Structured Review

Thermo Fisher pbmcs
Immune profiling of <t>ZIKV-infected</t> <t>PBMCs.</t> (A) Immune mediators in the culture supernatant of ZIKV-infected PBMCs and CD14-depleted PBMCs were quantified with a 45-plex microbead assay. Concentrations were scaled between 0 and 1. (B) Bar charts of three cytokines, levels of which were significantly affected by both the depletion of CD14 + monocytes and ZIKV infection. (C) Stimulatory capacity of the culture supernatants was further evaluated with freshly isolated PBMCs. Culture supernatant was added in a ratio of 1:10, and cells were harvested at 36 h poststimulation. Compiled percentages of CD107a-, IFN-γ-, and NKG2D-positive CD94 + CD56 + NK cells are shown as normalized to the respective mock sample. Data displayed were derived from seven donors. Lineage markers CD3, CD19, CD20, and CD14 have been included to rule out the presence of non-NK cells. All data are presented as means ± standard deviations. *, P
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1) Product Images from "Zika Virus Infection Preferentially Counterbalances Human Peripheral Monocyte and/or NK Cell Activity"

Article Title: Zika Virus Infection Preferentially Counterbalances Human Peripheral Monocyte and/or NK Cell Activity

Journal: mSphere

doi: 10.1128/mSphereDirect.00120-18

Immune profiling of ZIKV-infected PBMCs. (A) Immune mediators in the culture supernatant of ZIKV-infected PBMCs and CD14-depleted PBMCs were quantified with a 45-plex microbead assay. Concentrations were scaled between 0 and 1. (B) Bar charts of three cytokines, levels of which were significantly affected by both the depletion of CD14 + monocytes and ZIKV infection. (C) Stimulatory capacity of the culture supernatants was further evaluated with freshly isolated PBMCs. Culture supernatant was added in a ratio of 1:10, and cells were harvested at 36 h poststimulation. Compiled percentages of CD107a-, IFN-γ-, and NKG2D-positive CD94 + CD56 + NK cells are shown as normalized to the respective mock sample. Data displayed were derived from seven donors. Lineage markers CD3, CD19, CD20, and CD14 have been included to rule out the presence of non-NK cells. All data are presented as means ± standard deviations. *, P
Figure Legend Snippet: Immune profiling of ZIKV-infected PBMCs. (A) Immune mediators in the culture supernatant of ZIKV-infected PBMCs and CD14-depleted PBMCs were quantified with a 45-plex microbead assay. Concentrations were scaled between 0 and 1. (B) Bar charts of three cytokines, levels of which were significantly affected by both the depletion of CD14 + monocytes and ZIKV infection. (C) Stimulatory capacity of the culture supernatants was further evaluated with freshly isolated PBMCs. Culture supernatant was added in a ratio of 1:10, and cells were harvested at 36 h poststimulation. Compiled percentages of CD107a-, IFN-γ-, and NKG2D-positive CD94 + CD56 + NK cells are shown as normalized to the respective mock sample. Data displayed were derived from seven donors. Lineage markers CD3, CD19, CD20, and CD14 have been included to rule out the presence of non-NK cells. All data are presented as means ± standard deviations. *, P

Techniques Used: Infection, Isolation, Derivative Assay

Role of monocytes in NK cell activity. Full PBMCs and CD14-depleted PBMCs (2 × 10 6 cells per infection) were infected with Zika virus (ZIKV) at an MOI of 10 and harvested at 36 hpi. (A) Gating strategy of CD94 + CD56 + Lineage − NK cells and their expression of CD69, CD107a, and IFN-γ. Plots from one representative donor are shown. The red circle indicates the presence or absence of CD14 + monocytes. (B) Compiled percentages of CD107a- and IFN-γ-positive NK cells (depicted in panel A) as normalized to the respective mock sample. (C) Viral load in the infected cells. Data shown were derived from seven donors. Lineage markers CD3, CD19, CD20, and CD14 have been included to rule out the presence of non-NK cells. All data are presented as means ± standard deviations. *, P
Figure Legend Snippet: Role of monocytes in NK cell activity. Full PBMCs and CD14-depleted PBMCs (2 × 10 6 cells per infection) were infected with Zika virus (ZIKV) at an MOI of 10 and harvested at 36 hpi. (A) Gating strategy of CD94 + CD56 + Lineage − NK cells and their expression of CD69, CD107a, and IFN-γ. Plots from one representative donor are shown. The red circle indicates the presence or absence of CD14 + monocytes. (B) Compiled percentages of CD107a- and IFN-γ-positive NK cells (depicted in panel A) as normalized to the respective mock sample. (C) Viral load in the infected cells. Data shown were derived from seven donors. Lineage markers CD3, CD19, CD20, and CD14 have been included to rule out the presence of non-NK cells. All data are presented as means ± standard deviations. *, P

Techniques Used: Activity Assay, Infection, Expressing, Derivative Assay

2) Product Images from "Overexpression of Programmed Death Ligands in Naturally Occurring Postweaning Multisystemic Wasting Syndrome"

Article Title: Overexpression of Programmed Death Ligands in Naturally Occurring Postweaning Multisystemic Wasting Syndrome

Journal: Viral Immunology

doi: 10.1089/vim.2014.0097

Impaired PBMC proliferation with pigs suffering PMWS. PBMCs were labeled with carboxyfluorescein diacetate succinimidyl ester and then stimulated by concanavalin A. Cells were harvested after culture for 5 days at 37°C 5% CO 2 in RPMI-1640 culture medium supplemented with 10% fetal bovine serum. Cells were analyzed by flow cytometry. The mean fluorescence intensity of the proliferating PBMCs from diseased pigs (B) was remarkably decreased compared with that of uninfected pigs (A) ( p
Figure Legend Snippet: Impaired PBMC proliferation with pigs suffering PMWS. PBMCs were labeled with carboxyfluorescein diacetate succinimidyl ester and then stimulated by concanavalin A. Cells were harvested after culture for 5 days at 37°C 5% CO 2 in RPMI-1640 culture medium supplemented with 10% fetal bovine serum. Cells were analyzed by flow cytometry. The mean fluorescence intensity of the proliferating PBMCs from diseased pigs (B) was remarkably decreased compared with that of uninfected pigs (A) ( p

