Structured Review

Applichem brefeldin a
CD8 +  T cells primed in presence of TRC still produce IFNγ and kill target cells. The T cell activation assay (see legend of   Fig. 1 ) was performed without stroma (‘no stroma’) or in presence of the pLN2 TRC line for 4 days followed by flow cytometric analysis of OT-I T cell effector function. ( A ) Dot plots (left) show intracellular IFN γ  deposition versus CFSE dilution in OT-I T cells after  in vitro  re-stimulation with 1 µM SIINFEKL peptide in presence of brefeldin A. The right panel shows the median fluorescence intensity of IFN γ  expression in OT-I T cells. ( B ) The cytotoxic capacity of OT-I T cells was assessed by co-incubating OT-I T cells ( = effector cells, E) from the T cell activation assay in the indicated E∶T ratios with SIINFEKL-pulsed-eFluor670 high -labeled splenocytes ( = target cells, T) mixed 1∶1 with unpulsed-eFluor670 low -labeled splenocytes ( = internal control). After overnight culture the percentage of eFluor670 high  versus eFluor670 low  splenocytes was analyzed and plotted as histograms. Indicated as percentage is the survival index for the target cells, as based on the ratio of peptide-pulsed eFluor670 high  relative to unpulsed eFluor670 low  cells (± standard deviation). ( A, B ): n = 3, representative of 3 independent experiments. *  P
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1) Product Images from "Fibroblastic Reticular Cells From Lymph Nodes Attenuate T Cell Expansion by Producing Nitric Oxide"

Article Title: Fibroblastic Reticular Cells From Lymph Nodes Attenuate T Cell Expansion by Producing Nitric Oxide

Journal: PLoS ONE

doi: 10.1371/journal.pone.0027618

CD8 +  T cells primed in presence of TRC still produce IFNγ and kill target cells. The T cell activation assay (see legend of   Fig. 1 ) was performed without stroma (‘no stroma’) or in presence of the pLN2 TRC line for 4 days followed by flow cytometric analysis of OT-I T cell effector function. ( A ) Dot plots (left) show intracellular IFN γ  deposition versus CFSE dilution in OT-I T cells after  in vitro  re-stimulation with 1 µM SIINFEKL peptide in presence of brefeldin A. The right panel shows the median fluorescence intensity of IFN γ  expression in OT-I T cells. ( B ) The cytotoxic capacity of OT-I T cells was assessed by co-incubating OT-I T cells ( = effector cells, E) from the T cell activation assay in the indicated E∶T ratios with SIINFEKL-pulsed-eFluor670 high -labeled splenocytes ( = target cells, T) mixed 1∶1 with unpulsed-eFluor670 low -labeled splenocytes ( = internal control). After overnight culture the percentage of eFluor670 high  versus eFluor670 low  splenocytes was analyzed and plotted as histograms. Indicated as percentage is the survival index for the target cells, as based on the ratio of peptide-pulsed eFluor670 high  relative to unpulsed eFluor670 low  cells (± standard deviation). ( A, B ): n = 3, representative of 3 independent experiments. *  P
Figure Legend Snippet: CD8 + T cells primed in presence of TRC still produce IFNγ and kill target cells. The T cell activation assay (see legend of Fig. 1 ) was performed without stroma (‘no stroma’) or in presence of the pLN2 TRC line for 4 days followed by flow cytometric analysis of OT-I T cell effector function. ( A ) Dot plots (left) show intracellular IFN γ deposition versus CFSE dilution in OT-I T cells after in vitro re-stimulation with 1 µM SIINFEKL peptide in presence of brefeldin A. The right panel shows the median fluorescence intensity of IFN γ expression in OT-I T cells. ( B ) The cytotoxic capacity of OT-I T cells was assessed by co-incubating OT-I T cells ( = effector cells, E) from the T cell activation assay in the indicated E∶T ratios with SIINFEKL-pulsed-eFluor670 high -labeled splenocytes ( = target cells, T) mixed 1∶1 with unpulsed-eFluor670 low -labeled splenocytes ( = internal control). After overnight culture the percentage of eFluor670 high versus eFluor670 low splenocytes was analyzed and plotted as histograms. Indicated as percentage is the survival index for the target cells, as based on the ratio of peptide-pulsed eFluor670 high relative to unpulsed eFluor670 low cells (± standard deviation). ( A, B ): n = 3, representative of 3 independent experiments. * P

Techniques Used: Activation Assay, Flow Cytometry, In Vitro, Fluorescence, Expressing, Labeling, Standard Deviation

2) Product Images from "Cellular Mechanisms of Etrolizumab Treatment in Inflammatory Bowel Disease"

Article Title: Cellular Mechanisms of Etrolizumab Treatment in Inflammatory Bowel Disease

