated mice. * P values
Figure Legend Snippet: IFN-γ expression in the skin of mercury-exposed mice. B10.S were injected subcutaneously with PBS or 40 μg HgCl 2 in PBS twice/week for up to 28 days. Skin and spleen cells were isolated at the indicated times and treated for 8 h with PMA, ionomycin and Golgi plug. IFN-γ production in both skin and spleen cells was assessed by intracellular FACS staining. Top: Representative FACS profile (left panel) for intracellular expression of IFN-γ in the skin after 7 days of PBS (shaded histogram) or mercury (bold histogram) treatment. The average of viable IFN-γ positive cells in the skin of PBS or mercury-treated animals for each indicated times are summarized (right panel). Bottom: Representative FACS profile (left panel) for intracellular expression of IFN-γ in the spleen after 7 days of PBS (shaded histogram) or mercury (bold histogram) treatment. The average of viable IFN-γ positive cells in the spleen of PBS or mercury-treated animals for each indicated times are summarized ( right ). Data (mean ± SEM) are from 4 mice in each group. Statistical significance was assigned based on un-paired two tailed Mann–Whitney U test comparing PBS- and mercury-treated mice. * P values

Techniques Used: Expressing, Mouse Assay, Injection, Isolation, FACS, Staining, Two Tailed Test, MANN-WHITNEY

4) Product Images from "T cell and peripheral blood parameters define progression of autoimmune disease in the IL-2Rα KO model"

Article Title: T cell and peripheral blood parameters define progression of autoimmune disease in the IL-2Rα KO model

Journal: bioRxiv

doi: 10.1101/345512

IL-2Rα-KO T cells have increased capacity to produce IL-2 and IFN γ . Representative flow plots showing production of IL-2 and IFN γ in WT and IL-2Rα-KO LN T cells post PMA and ionomycin stimulation for 5 hours. Frequency of T cells producing IL-2 and/or IFN γ . n = 3-9 mice per experimental group over 7 independent experiments. Statistics: unpaired Student’s t-test with Benjamini-Hochberg alpha correction and Welch’s correction for variance where appropriate; * p
Figure Legend Snippet: IL-2Rα-KO T cells have increased capacity to produce IL-2 and IFN γ . Representative flow plots showing production of IL-2 and IFN γ in WT and IL-2Rα-KO LN T cells post PMA and ionomycin stimulation for 5 hours. Frequency of T cells producing IL-2 and/or IFN γ . n = 3-9 mice per experimental group over 7 independent experiments. Statistics: unpaired Student’s t-test with Benjamini-Hochberg alpha correction and Welch’s correction for variance where appropriate; * p

Techniques Used: Mouse Assay

5) Product Images from "From the Cover: Interplay Between IFN-γ and IL-6 Impacts the Inflammatory Response and Expression of Interferon-Regulated Genes in Environmental-Induced Autoimmunity"

Article Title: From the Cover: Interplay Between IFN-γ and IL-6 Impacts the Inflammatory Response and Expression of Interferon-Regulated Genes in Environmental-Induced Autoimmunity