Techniques Used: Labeling, Flow Cytometry, Cytometry, Fluorescence

3) Product Images from "New insights into the role and mechanism of macrophage migration inhibitory factor in steroid-resistant patients with systemic lupus erythematosus"

Article Title: New insights into the role and mechanism of macrophage migration inhibitory factor in steroid-resistant patients with systemic lupus erythematosus

Journal: Arthritis Research & Therapy

doi: 10.1186/ar3828

Cytosolic macrophage migration inhibitory factor and IκB expression after siRNA-mediated gene silencing in steroid-resistant patients . (A) Real-time PCR analysis of macrophage migration inhibitory factor (MIF) mRNA expression in peripheral blood mononuclear cells (PBMCs) from the steroid-resistant (SR) systemic lupus erythematosus group transfected with three siRNAs at different concentrations. C, control; NC, negative control; Si1, 100 nmol/l s8780; Si2, 100 nmol/l s194615; Si3, 100 nmol/l s194614; Si4, 200 nmol/l s8780; Si5, 200 nmol/l s194615; Si6, 200 nmol/l s194614. * P
Figure Legend Snippet: Cytosolic macrophage migration inhibitory factor and IκB expression after siRNA-mediated gene silencing in steroid-resistant patients . (A) Real-time PCR analysis of macrophage migration inhibitory factor (MIF) mRNA expression in peripheral blood mononuclear cells (PBMCs) from the steroid-resistant (SR) systemic lupus erythematosus group transfected with three siRNAs at different concentrations. C, control; NC, negative control; Si1, 100 nmol/l s8780; Si2, 100 nmol/l s194615; Si3, 100 nmol/l s194614; Si4, 200 nmol/l s8780; Si5, 200 nmol/l s194615; Si6, 200 nmol/l s194614. * P

Techniques Used: Migration, Expressing, Real-time Polymerase Chain Reaction, Transfection, Negative Control

4) Product Images from "Targeted Delivery of an Antigenic Peptide to the Endoplasmic Reticulum: Application for Development of a Peptide Therapy for Ankylosing Spondylitis"

Article Title: Targeted Delivery of an Antigenic Peptide to the Endoplasmic Reticulum: Application for Development of a Peptide Therapy for Ankylosing Spondylitis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0077451

Treatment with either THUC or THUB reduces the formation of (B27-HC) 2 . After treatment with each protein for the indicated time point, membrane proteins were extracted. 50 μg of extract was resolved by non-reducing SDS-PAGE (10%) and immunoblotted with a BH2 monoclonal antibody and anti-transferrin (Tf) receptor antibody. Tf receptor serves as an internal control. (A) THUC treatment for 12 h, but not THU or HUC treatment, significantly decreased the levels of (B27-HC) 2 . (B) The level of (B27-HC) 2 is significantly reduced in C1R-B2704 cells treated with THUB. (C) The production of (B27-HC) 2 is reduced when PBMCs isolated from AS patients are treated with THUC. (D) The results obtained in Figure 3C are plotted. The amount of immunostaining observed at 0 h was set to 100%. The results shown are the mean levels of (B27-HC) 2 immunostaining observed in membrane proteins independently extracted from the PBMCs of five AS patients (mean ± SD, n = 5).
Figure Legend Snippet: Treatment with either THUC or THUB reduces the formation of (B27-HC) 2 . After treatment with each protein for the indicated time point, membrane proteins were extracted. 50 μg of extract was resolved by non-reducing SDS-PAGE (10%) and immunoblotted with a BH2 monoclonal antibody and anti-transferrin (Tf) receptor antibody. Tf receptor serves as an internal control. (A) THUC treatment for 12 h, but not THU or HUC treatment, significantly decreased the levels of (B27-HC) 2 . (B) The level of (B27-HC) 2 is significantly reduced in C1R-B2704 cells treated with THUB. (C) The production of (B27-HC) 2 is reduced when PBMCs isolated from AS patients are treated with THUC. (D) The results obtained in Figure 3C are plotted. The amount of immunostaining observed at 0 h was set to 100%. The results shown are the mean levels of (B27-HC) 2 immunostaining observed in membrane proteins independently extracted from the PBMCs of five AS patients (mean ± SD, n = 5).

Techniques Used: SDS Page, Isolation, Immunostaining

Treatment with THUC, but not THUB or THU, enhances apoptosis mediated by CD8 + T-cell cytotoxicity. (A) Treatment with THUC, but not THU, increases apoptosis mediated by CD8 + T-cell cytotoxicity. TAP1-knockdown abolishes the THUC-induced increase in apoptosis. PBMCs isolated from AS patients (n = 9) were treated with THUC, and stimulated with IL-2. CD8 + T cells were isolated from THUC-stimulated PBMCs. C1R-B2704 cells were stained with phycoerythrin-conjugated anti-CD19 antibodies and the apoptotic cells were stained with FITC-conjugated anti-active caspase 3 antibody. (B) The results obtained in Figure 7A are plotted. (C) The THUC-induced cell apoptosis mediated by CD8 + T-cell cytotoxicity is CD8-dependent. (D) The results obtained in Figure 7C are plotted. (E) Treatment with THUB cannot enhance apoptosis mediated by CD8 + T-cell cytotoxicity. (F) The resulted obtained in Figure 7E are plotted.
Figure Legend Snippet: Treatment with THUC, but not THUB or THU, enhances apoptosis mediated by CD8 + T-cell cytotoxicity. (A) Treatment with THUC, but not THU, increases apoptosis mediated by CD8 + T-cell cytotoxicity. TAP1-knockdown abolishes the THUC-induced increase in apoptosis. PBMCs isolated from AS patients (n = 9) were treated with THUC, and stimulated with IL-2. CD8 + T cells were isolated from THUC-stimulated PBMCs. C1R-B2704 cells were stained with phycoerythrin-conjugated anti-CD19 antibodies and the apoptotic cells were stained with FITC-conjugated anti-active caspase 3 antibody. (B) The results obtained in Figure 7A are plotted. (C) The THUC-induced cell apoptosis mediated by CD8 + T-cell cytotoxicity is CD8-dependent. (D) The results obtained in Figure 7C are plotted. (E) Treatment with THUB cannot enhance apoptosis mediated by CD8 + T-cell cytotoxicity. (F) The resulted obtained in Figure 7E are plotted.