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2019.00039

Cell surface expression of β7 integrin is restored after removal of etrolizumab-s. (A) Left panels: Representative flow cytometry showing β7 integrin expression at baseline (day 0) and 24 (day 1), 48 (day 2), 72 (day 3) and 120 h (day 5) after treatment with etrolizumab-s for the first 24 h of the experiment. Right panels: Quantitative flow cytometry of β7 surface expression over time relative to day 0 ( n = 5–6 per group). (B) Left panels: Representative flow cytometry showing β7 integrin expression at baseline (day 0), and 24 (day 1), 48 (day 2), 72 (day 3) and 120 h (day 5) after treatment with etrolizumab-s for the first 24 h of the experiment and treatment with Brefeldin A from day 1 to day 5. Right panels: Quantitative flow cytometry of β7 surface expression over time relative to day 0 ( n = 5–6 per group).
Figure Legend Snippet: Cell surface expression of β7 integrin is restored after removal of etrolizumab-s. (A) Left panels: Representative flow cytometry showing β7 integrin expression at baseline (day 0) and 24 (day 1), 48 (day 2), 72 (day 3) and 120 h (day 5) after treatment with etrolizumab-s for the first 24 h of the experiment. Right panels: Quantitative flow cytometry of β7 surface expression over time relative to day 0 ( n = 5–6 per group). (B) Left panels: Representative flow cytometry showing β7 integrin expression at baseline (day 0), and 24 (day 1), 48 (day 2), 72 (day 3) and 120 h (day 5) after treatment with etrolizumab-s for the first 24 h of the experiment and treatment with Brefeldin A from day 1 to day 5. Right panels: Quantitative flow cytometry of β7 surface expression over time relative to day 0 ( n = 5–6 per group).

Techniques Used: Expressing, Flow Cytometry, Cytometry

3) Product Images from "Cellular Mechanisms of Etrolizumab Treatment in Inflammatory Bowel Disease"

Article Title: Cellular Mechanisms of Etrolizumab Treatment in Inflammatory Bowel Disease

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2019.00039

Cell surface expression of β7 integrin is restored after removal of etrolizumab-s. (A) Left panels: Representative flow cytometry showing β7 integrin expression at baseline (day 0) and 24 (day 1), 48 (day 2), 72 (day 3) and 120 h (day 5) after treatment with etrolizumab-s for the first 24 h of the experiment. Right panels: Quantitative flow cytometry of β7 surface expression over time relative to day 0 ( n = 5–6 per group). (B) Left panels: Representative flow cytometry showing β7 integrin expression at baseline (day 0), and 24 (day 1), 48 (day 2), 72 (day 3) and 120 h (day 5) after treatment with etrolizumab-s for the first 24 h of the experiment and treatment with Brefeldin A from day 1 to day 5. Right panels: Quantitative flow cytometry of β7 surface expression over time relative to day 0 ( n = 5–6 per group).
Figure Legend Snippet: Cell surface expression of β7 integrin is restored after removal of etrolizumab-s. (A) Left panels: Representative flow cytometry showing β7 integrin expression at baseline (day 0) and 24 (day 1), 48 (day 2), 72 (day 3) and 120 h (day 5) after treatment with etrolizumab-s for the first 24 h of the experiment. Right panels: Quantitative flow cytometry of β7 surface expression over time relative to day 0 ( n = 5–6 per group). (B) Left panels: Representative flow cytometry showing β7 integrin expression at baseline (day 0), and 24 (day 1), 48 (day 2), 72 (day 3) and 120 h (day 5) after treatment with etrolizumab-s for the first 24 h of the experiment and treatment with Brefeldin A from day 1 to day 5. Right panels: Quantitative flow cytometry of β7 surface expression over time relative to day 0 ( n = 5–6 per group).

Techniques Used: Expressing, Flow Cytometry, Cytometry

4) Product Images from "The novel BTB-kelch protein, KBTBD8, is located in the Golgi apparatus and translocates to the spindle apparatus during mitosis"

Article Title: The novel BTB-kelch protein, KBTBD8, is located in the Golgi apparatus and translocates to the spindle apparatus during mitosis