Journal: Toxicological Sciences

doi: 10.1093/toxsci/kfx083

IFN-γ expression in the skin of mercury-exposed mice. B10.S were injected subcutaneously with PBS or 40 μg HgCl 2 in PBS twice/week for up to 28 days. Skin and spleen cells were isolated at the indicated times and treated for 8 h with PMA, ionomycin and Golgi plug. IFN-γ production in both skin and spleen cells was assessed by intracellular FACS staining. Top: Representative FACS profile (left panel) for intracellular expression of IFN-γ in the skin after 7 days of PBS (shaded histogram) or mercury (bold histogram) treatment. The average of viable IFN-γ positive cells in the skin of PBS or mercury-treated animals for each indicated times are summarized (right panel). Bottom: Representative FACS profile (left panel) for intracellular expression of IFN-γ in the spleen after 7 days of PBS (shaded histogram) or mercury (bold histogram) treatment. The average of viable IFN-γ positive cells in the spleen of PBS or mercury-treated animals for each indicated times are summarized ( right ). Data (mean ± SEM) are from 4 mice in each group. Statistical significance was assigned based on un-paired two tailed Mann–Whitney U test comparing PBS- and mercury-treated mice. * P values
Figure Legend Snippet: IFN-γ expression in the skin of mercury-exposed mice. B10.S were injected subcutaneously with PBS or 40 μg HgCl 2 in PBS twice/week for up to 28 days. Skin and spleen cells were isolated at the indicated times and treated for 8 h with PMA, ionomycin and Golgi plug. IFN-γ production in both skin and spleen cells was assessed by intracellular FACS staining. Top: Representative FACS profile (left panel) for intracellular expression of IFN-γ in the skin after 7 days of PBS (shaded histogram) or mercury (bold histogram) treatment. The average of viable IFN-γ positive cells in the skin of PBS or mercury-treated animals for each indicated times are summarized (right panel). Bottom: Representative FACS profile (left panel) for intracellular expression of IFN-γ in the spleen after 7 days of PBS (shaded histogram) or mercury (bold histogram) treatment. The average of viable IFN-γ positive cells in the spleen of PBS or mercury-treated animals for each indicated times are summarized ( right ). Data (mean ± SEM) are from 4 mice in each group. Statistical significance was assigned based on un-paired two tailed Mann–Whitney U test comparing PBS- and mercury-treated mice. * P values

Techniques Used: Expressing, Mouse Assay, Injection, Isolation, FACS, Staining, Two Tailed Test, MANN-WHITNEY

6) Product Images from "Impact of bacteria on the phenotype, functions, and therapeutic activities of invariant NKT cells in mice"

Article Title: Impact of bacteria on the phenotype, functions, and therapeutic activities of invariant NKT cells in mice

Journal:

doi: 10.1172/JCI33071

Bacteria-induced iNKT cell hyporesponsiveness can be overcome by treatment with PMA plus ionomycin or with α-GalCer plus IL-2.
Figure Legend Snippet: Bacteria-induced iNKT cell hyporesponsiveness can be overcome by treatment with PMA plus ionomycin or with α-GalCer plus IL-2.

Techniques Used:

7) Product Images from "Nuclear calcium is required for human T cell activation"

Article Title: Nuclear calcium is required for human T cell activation

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201602001

Nuclear calcium controls the expression of activation markers and cytokines in primary human T cells. T cells transfected with expression vectors for CaMBP4.mCherry (CaMBP4) or mCherry either were left unstimulated (ctr) or were stimulated with a combination of antibodies to CD3 and CD28 or with a combination of PMA and ionomycin. Expression of CD25 (A), CD69 (B), IL-2 (C), and IFNγ (D) in T cells was analyzed by flow cytometry with FITC-conjugated antibodies. Dot plots (left) are representative examples of three independent experiments; histograms (right) show the mean percentage of FITC-positive cells (blue dots) from three independent experiments. Error bars represent SEM. Statistically significant differences are indicated with asterisks (*, P
Figure Legend Snippet: Nuclear calcium controls the expression of activation markers and cytokines in primary human T cells. T cells transfected with expression vectors for CaMBP4.mCherry (CaMBP4) or mCherry either were left unstimulated (ctr) or were stimulated with a combination of antibodies to CD3 and CD28 or with a combination of PMA and ionomycin. Expression of CD25 (A), CD69 (B), IL-2 (C), and IFNγ (D) in T cells was analyzed by flow cytometry with FITC-conjugated antibodies. Dot plots (left) are representative examples of three independent experiments; histograms (right) show the mean percentage of FITC-positive cells (blue dots) from three independent experiments. Error bars represent SEM. Statistically significant differences are indicated with asterisks (*, P

Techniques Used: Expressing, Activation Assay, Transfection, Flow Cytometry, Cytometry