Techniques Used: Isolation, Staining

5) Product Images from "Effects of anticancer agents on cell viability, proliferative activity and cytokine production of peripheral blood mononuclear cells"

Article Title: Effects of anticancer agents on cell viability, proliferative activity and cytokine production of peripheral blood mononuclear cells

Journal: Journal of Clinical Biochemistry and Nutrition

doi: 10.3164/jcbn.12-60

Effects of GEM on the frequency of IFN-γ-producing cells. After 2 h stimulation with GEM, PBMCs were incubated with PHA for 48 h. Cell types (CD4 and CD8) and intracellular staining for IFN-γ were evaluated by flow cytometry. In all panels, data are expressed as the mean ± SEM. * p
Figure Legend Snippet: Effects of GEM on the frequency of IFN-γ-producing cells. After 2 h stimulation with GEM, PBMCs were incubated with PHA for 48 h. Cell types (CD4 and CD8) and intracellular staining for IFN-γ were evaluated by flow cytometry. In all panels, data are expressed as the mean ± SEM. * p

Techniques Used: Incubation, Staining, Flow Cytometry, Cytometry

Effects of GEM on lymphocyte populations. After 2 h stimulation with GEM, PBMCs were incubated with PHA for 48 h. Cell types (CD4 and CD8) and intracellular staining for Foxp3 were evaluated by flow cytometry. In all panels, data are expressed as the mean ± SEM. * p
Figure Legend Snippet: Effects of GEM on lymphocyte populations. After 2 h stimulation with GEM, PBMCs were incubated with PHA for 48 h. Cell types (CD4 and CD8) and intracellular staining for Foxp3 were evaluated by flow cytometry. In all panels, data are expressed as the mean ± SEM. * p

Techniques Used: Incubation, Staining, Flow Cytometry, Cytometry

A representative result of CD4 and IFN-γ dual staining and flow cytometry. After 2 h stimulation with GEM, PBMCs were incubated with PHA for 48 h. CD4 and intracellular staining for IFN-γ were evaluated by flow cytometry.
Figure Legend Snippet: A representative result of CD4 and IFN-γ dual staining and flow cytometry. After 2 h stimulation with GEM, PBMCs were incubated with PHA for 48 h. CD4 and intracellular staining for IFN-γ were evaluated by flow cytometry.

Techniques Used: Staining, Flow Cytometry, Cytometry, Incubation

Effects of GEM on PHA-induced cytokine production. After 2 h chemical stimulation with GEM, PBMCs were then incubated with PHA for 48 h. Cell culture supernatant was assayed for TNF-α(a), IL-2(b), IL-4(c), IL-10(d) and IFN-γ(e) by using a cytokine-specific solid phase sandwich ELISA. In all panels, data are expressed as the mean ± SEM. * p
Figure Legend Snippet: Effects of GEM on PHA-induced cytokine production. After 2 h chemical stimulation with GEM, PBMCs were then incubated with PHA for 48 h. Cell culture supernatant was assayed for TNF-α(a), IL-2(b), IL-4(c), IL-10(d) and IFN-γ(e) by using a cytokine-specific solid phase sandwich ELISA. In all panels, data are expressed as the mean ± SEM. * p

Techniques Used: Incubation, Cell Culture, Sandwich ELISA

Effects of anticancer agents on proliferative activity of PBMCs. After stimulation with incremental concentrations of anticancer agents for 2 h, PBMCs were then stimulated with PHA (5 µg/ml) for 48 h. Cells were pulsed with bromodeoxyuridine (BrdU) solution and measured using a microplate reader. Data are expressed as the mean ± SEM. * p
Figure Legend Snippet: Effects of anticancer agents on proliferative activity of PBMCs. After stimulation with incremental concentrations of anticancer agents for 2 h, PBMCs were then stimulated with PHA (5 µg/ml) for 48 h. Cells were pulsed with bromodeoxyuridine (BrdU) solution and measured using a microplate reader. Data are expressed as the mean ± SEM. * p

Techniques Used: Activity Assay

Effects of 5-FU on PHA-induced cytokine production. After 2 h chemical stimulation with 5-FU, PBMCs were then incubated with PHA for 48 h. Cell culture supernatant was assayed for TNF-α(a), IL-2(b), IL-4(c), IL-10(d) and IFN-γ(e) by using a cytokine-specific solid phase sandwich ELISA. In all panels, data are expressed as the mean ± SEM. * p
Figure Legend Snippet: Effects of 5-FU on PHA-induced cytokine production. After 2 h chemical stimulation with 5-FU, PBMCs were then incubated with PHA for 48 h. Cell culture supernatant was assayed for TNF-α(a), IL-2(b), IL-4(c), IL-10(d) and IFN-γ(e) by using a cytokine-specific solid phase sandwich ELISA. In all panels, data are expressed as the mean ± SEM. * p