Journal: Cell Division

doi: 10.1186/1747-1028-8-3

Localization of KBTBD8 protein in NIH3T3 cell and after treatment with BFA or Nocodazole. NIH3T3 cells – with and without BFA or Nocodazole treatment - were fixed and co-stained with KBTBD8 antibody and Golgi matrix marker GM130 or microtubule marker protein α-Tubulin. Treatment with Brefeldin A (BFA) resulted in resorption of the Golgi membrane and Golgi marker proteins to the ER. For GM130 the expected punctuated cytoplasmic localization could be found indicating that the treatment worked. However, KBTBD8 was either no longer detectable, not co-localizing with GM130 or associated to the spindle apparatus of mitotic cells. Co-staining with α-Tubulin and Kbtbd8 in untreated cells showed no localisation of KBTBD8 at the cytoskeleton. After Nocodazole- treatment, which results in the depolarisation of microtubules and in the disperse of the Golgi stacks to the cytoplasm, GM130 and α-Tubulin showed the expected localisation whereas KBTBD8 was again either not detectable at all or no longer co-localizing with GM130. These results indicate that KBTBD8 is not a part of the Golgi matrix. Scale bar, 10 μm.
Figure Legend Snippet: Localization of KBTBD8 protein in NIH3T3 cell and after treatment with BFA or Nocodazole. NIH3T3 cells – with and without BFA or Nocodazole treatment - were fixed and co-stained with KBTBD8 antibody and Golgi matrix marker GM130 or microtubule marker protein α-Tubulin. Treatment with Brefeldin A (BFA) resulted in resorption of the Golgi membrane and Golgi marker proteins to the ER. For GM130 the expected punctuated cytoplasmic localization could be found indicating that the treatment worked. However, KBTBD8 was either no longer detectable, not co-localizing with GM130 or associated to the spindle apparatus of mitotic cells. Co-staining with α-Tubulin and Kbtbd8 in untreated cells showed no localisation of KBTBD8 at the cytoskeleton. After Nocodazole- treatment, which results in the depolarisation of microtubules and in the disperse of the Golgi stacks to the cytoplasm, GM130 and α-Tubulin showed the expected localisation whereas KBTBD8 was again either not detectable at all or no longer co-localizing with GM130. These results indicate that KBTBD8 is not a part of the Golgi matrix. Scale bar, 10 μm.

Techniques Used: Staining, Marker

5) Product Images from "Persistence of Candida albicans in the Oral Mucosa Induces a Curbed Inflammatory Host Response That Is Independent of Immunosuppression"

Article Title: Persistence of Candida albicans in the Oral Mucosa Induces a Curbed Inflammatory Host Response That Is Independent of Immunosuppression

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.00330

The absence of Tregs or IL-10 does not compromise persistence of strain 101 in the oral epithelium.  (A–C)  DEREG mice and control littermates were sublingually infected with  C. albicans  strain 101 and treated with diphtheria toxin on day 11 and 13 after the antifungal response was fully established. One day after the last treatment, the mice were sacrificed for analysis.  (A)  Treg depletion efficiency was analyzed in the cervical lymph nodes by flow cytometry. Data are the % of Foxp3 +  cells within the population of CD4 +  viable cells.  (B)  Lymph node cells were re-stimulated with MutuDC1940 cells that were pulsed with heat-killed  C. albicans  or left unpulsed for 5 h in the presence of Brefeldin A. IL-17A (left) and IFN-γ (right) production by CD3 + CD4 +  cells was analyzed by intracellular cytokine staining and flow cytometry.  (C)  The fungal burden was determined by plating tongue homogenates on YPD agar. Each bar represents the mean + SD of 3 to 4 mice per group. Data are from one out of two independent experiments.  (D,E)  IL-10-deficient mice and WT controls were sublingually infected with  C. albicans  strain 101 and analyzed on day 9 post-infection.  (D)  Lymph node cells were re-stimulated and analyzed for IL-17 (left) and IFN-γ (right) production as in B.  (E)  Tongue fungal burdens were analyzed as in C. Each bar represents the mean + SD of 8–9 mice per group pooled from two independent experiments. Statistics were calculated using unpaired t-Test. *** p
Figure Legend Snippet: The absence of Tregs or IL-10 does not compromise persistence of strain 101 in the oral epithelium. (A–C) DEREG mice and control littermates were sublingually infected with C. albicans strain 101 and treated with diphtheria toxin on day 11 and 13 after the antifungal response was fully established. One day after the last treatment, the mice were sacrificed for analysis. (A) Treg depletion efficiency was analyzed in the cervical lymph nodes by flow cytometry. Data are the % of Foxp3 + cells within the population of CD4 + viable cells. (B) Lymph node cells were re-stimulated with MutuDC1940 cells that were pulsed with heat-killed C. albicans or left unpulsed for 5 h in the presence of Brefeldin A. IL-17A (left) and IFN-γ (right) production by CD3 + CD4 + cells was analyzed by intracellular cytokine staining and flow cytometry. (C) The fungal burden was determined by plating tongue homogenates on YPD agar. Each bar represents the mean + SD of 3 to 4 mice per group. Data are from one out of two independent experiments. (D,E) IL-10-deficient mice and WT controls were sublingually infected with C. albicans strain 101 and analyzed on day 9 post-infection. (D) Lymph node cells were re-stimulated and analyzed for IL-17 (left) and IFN-γ (right) production as in B. (E) Tongue fungal burdens were analyzed as in C. Each bar represents the mean + SD of 8–9 mice per group pooled from two independent experiments. Statistics were calculated using unpaired t-Test. *** p