Analysis of nuclear calcium–regulated human T cell adaptogenomics. Representation of gene profiling data of human T cells transfected with expression vectors for CaMBP4.GFP (CaMBP4) or GFP and stimulated with a combination of PMA and ionomycin for 2, 6, and 16 h. (A) Heatmap with cluster dendrogram of 939 genes differentially expressed (log2 fold change) in at least one condition. Maximum value is set to 2.0. Fold ratio values greater than or equal to 2.0 are depicted with saturated red; values less than or equal to −2.0 are depicted with saturated green. (B and C) Number of genes induced by nuclear calcium signaling (i.e., down-regulated by CaMBP4.GFP; B) and repressed by nuclear calcium signaling (up-regulated by CaMBP4; C) at different time points on the basis of Illumina Human Sentrix-12 BeadChip arrays. (D–G) Validation of gene regulatory events by QRT-PCR. Gene expression was analyzed in T cells transfected with expression vectors for CaMBP4.GFP (CaMBP4) or GFP. Cells were stimulated with a combination of PMA and ionomycin for 2 h ( Egr2 and Egr3 ) and 6 h ( DUSP2 and Ikzf5 ) or were left unstimulated (ctr). mRNA levels are expressed relative to those in GFP-expressing T cells stimulated with PMA and ionomycin, which was set to 100% ( n = 3). Statistically significant differences are indicated with an asterisk (*, P
Figure Legend Snippet: Analysis of nuclear calcium–regulated human T cell adaptogenomics. Representation of gene profiling data of human T cells transfected with expression vectors for CaMBP4.GFP (CaMBP4) or GFP and stimulated with a combination of PMA and ionomycin for 2, 6, and 16 h. (A) Heatmap with cluster dendrogram of 939 genes differentially expressed (log2 fold change) in at least one condition. Maximum value is set to 2.0. Fold ratio values greater than or equal to 2.0 are depicted with saturated red; values less than or equal to −2.0 are depicted with saturated green. (B and C) Number of genes induced by nuclear calcium signaling (i.e., down-regulated by CaMBP4.GFP; B) and repressed by nuclear calcium signaling (up-regulated by CaMBP4; C) at different time points on the basis of Illumina Human Sentrix-12 BeadChip arrays. (D–G) Validation of gene regulatory events by QRT-PCR. Gene expression was analyzed in T cells transfected with expression vectors for CaMBP4.GFP (CaMBP4) or GFP. Cells were stimulated with a combination of PMA and ionomycin for 2 h ( Egr2 and Egr3 ) and 6 h ( DUSP2 and Ikzf5 ) or were left unstimulated (ctr). mRNA levels are expressed relative to those in GFP-expressing T cells stimulated with PMA and ionomycin, which was set to 100% ( n = 3). Statistically significant differences are indicated with an asterisk (*, P

Techniques Used: Transfection, Expressing, Quantitative RT-PCR

Nuclear calcium signaling does not regulate nuclear translocation of NFATc1 in primary human T cells. (A) T cells transfected with expression vectors for CaMBP4.mCherry (CaMBP4) or mCherry were left unstimulated (ctr), were stimulated with a combination of antibodies to CD3 and CD28 in the presence or absence of cyclosporine A (CsA) and FK506, or were stimulated with ionomycin (iono) for 30 min. Cells were stained with antibodies against NFATc1. Nuclei were stained with Hoechst. Bar, 5 µm. (B) Quantitative analysis of NFATc1 nuclear localization ( n = 3). Error bars represent SEM.
Figure Legend Snippet: Nuclear calcium signaling does not regulate nuclear translocation of NFATc1 in primary human T cells. (A) T cells transfected with expression vectors for CaMBP4.mCherry (CaMBP4) or mCherry were left unstimulated (ctr), were stimulated with a combination of antibodies to CD3 and CD28 in the presence or absence of cyclosporine A (CsA) and FK506, or were stimulated with ionomycin (iono) for 30 min. Cells were stained with antibodies against NFATc1. Nuclei were stained with Hoechst. Bar, 5 µm. (B) Quantitative analysis of NFATc1 nuclear localization ( n = 3). Error bars represent SEM.