Techniques Used: Incubation, Cell Culture, Sandwich ELISA

Effects of CDDP on PHA-induced cytokine production. After 2 h chemical stimulation with CDDP, PBMCs were then incubated with PHA for 48 h. Cell culture supernatant was assayed for TNF-α(a), IL-2(b), IL-4(c), IL-10(d) and IFN-γ(e) by using a cytokine-specific solid phase sandwich ELISA. In all panels, data are expressed as the mean ± SEM. * p
Figure Legend Snippet: Effects of CDDP on PHA-induced cytokine production. After 2 h chemical stimulation with CDDP, PBMCs were then incubated with PHA for 48 h. Cell culture supernatant was assayed for TNF-α(a), IL-2(b), IL-4(c), IL-10(d) and IFN-γ(e) by using a cytokine-specific solid phase sandwich ELISA. In all panels, data are expressed as the mean ± SEM. * p

Techniques Used: Incubation, Cell Culture, Sandwich ELISA

A representative result of CD4 and Foxp3 dual staining and flow cytometry. After 2 h stimulation with GEM, PBMCs were incubated with PHA for 48 h. CD4 and intracellular staining for Foxp3 were evaluated by flow cytometry.
Figure Legend Snippet: A representative result of CD4 and Foxp3 dual staining and flow cytometry. After 2 h stimulation with GEM, PBMCs were incubated with PHA for 48 h. CD4 and intracellular staining for Foxp3 were evaluated by flow cytometry.

Techniques Used: Staining, Flow Cytometry, Cytometry, Incubation

Effects of CPT-11 on PHA-induced cytokine production. After 2 h chemical stimulation with CPT-11, PBMCs were then incubated with PHA for 48 h. Cell culture supernatant was assayed for TNF-α(a), IL-2(b), IL-4(c), IL-10(d) and IFN-γ(e) by using a cytokine-specific solid phase sandwich ELISA. In all panels, data are expressed as the mean ± SEM. * p
Figure Legend Snippet: Effects of CPT-11 on PHA-induced cytokine production. After 2 h chemical stimulation with CPT-11, PBMCs were then incubated with PHA for 48 h. Cell culture supernatant was assayed for TNF-α(a), IL-2(b), IL-4(c), IL-10(d) and IFN-γ(e) by using a cytokine-specific solid phase sandwich ELISA. In all panels, data are expressed as the mean ± SEM. * p

Techniques Used: Cycling Probe Technology, Incubation, Cell Culture, Sandwich ELISA

6) Product Images from "Anti-melanoma activity of T cells redirected with a TCR-like chimeric antigen receptor"

Article Title: Anti-melanoma activity of T cells redirected with a TCR-like chimeric antigen receptor

Journal: Scientific Reports

doi: 10.1038/srep03571

Co-culture of GPA7-CD28/ζ transduced PBMC with gp100-pulsed T2 cells. (a) Schematic illustration of PBMC stimulation, transduction and expansion protocol. After lentiviral transduction, PBMC were cultured and re-stimulated with irradiated antigen-loaded T2 cells from day 4. (b) Cell surface phenotype of transduced T-cell cultures after a round of re-stimulation with gp100-loaded T2 cells. Anti-CD3-PE, anti-CD4-FITC, anti-CD8-FITC and PE-labeled gp100-HLA-A2 tetramer were used for characterization. Control was isotype-stained cells. Frequencies for each population were indicated above panels. A representative of three independent repeats from flow cytometry plots is shown.
Figure Legend Snippet: Co-culture of GPA7-CD28/ζ transduced PBMC with gp100-pulsed T2 cells. (a) Schematic illustration of PBMC stimulation, transduction and expansion protocol. After lentiviral transduction, PBMC were cultured and re-stimulated with irradiated antigen-loaded T2 cells from day 4. (b) Cell surface phenotype of transduced T-cell cultures after a round of re-stimulation with gp100-loaded T2 cells. Anti-CD3-PE, anti-CD4-FITC, anti-CD8-FITC and PE-labeled gp100-HLA-A2 tetramer were used for characterization. Control was isotype-stained cells. Frequencies for each population were indicated above panels. A representative of three independent repeats from flow cytometry plots is shown.

Techniques Used: Co-Culture Assay, Transduction, Cell Culture, Irradiation, Labeling, Staining, Flow Cytometry, Cytometry

7) Product Images from "Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies"

Article Title: Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-9-3

PCR efficiency and PreAmp uniformity in PBMCs using T-PreAmp . a) Comparison of PCR efficiency of the investigated candidate gene assays calculated from PBMCs' cDNA (25 ng/μl) and 7 dilution steps (up to 1:1,024) by direct (cDNA) and T-PreAmp real-time RT-PCR (T-PreAmp). Taqman ® ABI inventoried gene assays are indicated by red bars, self-designed gene assays designed by coworkers of the Charité are indicated by blue bars. b) PreAmp uniformity of the investigated candidate gene assays at 7 dilution steps from 25 ng/μl PBMCs' cDNA (1) to 1:1,024. The red bars indicate the means ± SD of the 8 reactions for each gene assay.
Figure Legend Snippet: PCR efficiency and PreAmp uniformity in PBMCs using T-PreAmp . a) Comparison of PCR efficiency of the investigated candidate gene assays calculated from PBMCs' cDNA (25 ng/μl) and 7 dilution steps (up to 1:1,024) by direct (cDNA) and T-PreAmp real-time RT-PCR (T-PreAmp). Taqman ® ABI inventoried gene assays are indicated by red bars, self-designed gene assays designed by coworkers of the Charité are indicated by blue bars. b) PreAmp uniformity of the investigated candidate gene assays at 7 dilution steps from 25 ng/μl PBMCs' cDNA (1) to 1:1,024. The red bars indicate the means ± SD of the 8 reactions for each gene assay.