Techniques Used: Mouse Assay, Infection, Flow Cytometry, Cytometry, Staining

The Treg response during persistent colonization of the oral mucosa with  C. albicans .  (A–F)  WT mice were sublingually infected with strain 101 or SC5314 and cervical lymph node cells were analyzed on day 7 or day 18–21 as indicated.  (A,B)  Lymph node cells were re-stimulated with MutuDC1940 cells that were pulsed with heat-killed  C. albicans  or left unpulsed for 5 h in the presence of Brefeldin A. IL-17A production by CD3 + CD4 +  cells was analyzed by intracellular cytokine staining and flow cytometry.  (C,D)  The frequency of Foxp3-expressing cells within the CD4 +  lymphocyte compartment was assessed by flow cytometry.  (E,F)  PD-1, TIGIT, and Tim-3 expression by CD4 + Foxp3 +  Treg cells was analyzed by flow cytometry. ( G–J )  Il10 -Thy1.1 reporter mice were sublingually infected with strain 101 or SC5314 or left naive. IL-10 expression by Foxp3 +  Tregs and Foxp3 −  effector T cells was assessed in the cervical lymph nodes  (G,H)  and in the tongue  (I,J)  on day 21 post-infection by flow cytometry. Cells were pregated on CD90 + CD4 + (G,H)  or on CD45.2 + CD3 + (I,J) , respectively. Representative FACS plots are shown in (A, C, G, I); summary plots with data pooled from at least 2 experiments with 6–9 animals per infected group and 3–4 animals per naive group are shown in (B, D–F, H, J), with the exception of the right plot in  (D) , where data are from a single experiment with 3 animals per group. In B, Statistics were calculated using  t -test. In  (D–F, H, J) , statistics were calculated using one-way ANOVA. * p
Figure Legend Snippet: The Treg response during persistent colonization of the oral mucosa with C. albicans . (A–F) WT mice were sublingually infected with strain 101 or SC5314 and cervical lymph node cells were analyzed on day 7 or day 18–21 as indicated. (A,B) Lymph node cells were re-stimulated with MutuDC1940 cells that were pulsed with heat-killed C. albicans or left unpulsed for 5 h in the presence of Brefeldin A. IL-17A production by CD3 + CD4 + cells was analyzed by intracellular cytokine staining and flow cytometry. (C,D) The frequency of Foxp3-expressing cells within the CD4 + lymphocyte compartment was assessed by flow cytometry. (E,F) PD-1, TIGIT, and Tim-3 expression by CD4 + Foxp3 + Treg cells was analyzed by flow cytometry. ( G–J ) Il10 -Thy1.1 reporter mice were sublingually infected with strain 101 or SC5314 or left naive. IL-10 expression by Foxp3 + Tregs and Foxp3 − effector T cells was assessed in the cervical lymph nodes (G,H) and in the tongue (I,J) on day 21 post-infection by flow cytometry. Cells were pregated on CD90 + CD4 + (G,H) or on CD45.2 + CD3 + (I,J) , respectively. Representative FACS plots are shown in (A, C, G, I); summary plots with data pooled from at least 2 experiments with 6–9 animals per infected group and 3–4 animals per naive group are shown in (B, D–F, H, J), with the exception of the right plot in (D) , where data are from a single experiment with 3 animals per group. In B, Statistics were calculated using t -test. In (D–F, H, J) , statistics were calculated using one-way ANOVA. * p

Techniques Used: Mouse Assay, Infection, Staining, Flow Cytometry, Cytometry, Expressing, FACS

6) Product Images from "The contact allergen nickel sensitizes primary human endothelial cells and keratinocytes to TRAIL-mediated apoptosis"

Article Title: The contact allergen nickel sensitizes primary human endothelial cells and keratinocytes to TRAIL-mediated apoptosis