Techniques Used: Translocation Assay, Transfection, Expressing, Staining

Nuclear calcium signaling differentially regulates CREB activation and CREM expression in primary human T cells. (A, left) Immunoblot analysis of CREB and the serine 133–phosphorylated form of CREB (pCREB) in T cells transfected with expression vectors for CaMBP4.GFP (CaMBP4) or GFP and stimulated with a combination of PMA and ionomycin for 30 or 120 min. A representative example is shown. (right) Quantitative analysis of three experiments; fold induction is relative to the pCREB/CREB ratio in unstimulated GFP-expressing T cells. (B) QRT-PCR analysis of Crem mRNA in T cells transfected with expression vectors for CaMBP4.GFP (CaMBP4) or GFP. Cells were stimulated with a combination of PMA and ionomycin for 6 h or were left unstimulated (ctr). mRNA levels are expressed relative to the mRNA level in GFP-expressing T cells stimulated with a combination of PMA and ionomycin, which was set to 100%. n = 3. Statistically significant differences are indicated with an asterisk (*, P
Figure Legend Snippet: Nuclear calcium signaling differentially regulates CREB activation and CREM expression in primary human T cells. (A, left) Immunoblot analysis of CREB and the serine 133–phosphorylated form of CREB (pCREB) in T cells transfected with expression vectors for CaMBP4.GFP (CaMBP4) or GFP and stimulated with a combination of PMA and ionomycin for 30 or 120 min. A representative example is shown. (right) Quantitative analysis of three experiments; fold induction is relative to the pCREB/CREB ratio in unstimulated GFP-expressing T cells. (B) QRT-PCR analysis of Crem mRNA in T cells transfected with expression vectors for CaMBP4.GFP (CaMBP4) or GFP. Cells were stimulated with a combination of PMA and ionomycin for 6 h or were left unstimulated (ctr). mRNA levels are expressed relative to the mRNA level in GFP-expressing T cells stimulated with a combination of PMA and ionomycin, which was set to 100%. n = 3. Statistically significant differences are indicated with an asterisk (*, P

Techniques Used: Activation Assay, Expressing, Transfection, Quantitative RT-PCR

TCR activation induces calcium transients in the cytoplasm and the nucleus in primary human T cells. (A) Confocal images showing the subcellular localization of the genetically encoded calcium indicators GCaMP3 (cytoplasm and nucleus), GCaMP3.NLS (nucleus), and GCaMP3.NES (cytoplasm). Bar, 5 µm. (B–D) Representative experiments showing calcium transients in human T cells transfected with the indicated calcium sensors and stimulated with CD3 antibodies. Anti–mouse IgG (black arrows) was added for antibody cross-linking, and calcium signals were measured for 9 min. Ionomycin (red arrows) and EGTA (blue arrows) were added to obtain the maximum ( F max ) and minimum ( F min ) signal, respectively. Calcium signals were analyzed by monitoring changes in GFP mean fluorescence as a function of time. The dot plots (left) are representative examples of at least four independent experiments (a.u., arbitrary units). Histograms (right) show mean GFP fluorescence (Δ F / F 0 ) from seven (GCaMP3), four (GCaMP3.NLS), and four (GCaMP3.NES) independent experiments. Error bars represent SEM.
Figure Legend Snippet: TCR activation induces calcium transients in the cytoplasm and the nucleus in primary human T cells. (A) Confocal images showing the subcellular localization of the genetically encoded calcium indicators GCaMP3 (cytoplasm and nucleus), GCaMP3.NLS (nucleus), and GCaMP3.NES (cytoplasm). Bar, 5 µm. (B–D) Representative experiments showing calcium transients in human T cells transfected with the indicated calcium sensors and stimulated with CD3 antibodies. Anti–mouse IgG (black arrows) was added for antibody cross-linking, and calcium signals were measured for 9 min. Ionomycin (red arrows) and EGTA (blue arrows) were added to obtain the maximum ( F max ) and minimum ( F min ) signal, respectively. Calcium signals were analyzed by monitoring changes in GFP mean fluorescence as a function of time. The dot plots (left) are representative examples of at least four independent experiments (a.u., arbitrary units). Histograms (right) show mean GFP fluorescence (Δ F / F 0 ) from seven (GCaMP3), four (GCaMP3.NLS), and four (GCaMP3.NES) independent experiments. Error bars represent SEM.