Techniques Used: Polymerase Chain Reaction, Quantitative RT-PCR, Gene Assay

8) Product Images from "The CD16+ (Fc?RIII+) Subset of Human Monocytes Preferentially Becomes Migratory Dendritic Cells in a Model Tissue Setting"

Article Title: The CD16+ (Fc?RIII+) Subset of Human Monocytes Preferentially Becomes Migratory Dendritic Cells in a Model Tissue Setting

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20011608

Effect of TGFβ1 on the phenotype, migratory ability, and survival of CD16 − monocytes. PBMCs were depleted of CD16 + cells using miniMACS magnetic selection. Then remaining cells were cultured for up to 3 d in TGFβ1 (bold lines) or anti-TGFβ (thin lines) to block activity of endogenous TGFβ. (A) The cell surface phenotype of these cultured cells was assessed by flow cytometry. Control mAb staining is depicted as a dashed line in top left histogram. (B) Monocytes cultured overnight in TGFβ1 or anti-TGFβ were applied to endothelial monolayers grown on collagen gels lacking zymosan and apical-to-basal transendothelial migration was quantified after a 1.5 incubation. (C) Reverse transmigration was quantified at 48 h as the percent of cells that originally migrated across the endothelium in each condition and then later retraversed the endothelium in ablumenal-to-lumenal direction. (D) Percentage of live cells in the reverse transmigrated populations derived from TGFβ1 or anti-TGFβ treated monocytes incubated with endothelial cultures lacking or containing zymosan within the subendothelium was assessed by trypan blue exclusion.
Figure Legend Snippet: Effect of TGFβ1 on the phenotype, migratory ability, and survival of CD16 − monocytes. PBMCs were depleted of CD16 + cells using miniMACS magnetic selection. Then remaining cells were cultured for up to 3 d in TGFβ1 (bold lines) or anti-TGFβ (thin lines) to block activity of endogenous TGFβ. (A) The cell surface phenotype of these cultured cells was assessed by flow cytometry. Control mAb staining is depicted as a dashed line in top left histogram. (B) Monocytes cultured overnight in TGFβ1 or anti-TGFβ were applied to endothelial monolayers grown on collagen gels lacking zymosan and apical-to-basal transendothelial migration was quantified after a 1.5 incubation. (C) Reverse transmigration was quantified at 48 h as the percent of cells that originally migrated across the endothelium in each condition and then later retraversed the endothelium in ablumenal-to-lumenal direction. (D) Percentage of live cells in the reverse transmigrated populations derived from TGFβ1 or anti-TGFβ treated monocytes incubated with endothelial cultures lacking or containing zymosan within the subendothelium was assessed by trypan blue exclusion.

Techniques Used: Selection, Cell Culture, Blocking Assay, Activity Assay, Flow Cytometry, Cytometry, Staining, Migration, Incubation, Transmigration Assay, Derivative Assay

Transendothelial migration of blood DC precursors across unstimulated endothelium. The entire fraction of freshly isolated PBMCs were incubated with endothelial cell/collagen cultures for 1.5 h to permit transmigration. The apical surface of the cultures was washed to collect nonmigrated cells, and the migrated population was recovered from the subendothelial collagen using collagenase D. Cells considered for quantitative evaluation were large mononuclear cells (LMC) uniformly positive for HLA-DR and negative for the B cell marker CD19 or T cell marker CD3. The plot shows the percent distribution of three distinct populations: CD14 + CD16 − , CD14 + CD16 + , and CD14 − CD16 − cells. The total height of each bar represents the relative distribution of these populations in freshly isolated PBMCs. The filled portion of each bar indicates the fraction of each population that emigrated beneath the endothelium, and the open portion of each bar represents the portion of the population that was recovered in the nonmigrated fraction. These data are representative of results obtained using PBMCs from three different blood donors.
Figure Legend Snippet: Transendothelial migration of blood DC precursors across unstimulated endothelium. The entire fraction of freshly isolated PBMCs were incubated with endothelial cell/collagen cultures for 1.5 h to permit transmigration. The apical surface of the cultures was washed to collect nonmigrated cells, and the migrated population was recovered from the subendothelial collagen using collagenase D. Cells considered for quantitative evaluation were large mononuclear cells (LMC) uniformly positive for HLA-DR and negative for the B cell marker CD19 or T cell marker CD3. The plot shows the percent distribution of three distinct populations: CD14 + CD16 − , CD14 + CD16 + , and CD14 − CD16 − cells. The total height of each bar represents the relative distribution of these populations in freshly isolated PBMCs. The filled portion of each bar indicates the fraction of each population that emigrated beneath the endothelium, and the open portion of each bar represents the portion of the population that was recovered in the nonmigrated fraction. These data are representative of results obtained using PBMCs from three different blood donors.

Techniques Used: Migration, Isolation, Incubation, Transmigration Assay, Marker

Capacity of reverse-transmigrated cells derived from monocyte subsets to stimulate allogeneic T cell proliferation. Monocytes were sorted according to their level of expression of CD14. First lymphocytes were removed from the PBMC fraction to generate a “presort” population of monocytes (a). Monocytes were then sorted into CD14 hi (b, c) and CD14 med (d) expressing cells. Some of the CD14 hi monocytes were then treated with anti-CD16 miniMACs beads to remove any remaining CD16 + cells (c). These populations were added to endothelial cultures in which zymosan was present in the subendothelial matrix, and then live reverse transmigrated cells collected 2 d later were tested for their ability to induce allogeneic T cell proliferation at a ratio of 1 antigen-presenting cell: 80 T cells. In some conditions, a 1:1 mix of some reverse-transmigrated populations (d + b, or d + c) were used as APCs. Background counts for T cells cultured alone were
Figure Legend Snippet: Capacity of reverse-transmigrated cells derived from monocyte subsets to stimulate allogeneic T cell proliferation. Monocytes were sorted according to their level of expression of CD14. First lymphocytes were removed from the PBMC fraction to generate a “presort” population of monocytes (a). Monocytes were then sorted into CD14 hi (b, c) and CD14 med (d) expressing cells. Some of the CD14 hi monocytes were then treated with anti-CD16 miniMACs beads to remove any remaining CD16 + cells (c). These populations were added to endothelial cultures in which zymosan was present in the subendothelial matrix, and then live reverse transmigrated cells collected 2 d later were tested for their ability to induce allogeneic T cell proliferation at a ratio of 1 antigen-presenting cell: 80 T cells. In some conditions, a 1:1 mix of some reverse-transmigrated populations (d + b, or d + c) were used as APCs. Background counts for T cells cultured alone were