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2009.00823.x

Ni 2+ leads to altered TRAIL-receptor expression and rapid Caspase-8 and -3 cleavage upon TRAIL stimulation. (A) Flow cytometric analysis of TRAIL-R1, -R2, -R3 and -R4 cell surface expression of non-stimulated or Ni 2+ -stimulated (16 hrs) HUVEC using specific antibodies to TRAIL-R1-R4 (filled lines). Grey lines indicate staining with the respective isotype-specific control antibodies. One of four independent experiments is shown with the respective background-subtracted median fluorescence intensity (MFI). Additionally, MFI ratios of Ni 2+ -treated samples and its respective controls are shown. (B) HUVEC were left untreated or pre-incubated with Ni 2+ for 16 hrs and subsequently incubated with TRAIL as indicated. Lysates were analysed for cleavage of Caspase-8 (p43/p41/p18), and Caspase-3 (p20/p17) by Western blot. Membranes were rehybridized with an Ab to tubulin to control for equal protein loading. (C) EC were pre-treated for 16 hrs with Brefeldin A (2 μg/ml) in the presence or absence of Ni 2+ (1.5 mM) and subsequently treated with TRAIL (100 ng/ml) for 6 hrs. The viability was subsequently determined by crystal violet assay. Statistical significance of the detected effects was evaluated by Student’s t-test. Statistically significant changes ( P
Figure Legend Snippet: Ni 2+ leads to altered TRAIL-receptor expression and rapid Caspase-8 and -3 cleavage upon TRAIL stimulation. (A) Flow cytometric analysis of TRAIL-R1, -R2, -R3 and -R4 cell surface expression of non-stimulated or Ni 2+ -stimulated (16 hrs) HUVEC using specific antibodies to TRAIL-R1-R4 (filled lines). Grey lines indicate staining with the respective isotype-specific control antibodies. One of four independent experiments is shown with the respective background-subtracted median fluorescence intensity (MFI). Additionally, MFI ratios of Ni 2+ -treated samples and its respective controls are shown. (B) HUVEC were left untreated or pre-incubated with Ni 2+ for 16 hrs and subsequently incubated with TRAIL as indicated. Lysates were analysed for cleavage of Caspase-8 (p43/p41/p18), and Caspase-3 (p20/p17) by Western blot. Membranes were rehybridized with an Ab to tubulin to control for equal protein loading. (C) EC were pre-treated for 16 hrs with Brefeldin A (2 μg/ml) in the presence or absence of Ni 2+ (1.5 mM) and subsequently treated with TRAIL (100 ng/ml) for 6 hrs. The viability was subsequently determined by crystal violet assay. Statistical significance of the detected effects was evaluated by Student’s t-test. Statistically significant changes ( P

Techniques Used: Expressing, Flow Cytometry, Staining, Fluorescence, Incubation, Western Blot, Crystal Violet Assay

7) Product Images from "Cellular Mechanisms of Etrolizumab Treatment in Inflammatory Bowel Disease"

Article Title: Cellular Mechanisms of Etrolizumab Treatment in Inflammatory Bowel Disease

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2019.00039

Cell surface expression of β7 integrin is restored after removal of etrolizumab-s. (A) Left panels: Representative flow cytometry showing β7 integrin expression at baseline (day 0) and 24 (day 1), 48 (day 2), 72 (day 3) and 120 h (day 5) after treatment with etrolizumab-s for the first 24 h of the experiment. Right panels: Quantitative flow cytometry of β7 surface expression over time relative to day 0 ( n = 5–6 per group). (B) Left panels: Representative flow cytometry showing β7 integrin expression at baseline (day 0), and 24 (day 1), 48 (day 2), 72 (day 3) and 120 h (day 5) after treatment with etrolizumab-s for the first 24 h of the experiment and treatment with Brefeldin A from day 1 to day 5. Right panels: Quantitative flow cytometry of β7 surface expression over time relative to day 0 ( n = 5–6 per group).
Figure Legend Snippet: Cell surface expression of β7 integrin is restored after removal of etrolizumab-s. (A) Left panels: Representative flow cytometry showing β7 integrin expression at baseline (day 0) and 24 (day 1), 48 (day 2), 72 (day 3) and 120 h (day 5) after treatment with etrolizumab-s for the first 24 h of the experiment. Right panels: Quantitative flow cytometry of β7 surface expression over time relative to day 0 ( n = 5–6 per group). (B) Left panels: Representative flow cytometry showing β7 integrin expression at baseline (day 0), and 24 (day 1), 48 (day 2), 72 (day 3) and 120 h (day 5) after treatment with etrolizumab-s for the first 24 h of the experiment and treatment with Brefeldin A from day 1 to day 5. Right panels: Quantitative flow cytometry of β7 surface expression over time relative to day 0 ( n = 5–6 per group).

Techniques Used: Expressing, Flow Cytometry, Cytometry

8) Product Images from "Cellular Mechanisms of Etrolizumab Treatment in Inflammatory Bowel Disease"

Article Title: Cellular Mechanisms of Etrolizumab Treatment in Inflammatory Bowel Disease

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2019.00039

Cell surface expression of β7 integrin is restored after removal of etrolizumab-s. (A) Left panels: Representative flow cytometry showing β7 integrin expression at baseline (day 0) and 24 (day 1), 48 (day 2), 72 (day 3) and 120 h (day 5) after treatment with etrolizumab-s for the first 24 h of the experiment. Right panels: Quantitative flow cytometry of β7 surface expression over time relative to day 0 ( n = 5–6 per group). (B) Left panels: Representative flow cytometry showing β7 integrin expression at baseline (day 0), and 24 (day 1), 48 (day 2), 72 (day 3) and 120 h (day 5) after treatment with etrolizumab-s for the first 24 h of the experiment and treatment with Brefeldin A from day 1 to day 5. Right panels: Quantitative flow cytometry of β7 surface expression over time relative to day 0 ( n = 5–6 per group).
Figure Legend Snippet: Cell surface expression of β7 integrin is restored after removal of etrolizumab-s. (A) Left panels: Representative flow cytometry showing β7 integrin expression at baseline (day 0) and 24 (day 1), 48 (day 2), 72 (day 3) and 120 h (day 5) after treatment with etrolizumab-s for the first 24 h of the experiment. Right panels: Quantitative flow cytometry of β7 surface expression over time relative to day 0 ( n = 5–6 per group). (B) Left panels: Representative flow cytometry showing β7 integrin expression at baseline (day 0), and 24 (day 1), 48 (day 2), 72 (day 3) and 120 h (day 5) after treatment with etrolizumab-s for the first 24 h of the experiment and treatment with Brefeldin A from day 1 to day 5. Right panels: Quantitative flow cytometry of β7 surface expression over time relative to day 0 ( n = 5–6 per group).