Techniques Used: Activation Assay, Transfection, Fluorescence

8) Product Images from "A Skin-selective Homing Mechanism for Human Immune Surveillance T Cells"

Article Title: A Skin-selective Homing Mechanism for Human Immune Surveillance T Cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20032177

CCR8 + T cells predominate in normal human skin. Analyses were performed with freshly isolated cells. (a) Flow cytometric detection of CCR8 (gray histograms) and CCR9 (bold line) in T cells isolated from human skin, lamina propria of small intestine, and peripheral blood. Peptide-blocked control for CCR8 staining is shown as a thin line. CD4, CD8, and CD3 denote the respective populations analyzed within the αβTCR + cell gate, and the numbers indicate percentages of CCR8- and CCR9-positive cells, respectively. Data for skin and intestinal cells are representative for nine and three donors, respectively. (b) Chemotactic migration of skin T cells, gated on CD4 + cells (black bars) and CD8 + (white bars), in response to chemokines as indicated. Values represent the percentage of migrated cells as a proportion of input cells, and control denotes the level of migration in the absence of chemokines. (c) Expression of CLA and CD45RA on CCR8 + skin T cells. Gates were set for CD4 + αβTCR + cells (CD4) and CD8 + αβTCR + cells (CD8), and the numbers refer to the percentage of cells within each quadrant. (d) Production of various cytokines by CD4 + (black bars) or CD8 + (white bars) CCR8 + αβTCR + skin T cells. Intracellular accumulation of cytokines in response to PMA/ionomycin was measured by flow cytometry and is expressed as percent cytokine-positive cells. Chemotaxis and cytokine production data are representative of four independent experiments.
Figure Legend Snippet: CCR8 + T cells predominate in normal human skin. Analyses were performed with freshly isolated cells. (a) Flow cytometric detection of CCR8 (gray histograms) and CCR9 (bold line) in T cells isolated from human skin, lamina propria of small intestine, and peripheral blood. Peptide-blocked control for CCR8 staining is shown as a thin line. CD4, CD8, and CD3 denote the respective populations analyzed within the αβTCR + cell gate, and the numbers indicate percentages of CCR8- and CCR9-positive cells, respectively. Data for skin and intestinal cells are representative for nine and three donors, respectively. (b) Chemotactic migration of skin T cells, gated on CD4 + cells (black bars) and CD8 + (white bars), in response to chemokines as indicated. Values represent the percentage of migrated cells as a proportion of input cells, and control denotes the level of migration in the absence of chemokines. (c) Expression of CLA and CD45RA on CCR8 + skin T cells. Gates were set for CD4 + αβTCR + cells (CD4) and CD8 + αβTCR + cells (CD8), and the numbers refer to the percentage of cells within each quadrant. (d) Production of various cytokines by CD4 + (black bars) or CD8 + (white bars) CCR8 + αβTCR + skin T cells. Intracellular accumulation of cytokines in response to PMA/ionomycin was measured by flow cytometry and is expressed as percent cytokine-positive cells. Chemotaxis and cytokine production data are representative of four independent experiments.

Techniques Used: Isolation, Flow Cytometry, Staining, Migration, Expressing, Cytometry, Chemotaxis Assay