Techniques Used: Derivative Assay, Expressing, Cell Culture

Distribution of CD16 + monocyte-derived cells after coculture with endothelial cells grown on collagen. (A) Expression of CD16 was monitored in monocyte/endothelial cocultures at 48 h, when the majority of cells that will reverse transmigrate have done so. Assessments were made in reverse transmigrated (R/T, thin-lined profile) and subendothelial (S/E, bolded line profile) monocyte-derived cells from cultures that received no activation stimuli such as exogenous cytokines or phagocytic particles. Dotted line demarcates the staining intensity of cells incubated with isotype-matched mAbs to an irrelevant antigen. Filled profile represents the expression of CD16 in freshly isolated monocytes. (B) In other cultures, zymosan was included in the collagen to promote DC maturation. In some unstimulated endothelial cultures, the whole fraction of PBMCs (C) or CD16 + monocytes sorted using flow cytometry (D), were labeled with CFSE. Transendothelial migration into the collagen and subsequent reverse transmigration was evaluated to assess the distribution of CFSE + cells. CFSE-labeled CD16 + monocytes were remixed with CD16 − PBMCs so that CD16 + CFSE + cells represented 15% of the total population (D).
Figure Legend Snippet: Distribution of CD16 + monocyte-derived cells after coculture with endothelial cells grown on collagen. (A) Expression of CD16 was monitored in monocyte/endothelial cocultures at 48 h, when the majority of cells that will reverse transmigrate have done so. Assessments were made in reverse transmigrated (R/T, thin-lined profile) and subendothelial (S/E, bolded line profile) monocyte-derived cells from cultures that received no activation stimuli such as exogenous cytokines or phagocytic particles. Dotted line demarcates the staining intensity of cells incubated with isotype-matched mAbs to an irrelevant antigen. Filled profile represents the expression of CD16 in freshly isolated monocytes. (B) In other cultures, zymosan was included in the collagen to promote DC maturation. In some unstimulated endothelial cultures, the whole fraction of PBMCs (C) or CD16 + monocytes sorted using flow cytometry (D), were labeled with CFSE. Transendothelial migration into the collagen and subsequent reverse transmigration was evaluated to assess the distribution of CFSE + cells. CFSE-labeled CD16 + monocytes were remixed with CD16 − PBMCs so that CD16 + CFSE + cells represented 15% of the total population (D).

Techniques Used: Derivative Assay, Expressing, Activation Assay, Staining, Incubation, Isolation, Flow Cytometry, Cytometry, Labeling, Migration, Transmigration Assay

Effect of a reverse transmigration antagonist on the distribution of CD16 + cells in endothelial cell cultures. PBMCs were incubated with endothelial/collagen cultures for 1.5 h, then washed to remove nonadherent, nonmigrated cells. Cultures were fed with medium containing mAb to MDR-1 or isotype-matched control mAb UPC10, and incubation was continued for 48 h to allow reverse transmigration. The presence of CD16 + cells in reverse-transmigrated and subendothelial leukocytes was monitored after 48 h by flow cytometry. The total number of CD16 + cells recovered from cultures in the presence and absence of anti-MDR-1 is shown. The fraction of such cells that had reverse transmigrated (R/T) is shown by the open portion of the bars, whereas the fraction that remained in the subendothelium (S/E) is shown by the filled portion of the bars.
Figure Legend Snippet: Effect of a reverse transmigration antagonist on the distribution of CD16 + cells in endothelial cell cultures. PBMCs were incubated with endothelial/collagen cultures for 1.5 h, then washed to remove nonadherent, nonmigrated cells. Cultures were fed with medium containing mAb to MDR-1 or isotype-matched control mAb UPC10, and incubation was continued for 48 h to allow reverse transmigration. The presence of CD16 + cells in reverse-transmigrated and subendothelial leukocytes was monitored after 48 h by flow cytometry. The total number of CD16 + cells recovered from cultures in the presence and absence of anti-MDR-1 is shown. The fraction of such cells that had reverse transmigrated (R/T) is shown by the open portion of the bars, whereas the fraction that remained in the subendothelium (S/E) is shown by the filled portion of the bars.

Techniques Used: Transmigration Assay, Incubation, Flow Cytometry, Cytometry

Cell surface markers distinguish CD16 + and CD16 − HLA-DR + PBMCs. Freshly isolated PBMCs were depleted of CD56 + NK cells. Two-color flow cytometry was conducted with a gate set to exclude smaller PBMCs of the lymphocyte lineage. Expression of macrophage-associated markers CD14, CD36, and CD64 or DC-associated markers HLA-DR, HLA-DP, and CD86 (all x-axis) were examined. Cells were counterstained with a mAb to CD16 (y-axis). Some cells were stained with M-DC8 mAb to identify a subset of CD16 + cells (reference 11 ). Quadrant markers (shown in CD14 and CD123 panels) are positioned according to the level of fluorescence observed in cells stained with nonbinding isotype-matched control mAbs (lower left quadrant, negative staining). The phenotype illustrated was similarly observed among four donors examined.
Figure Legend Snippet: Cell surface markers distinguish CD16 + and CD16 − HLA-DR + PBMCs. Freshly isolated PBMCs were depleted of CD56 + NK cells. Two-color flow cytometry was conducted with a gate set to exclude smaller PBMCs of the lymphocyte lineage. Expression of macrophage-associated markers CD14, CD36, and CD64 or DC-associated markers HLA-DR, HLA-DP, and CD86 (all x-axis) were examined. Cells were counterstained with a mAb to CD16 (y-axis). Some cells were stained with M-DC8 mAb to identify a subset of CD16 + cells (reference 11 ). Quadrant markers (shown in CD14 and CD123 panels) are positioned according to the level of fluorescence observed in cells stained with nonbinding isotype-matched control mAbs (lower left quadrant, negative staining). The phenotype illustrated was similarly observed among four donors examined.