Techniques Used: Expressing, Flow Cytometry, Cytometry

Related Articles

Flow Cytometry:

Article Title: Cellular Mechanisms of Etrolizumab Treatment in Inflammatory Bowel Disease
Article Snippet: .. Cells were left untreated or treated with etrolizumab-s or PMA/ionomycin for 2 h. Subsequently, Brefeldin A was added for additional 4 h and the expression of cytokines was assessed by flow cytometry. .. While increased expression of IFN-γ, IL-4, IL-9, and IL-17A could be observed in PMA/ionomycin-treated CD4+ T cells (Figure ), only very low and comparable production was found in untreated cells and etrolizumab-s-treated cells.

In Vitro:

Article Title: Fibroblastic Reticular Cells From Lymph Nodes Attenuate T Cell Expansion by Producing Nitric Oxide
Article Snippet: .. For IFN γ staining: cells were re-stimulated in vitro with 1 µM SIINFEKL peptide in the presence of Brefeldin-A (10 µg/ml, AppliChem) for 3–4 h at 37°C. .. Data were acquired on a FACSCanto or LSR II flow cytometer (both BectonDickinson) and were analyzed with FlowJo software (TreeStar).

Cytometry:

Article Title: Cellular Mechanisms of Etrolizumab Treatment in Inflammatory Bowel Disease
Article Snippet: .. Cells were left untreated or treated with etrolizumab-s or PMA/ionomycin for 2 h. Subsequently, Brefeldin A was added for additional 4 h and the expression of cytokines was assessed by flow cytometry. .. While increased expression of IFN-γ, IL-4, IL-9, and IL-17A could be observed in PMA/ionomycin-treated CD4+ T cells (Figure ), only very low and comparable production was found in untreated cells and etrolizumab-s-treated cells.

Cell Culture:

Article Title: Cellular Mechanisms of Etrolizumab Treatment in Inflammatory Bowel Disease
Article Snippet: .. Subsequently, these PBMCs were cultured in the presence of etrolizumab-s at 37°C for 24 h. Next, cells were harvested, washed and then re-seeded in cell culture plates for a further 96 h in the presence or absence of Brefeldin A. .. The time course of β7 integrin re-expression was determined by flow cytometric analysis of aliquots cells harvested at 24, 48, 72, and 120 h from baseline.

other:

Article Title: Cellular Mechanisms of Etrolizumab Treatment in Inflammatory Bowel Disease
Article Snippet: To address whether this was due to recycling of internalized β7 integrin or resulting from de novo synthesis, we performed an additional series of experiments, in which etrolizumab-s treatment was followed by application of Brefeldin A to inhibit Golgi transport of freshly translated β7 protein.

Expressing:

Article Title: Cellular Mechanisms of Etrolizumab Treatment in Inflammatory Bowel Disease
Article Snippet: .. Cells were left untreated or treated with etrolizumab-s or PMA/ionomycin for 2 h. Subsequently, Brefeldin A was added for additional 4 h and the expression of cytokines was assessed by flow cytometry. .. While increased expression of IFN-γ, IL-4, IL-9, and IL-17A could be observed in PMA/ionomycin-treated CD4+ T cells (Figure ), only very low and comparable production was found in untreated cells and etrolizumab-s-treated cells.

Article Title: Cellular Mechanisms of Etrolizumab Treatment in Inflammatory Bowel Disease
Article Snippet: .. After 2 h of culture, all cells were treated with 10 ng/μL Brefeldin A (Applichem) for the remaining 4 h. For the analysis of β7 integrin re-expression, baseline expression of β7 was determined in PBMCs. .. Subsequently, these PBMCs were cultured in the presence of etrolizumab-s at 37°C for 24 h. Next, cells were harvested, washed and then re-seeded in cell culture plates for a further 96 h in the presence or absence of Brefeldin A.

Staining:

Article Title: Fibroblastic Reticular Cells From Lymph Nodes Attenuate T Cell Expansion by Producing Nitric Oxide
Article Snippet: .. For IFN γ staining: cells were re-stimulated in vitro with 1 µM SIINFEKL peptide in the presence of Brefeldin-A (10 µg/ml, AppliChem) for 3–4 h at 37°C. .. Data were acquired on a FACSCanto or LSR II flow cytometer (both BectonDickinson) and were analyzed with FlowJo software (TreeStar).