Analysis of T cell clones derived from skin CCR8 + T cells. Sorted CCR8 + T cells isolated from normal skin were cloned under nonpolarizing conditions. (a) For cytokine and (b) CCL1 secretion analysis, 6 CD4 + and 26 CD8 + T cell clones were stimulated with PMA/ionomycin for 24 h, and cell-free supernatants were tested in ELISA. (c) Expression of CCR8 by T cell clones, as determined by flow cytometry. Percent (%) CCR8 + refers to the fraction of CCR8-positive cells present within individual clones. (d) Detailed analysis of three clones representing high, intermediate, and low level expression of CCR8. Cells stained with anti-CCR8 antibodies (left, shaded histogram) or peptide-blocked anti-CCR8 (unshaded histogram). Numbers in parentheses indicate the clone number, whereas numbers above the gate lines refer to the percentage of cells positive for CCR8. The corresponding center panels show chemotactic responses of the same clones toward CCL1, expressed as the number of migrated cells counted (mean of triplicate wells ± SEM) in five high-power fields. Background migration in the absence of chemokine is indicated by open circles. The expression of CLA versus CD45RA, with the percentage of cells positive within each quadrant indicated (right).
Figure Legend Snippet: Analysis of T cell clones derived from skin CCR8 + T cells. Sorted CCR8 + T cells isolated from normal skin were cloned under nonpolarizing conditions. (a) For cytokine and (b) CCL1 secretion analysis, 6 CD4 + and 26 CD8 + T cell clones were stimulated with PMA/ionomycin for 24 h, and cell-free supernatants were tested in ELISA. (c) Expression of CCR8 by T cell clones, as determined by flow cytometry. Percent (%) CCR8 + refers to the fraction of CCR8-positive cells present within individual clones. (d) Detailed analysis of three clones representing high, intermediate, and low level expression of CCR8. Cells stained with anti-CCR8 antibodies (left, shaded histogram) or peptide-blocked anti-CCR8 (unshaded histogram). Numbers in parentheses indicate the clone number, whereas numbers above the gate lines refer to the percentage of cells positive for CCR8. The corresponding center panels show chemotactic responses of the same clones toward CCL1, expressed as the number of migrated cells counted (mean of triplicate wells ± SEM) in five high-power fields. Background migration in the absence of chemokine is indicated by open circles. The expression of CLA versus CD45RA, with the percentage of cells positive within each quadrant indicated (right).

Techniques Used: Clone Assay, Derivative Assay, Isolation, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Cytometry, Staining, Migration

Related Articles

Clone Assay:

Article Title: A Skin-selective Homing Mechanism for Human Immune Surveillance T Cells
Article Snippet: .. Sorted αβTCR+ CCR8+ CD8+ or αβTCR+ CCR8+ CD8− skin T cells, or skin T cell clones, were stimulated with 10 ng/ml PMA, 1 μg/ml ionomycin, and 10 μg/ml brefeldin A (Qbiogene) for 4.5–10 h. After fixing with 2% paraformaldehyde for 20 min at 4°C and permeabilization with 0.2% saponin/2% FCS in PBS− , cells were stained with antibodies and analyzed by flow cytometry. .. Perforin was examined without prior cell activation.

Flow Cytometry:

Article Title: A Skin-selective Homing Mechanism for Human Immune Surveillance T Cells
Article Snippet: .. Sorted αβTCR+ CCR8+ CD8+ or αβTCR+ CCR8+ CD8− skin T cells, or skin T cell clones, were stimulated with 10 ng/ml PMA, 1 μg/ml ionomycin, and 10 μg/ml brefeldin A (Qbiogene) for 4.5–10 h. After fixing with 2% paraformaldehyde for 20 min at 4°C and permeabilization with 0.2% saponin/2% FCS in PBS− , cells were stained with antibodies and analyzed by flow cytometry. .. Perforin was examined without prior cell activation.

Article Title: From the Cover: Interplay Between IFN-γ and IL-6 Impacts the Inflammatory Response and Expression of Interferon-Regulated Genes in Environmental-Induced Autoimmunity
Article Snippet: .. Cells were counted and seeded at 2× 106 cells in RPMI1640 containing 10% FBS/ml and allowed to rest for 4 h. Stimulation was performed for 8 h with 40 ng/ml of PMA (Fisher Scientific, Pittsburgh, PA), 2 μg/ml of ionomycin (MP Biomedicals, Solon, OH), and 1 μl/ml of Golgi Plug (BD Biosciences) before analysis by flow cytometry. ..