Techniques Used: Isolation, Flow Cytometry, Cytometry, Expressing, Staining, Fluorescence, Negative Staining

Evaluation of reverse transmigration and expression of CD16 after depletion of peripheral blood CD16 + monocytes. CD56 + NK cells were depleted from the starting PBMC fraction, leaving a fraction of PBMCs that included both CD16 − and CD16 + (CD16 mix ) CD14 + monocytes. In some samples, the remaining CD16 + cells were depleted, leaving CD56 − CD16 − PBMCs. CD16 mix or CD16 − PBMCs were applied to endothelial/collagen cultures at the same starting density. The number of reverse transmigrated cells in the CD16 − fraction was evaluated after 2 d and compared in five independent experiments to the number of reverse transmigrated cells in the control CD16 mix population of PBMCs (A). The relative recovery was calculated by setting equal to 1.0 the number of reverse transmigrated cells recovered per well of cultured endothelium after application of CD56 − CD16 mix PBMCs and then determining the fractional recovery in each experiment when CD16-depleted PBMCs were applied. (B) The possibility that CD16 might be upregulated on peripheral blood cells that originally lacked CD16 was tested by examining the expression of CD16 in reverse-transmigrated and subendothelial populations after full depletion of CD16 + blood cells. Flow cytometric evaluation of CD16 expression in reverse transmigrated (R/T) and subendothelial (S/E) cells that arose from CD16-depleted PBMCs is plotted as a histogram. (C) Phenotyping for CD86 and HLA-DR expression was conducted in reverse-transmigrated populations derived in the presence or absence of CD16 + monocytes. The dot plots shown in panel C represent the number and phenotype of cells recovered from equal numbers of zymosan-containing endothelial cell microtiter cultures to which equivalent densities of CD16 mix and CD16 − monocytes were applied.
Figure Legend Snippet: Evaluation of reverse transmigration and expression of CD16 after depletion of peripheral blood CD16 + monocytes. CD56 + NK cells were depleted from the starting PBMC fraction, leaving a fraction of PBMCs that included both CD16 − and CD16 + (CD16 mix ) CD14 + monocytes. In some samples, the remaining CD16 + cells were depleted, leaving CD56 − CD16 − PBMCs. CD16 mix or CD16 − PBMCs were applied to endothelial/collagen cultures at the same starting density. The number of reverse transmigrated cells in the CD16 − fraction was evaluated after 2 d and compared in five independent experiments to the number of reverse transmigrated cells in the control CD16 mix population of PBMCs (A). The relative recovery was calculated by setting equal to 1.0 the number of reverse transmigrated cells recovered per well of cultured endothelium after application of CD56 − CD16 mix PBMCs and then determining the fractional recovery in each experiment when CD16-depleted PBMCs were applied. (B) The possibility that CD16 might be upregulated on peripheral blood cells that originally lacked CD16 was tested by examining the expression of CD16 in reverse-transmigrated and subendothelial populations after full depletion of CD16 + blood cells. Flow cytometric evaluation of CD16 expression in reverse transmigrated (R/T) and subendothelial (S/E) cells that arose from CD16-depleted PBMCs is plotted as a histogram. (C) Phenotyping for CD86 and HLA-DR expression was conducted in reverse-transmigrated populations derived in the presence or absence of CD16 + monocytes. The dot plots shown in panel C represent the number and phenotype of cells recovered from equal numbers of zymosan-containing endothelial cell microtiter cultures to which equivalent densities of CD16 mix and CD16 − monocytes were applied.

Techniques Used: Transmigration Assay, Expressing, Cell Culture, Flow Cytometry, Derivative Assay

9) Product Images from "New insights into Lyme disease"

Article Title: New insights into Lyme disease

Journal: Redox Biology

doi: 10.1016/j.redox.2015.03.002

Cytosolic ionized calcium levels of PBMCs. Dashed blue lines (left) represent healthy controls ( x̄ =46 nM), where red dots (right) represent Lyme borreliosis patients ( x̄ =26 nM). Significant difference between groups was measured by Student's t -test (**** p
Figure Legend Snippet: Cytosolic ionized calcium levels of PBMCs. Dashed blue lines (left) represent healthy controls ( x̄ =46 nM), where red dots (right) represent Lyme borreliosis patients ( x̄ =26 nM). Significant difference between groups was measured by Student's t -test (**** p

Techniques Used:

10) Product Images from "Human CD103+ dendritic cells promote the differentiation of Porphyromonas gingivalis heat shock protein peptide-specific regulatory T cells"

Article Title: Human CD103+ dendritic cells promote the differentiation of Porphyromonas gingivalis heat shock protein peptide-specific regulatory T cells