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    Applichem brefeldin a
    CD8 +  T cells primed in presence of TRC still produce IFNγ and kill target cells. The T cell activation assay (see legend of   Fig. 1 ) was performed without stroma (‘no stroma’) or in presence of the pLN2 TRC line for 4 days followed by flow cytometric analysis of OT-I T cell effector function. ( A ) Dot plots (left) show intracellular IFN γ  deposition versus CFSE dilution in OT-I T cells after  in vitro  re-stimulation with 1 µM SIINFEKL peptide in presence of brefeldin A. The right panel shows the median fluorescence intensity of IFN γ  expression in OT-I T cells. ( B ) The cytotoxic capacity of OT-I T cells was assessed by co-incubating OT-I T cells ( = effector cells, E) from the T cell activation assay in the indicated E∶T ratios with SIINFEKL-pulsed-eFluor670 high -labeled splenocytes ( = target cells, T) mixed 1∶1 with unpulsed-eFluor670 low -labeled splenocytes ( = internal control). After overnight culture the percentage of eFluor670 high  versus eFluor670 low  splenocytes was analyzed and plotted as histograms. Indicated as percentage is the survival index for the target cells, as based on the ratio of peptide-pulsed eFluor670 high  relative to unpulsed eFluor670 low  cells (± standard deviation). ( A, B ): n = 3, representative of 3 independent experiments. *  P
    Brefeldin A, supplied by Applichem, used in various techniques. Bioz Stars score: 92/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CD8 +  T cells primed in presence of TRC still produce IFNγ and kill target cells. The T cell activation assay (see legend of   Fig. 1 ) was performed without stroma (‘no stroma’) or in presence of the pLN2 TRC line for 4 days followed by flow cytometric analysis of OT-I T cell effector function. ( A ) Dot plots (left) show intracellular IFN γ  deposition versus CFSE dilution in OT-I T cells after  in vitro  re-stimulation with 1 µM SIINFEKL peptide in presence of brefeldin A. The right panel shows the median fluorescence intensity of IFN γ  expression in OT-I T cells. ( B ) The cytotoxic capacity of OT-I T cells was assessed by co-incubating OT-I T cells ( = effector cells, E) from the T cell activation assay in the indicated E∶T ratios with SIINFEKL-pulsed-eFluor670 high -labeled splenocytes ( = target cells, T) mixed 1∶1 with unpulsed-eFluor670 low -labeled splenocytes ( = internal control). After overnight culture the percentage of eFluor670 high  versus eFluor670 low  splenocytes was analyzed and plotted as histograms. Indicated as percentage is the survival index for the target cells, as based on the ratio of peptide-pulsed eFluor670 high  relative to unpulsed eFluor670 low  cells (± standard deviation). ( A, B ): n = 3, representative of 3 independent experiments. *  P

    Journal: PLoS ONE

    Article Title: Fibroblastic Reticular Cells From Lymph Nodes Attenuate T Cell Expansion by Producing Nitric Oxide

    doi: 10.1371/journal.pone.0027618

    Figure Lengend Snippet: CD8 + T cells primed in presence of TRC still produce IFNγ and kill target cells. The T cell activation assay (see legend of Fig. 1 ) was performed without stroma (‘no stroma’) or in presence of the pLN2 TRC line for 4 days followed by flow cytometric analysis of OT-I T cell effector function. ( A ) Dot plots (left) show intracellular IFN γ deposition versus CFSE dilution in OT-I T cells after in vitro re-stimulation with 1 µM SIINFEKL peptide in presence of brefeldin A. The right panel shows the median fluorescence intensity of IFN γ expression in OT-I T cells. ( B ) The cytotoxic capacity of OT-I T cells was assessed by co-incubating OT-I T cells ( = effector cells, E) from the T cell activation assay in the indicated E∶T ratios with SIINFEKL-pulsed-eFluor670 high -labeled splenocytes ( = target cells, T) mixed 1∶1 with unpulsed-eFluor670 low -labeled splenocytes ( = internal control). After overnight culture the percentage of eFluor670 high versus eFluor670 low splenocytes was analyzed and plotted as histograms. Indicated as percentage is the survival index for the target cells, as based on the ratio of peptide-pulsed eFluor670 high relative to unpulsed eFluor670 low cells (± standard deviation). ( A, B ): n = 3, representative of 3 independent experiments. * P

    Article Snippet: For IFN γ staining: cells were re-stimulated in vitro with 1 µM SIINFEKL peptide in the presence of Brefeldin-A (10 µg/ml, AppliChem) for 3–4 h at 37°C.