Article Title: From the Cover: Interplay Between IFN-γ and IL-6 Impacts the Inflammatory Response and Expression of Interferon-Regulated Genes in Environmental-Induced Autoimmunity
Article Snippet: .. Cells were then washed, counted and seeded at 1× 106 cells/ml in RPMI1640 containing 10% FBS for 6 h. Stimulation was performed for 8 h with 40 ng/ml of PMA (Fisher Scientific), 2 μg/ml of ionomycin (MP Biomedicals), and 1 μl/ml of Golgi Plug (BD Biosciences) before analysis by flow cytometry. .. Flow cytometry analysis.

Cytometry:

Article Title: A Skin-selective Homing Mechanism for Human Immune Surveillance T Cells
Article Snippet: .. Sorted αβTCR+ CCR8+ CD8+ or αβTCR+ CCR8+ CD8− skin T cells, or skin T cell clones, were stimulated with 10 ng/ml PMA, 1 μg/ml ionomycin, and 10 μg/ml brefeldin A (Qbiogene) for 4.5–10 h. After fixing with 2% paraformaldehyde for 20 min at 4°C and permeabilization with 0.2% saponin/2% FCS in PBS− , cells were stained with antibodies and analyzed by flow cytometry. .. Perforin was examined without prior cell activation.

Article Title: From the Cover: Interplay Between IFN-γ and IL-6 Impacts the Inflammatory Response and Expression of Interferon-Regulated Genes in Environmental-Induced Autoimmunity
Article Snippet: .. Cells were counted and seeded at 2× 106 cells in RPMI1640 containing 10% FBS/ml and allowed to rest for 4 h. Stimulation was performed for 8 h with 40 ng/ml of PMA (Fisher Scientific, Pittsburgh, PA), 2 μg/ml of ionomycin (MP Biomedicals, Solon, OH), and 1 μl/ml of Golgi Plug (BD Biosciences) before analysis by flow cytometry. ..

Article Title: From the Cover: Interplay Between IFN-γ and IL-6 Impacts the Inflammatory Response and Expression of Interferon-Regulated Genes in Environmental-Induced Autoimmunity
Article Snippet: .. Cells were then washed, counted and seeded at 1× 106 cells/ml in RPMI1640 containing 10% FBS for 6 h. Stimulation was performed for 8 h with 40 ng/ml of PMA (Fisher Scientific), 2 μg/ml of ionomycin (MP Biomedicals), and 1 μl/ml of Golgi Plug (BD Biosciences) before analysis by flow cytometry. .. Flow cytometry analysis.

Incubation:

Article Title: Nuclear calcium is required for human T cell activation
Article Snippet: .. Alternatively, cells were incubated with 1 nM PMA (Sigma-Aldrich) and 1 µM ionomycin (MP Biomedicals) for the indicated time. .. For anergy induction in vitro, T cells were pretreated overnight with plate-bound CD3 antibodies.

Staining:

Article Title: A Skin-selective Homing Mechanism for Human Immune Surveillance T Cells
Article Snippet: .. Sorted αβTCR+ CCR8+ CD8+ or αβTCR+ CCR8+ CD8− skin T cells, or skin T cell clones, were stimulated with 10 ng/ml PMA, 1 μg/ml ionomycin, and 10 μg/ml brefeldin A (Qbiogene) for 4.5–10 h. After fixing with 2% paraformaldehyde for 20 min at 4°C and permeabilization with 0.2% saponin/2% FCS in PBS− , cells were stained with antibodies and analyzed by flow cytometry. .. Perforin was examined without prior cell activation.

Recombinant:

Article Title: Impact of bacteria on the phenotype, functions, and therapeutic activities of invariant NKT cells in mice
Article Snippet: .. Mouse recombinant IL-2 was also obtained from BD Biosciences — Pharmingen; PMA and ionomycin from MP Biomedicals; complete and incomplete Freund’s adjuvant from BD Biosciences — Pharmingen; CFSE from Invitrogen; ConA from MP Biomedicals; lipoteichoic acid, E. coli , and Salmonella LPS from Sigma-Aldrich; CpG, imiquimod, and Salmonella flagellin from Invivogen; and polyinosinic acid–polycytidylic acid from Amersham Pharmacia. .. E. coli flagellin was purified from cultures as described previously ( ).