    Techniques: Activation Assay, Flow Cytometry, In Vitro, Fluorescence, Expressing, Labeling, Standard Deviation

    Cell surface expression of β7 integrin is restored after removal of etrolizumab-s. (A) Left panels: Representative flow cytometry showing β7 integrin expression at baseline (day 0) and 24 (day 1), 48 (day 2), 72 (day 3) and 120 h (day 5) after treatment with etrolizumab-s for the first 24 h of the experiment. Right panels: Quantitative flow cytometry of β7 surface expression over time relative to day 0 ( n = 5–6 per group). (B) Left panels: Representative flow cytometry showing β7 integrin expression at baseline (day 0), and 24 (day 1), 48 (day 2), 72 (day 3) and 120 h (day 5) after treatment with etrolizumab-s for the first 24 h of the experiment and treatment with Brefeldin A from day 1 to day 5. Right panels: Quantitative flow cytometry of β7 surface expression over time relative to day 0 ( n = 5–6 per group).

    Journal: Frontiers in Pharmacology

    Article Title: Cellular Mechanisms of Etrolizumab Treatment in Inflammatory Bowel Disease

    doi: 10.3389/fphar.2019.00039

    Figure Lengend Snippet: Cell surface expression of β7 integrin is restored after removal of etrolizumab-s. (A) Left panels: Representative flow cytometry showing β7 integrin expression at baseline (day 0) and 24 (day 1), 48 (day 2), 72 (day 3) and 120 h (day 5) after treatment with etrolizumab-s for the first 24 h of the experiment. Right panels: Quantitative flow cytometry of β7 surface expression over time relative to day 0 ( n = 5–6 per group). (B) Left panels: Representative flow cytometry showing β7 integrin expression at baseline (day 0), and 24 (day 1), 48 (day 2), 72 (day 3) and 120 h (day 5) after treatment with etrolizumab-s for the first 24 h of the experiment and treatment with Brefeldin A from day 1 to day 5. Right panels: Quantitative flow cytometry of β7 surface expression over time relative to day 0 ( n = 5–6 per group).

    Article Snippet: To address whether this was due to recycling of internalized β7 integrin or resulting from de novo synthesis, we performed an additional series of experiments, in which etrolizumab-s treatment was followed by application of Brefeldin A to inhibit Golgi transport of freshly translated β7 protein.

    Techniques: Expressing, Flow Cytometry, Cytometry

    Localization of KBTBD8 protein in NIH3T3 cell and after treatment with BFA or Nocodazole. NIH3T3 cells – with and without BFA or Nocodazole treatment - were fixed and co-stained with KBTBD8 antibody and Golgi matrix marker GM130 or microtubule marker protein α-Tubulin. Treatment with Brefeldin A (BFA) resulted in resorption of the Golgi membrane and Golgi marker proteins to the ER. For GM130 the expected punctuated cytoplasmic localization could be found indicating that the treatment worked. However, KBTBD8 was either no longer detectable, not co-localizing with GM130 or associated to the spindle apparatus of mitotic cells. Co-staining with α-Tubulin and Kbtbd8 in untreated cells showed no localisation of KBTBD8 at the cytoskeleton. After Nocodazole- treatment, which results in the depolarisation of microtubules and in the disperse of the Golgi stacks to the cytoplasm, GM130 and α-Tubulin showed the expected localisation whereas KBTBD8 was again either not detectable at all or no longer co-localizing with GM130. These results indicate that KBTBD8 is not a part of the Golgi matrix. Scale bar, 10 μm.

    Journal: Cell Division

    Article Title: The novel BTB-kelch protein, KBTBD8, is located in the Golgi apparatus and translocates to the spindle apparatus during mitosis

    doi: 10.1186/1747-1028-8-3

    Figure Lengend Snippet: Localization of KBTBD8 protein in NIH3T3 cell and after treatment with BFA or Nocodazole. NIH3T3 cells – with and without BFA or Nocodazole treatment - were fixed and co-stained with KBTBD8 antibody and Golgi matrix marker GM130 or microtubule marker protein α-Tubulin. Treatment with Brefeldin A (BFA) resulted in resorption of the Golgi membrane and Golgi marker proteins to the ER. For GM130 the expected punctuated cytoplasmic localization could be found indicating that the treatment worked. However, KBTBD8 was either no longer detectable, not co-localizing with GM130 or associated to the spindle apparatus of mitotic cells. Co-staining with α-Tubulin and Kbtbd8 in untreated cells showed no localisation of KBTBD8 at the cytoskeleton. After Nocodazole- treatment, which results in the depolarisation of microtubules and in the disperse of the Golgi stacks to the cytoplasm, GM130 and α-Tubulin showed the expected localisation whereas KBTBD8 was again either not detectable at all or no longer co-localizing with GM130. These results indicate that KBTBD8 is not a part of the Golgi matrix. Scale bar, 10 μm.

    Article Snippet: Brefeldin A was purchased from AppliChem (Darmstadt, Germany).

    Techniques: Staining, Marker