Journal: Journal of Periodontal & Implant Science

doi: 10.5051/jpis.2014.44.5.235

Fluorescence activated cell sorter profiles of peptide 19-specific regulatory T cells (Tregs) (CD4 + , CD25 + , and Foxp3 + ) in peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) from two representative subjects (A, B) demonstrating the capability of DCs to enhance Tregs from 0.07% to 22.89% in subject 1 and from 0.59% to 34.83% in subject 2, respectively. The bar graph shows the mean percentage of peptide 19-specific Tregs of 10 subjects (C). The difference between PMBCs and DCs was not statistically significant. Phosphate buffered saline (PBS) was used as a control antigen. UL: upper left panel, UR: upper right panel, LL: lower left panel, LR: lower right panel.
Figure Legend Snippet: Fluorescence activated cell sorter profiles of peptide 19-specific regulatory T cells (Tregs) (CD4 + , CD25 + , and Foxp3 + ) in peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) from two representative subjects (A, B) demonstrating the capability of DCs to enhance Tregs from 0.07% to 22.89% in subject 1 and from 0.59% to 34.83% in subject 2, respectively. The bar graph shows the mean percentage of peptide 19-specific Tregs of 10 subjects (C). The difference between PMBCs and DCs was not statistically significant. Phosphate buffered saline (PBS) was used as a control antigen. UL: upper left panel, UR: upper right panel, LL: lower left panel, LR: lower right panel.

Techniques Used: Fluorescence

Stimulation of peptide-specific regulatory T cells (Tregs) by peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs). (A) Peptide number 19 from Streptococcus sanguinis heat shock protein 60 has shown a minimal capability (0.05%) to convert T cells into Tregs when PBMCs are used as antigen-presenting cells. (B) DCs were used for presenting peptide antigens, which enhanced induction of Tregs (17.02%). (C) The bar graph shows the percentage of peptide-specific Tregs among T cells in one subject. UL: upper left panel, UR: upper right panel, LL: lower left panel, LR: lower right panel.
Figure Legend Snippet: Stimulation of peptide-specific regulatory T cells (Tregs) by peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs). (A) Peptide number 19 from Streptococcus sanguinis heat shock protein 60 has shown a minimal capability (0.05%) to convert T cells into Tregs when PBMCs are used as antigen-presenting cells. (B) DCs were used for presenting peptide antigens, which enhanced induction of Tregs (17.02%). (C) The bar graph shows the percentage of peptide-specific Tregs among T cells in one subject. UL: upper left panel, UR: upper right panel, LL: lower left panel, LR: lower right panel.

Techniques Used:

Fluorescence activated cell sorter profiles of peptide 14-specific regulatory T cells (Tregs) (CD4 + , CD25 + , and Foxp3 + ) in peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) from two representative subjects (A, B) demonstrating the capability of DCs to enhance Tregs from 0.07% to 32.54% in subject 1 and from 0.15% to 34.46% in subject 2. The bar graph shows the mean percentage of peptide 14-specific Tregs of 10 subjects (C), evidencing a statistically significant difference between PMBCs and DCs ( * P
Figure Legend Snippet: Fluorescence activated cell sorter profiles of peptide 14-specific regulatory T cells (Tregs) (CD4 + , CD25 + , and Foxp3 + ) in peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) from two representative subjects (A, B) demonstrating the capability of DCs to enhance Tregs from 0.07% to 32.54% in subject 1 and from 0.15% to 34.46% in subject 2. The bar graph shows the mean percentage of peptide 14-specific Tregs of 10 subjects (C), evidencing a statistically significant difference between PMBCs and DCs ( * P

Techniques Used: Fluorescence

11) Product Images from "T Follicular Helper Cells and Regulatory B Cells Dynamics in Systemic Lupus Erythematosus"

Article Title: T Follicular Helper Cells and Regulatory B Cells Dynamics in Systemic Lupus Erythematosus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0088441

Expansion of circulating Breg cells in SLE patients. (A) Human PBMCs were labeled with lymphocyte-specific antibodies (CD19, CD5, and CD1d). The percentage of CD5 + CD1d high cells among a CD19 gate was determined by flow cytometry. (B) Results of flow cytometric analysis of Breg cells in patients with active SLE (n = 16), patients with inactive SLE (n = 14), and control subject (n = 15). (C) A positive correlation between the proportion of CD5 + CD1d high cells among CD19 + B cells and the clinical severity of the flare as scored using the SLEDAI (n = 30) was observed.
Figure Legend Snippet: Expansion of circulating Breg cells in SLE patients. (A) Human PBMCs were labeled with lymphocyte-specific antibodies (CD19, CD5, and CD1d). The percentage of CD5 + CD1d high cells among a CD19 gate was determined by flow cytometry. (B) Results of flow cytometric analysis of Breg cells in patients with active SLE (n = 16), patients with inactive SLE (n = 14), and control subject (n = 15). (C) A positive correlation between the proportion of CD5 + CD1d high cells among CD19 + B cells and the clinical severity of the flare as scored using the SLEDAI (n = 30) was observed.

Techniques Used: Labeling, Flow Cytometry, Cytometry

IL-10 production in Breg cells of SLE patients. (A) Real-time RT-PCR analysis of IL-10 mRNA expression in PBMCs from patients with active SLE (n = 16), patients with inactive SLE (n = 14), and control subject (n = 15). (B) Serum levels of IL-10 were detected in patients with active SLE (n = 16), patients with inactive SLE (n = 14), and control subject (n = 15) by ELISA. (C) PBMCs were isolated and stimulated with LPS for 24 hours and PIB for the final 5 hours. CD19 + IL-10 + cells were detected by flow cytometry analysis in a CD19 gate (left). Results of flow cytometric analysis of CD19 + IL-10 + cells (right, n = 6 for each group). (D) Sorted CD19 + CD5 + CD1d high B cells from SLE patients and healthy controls were stimulated with LPS for 24 hours and PIB for the last 5 hours. IL-10 mRNA expression was detected by real-time RT-PCR. Results shown are representative of at least three independent experiments. (E) Sorted CD19 + CD5 + CD1d high B cells from SLE patients and healthy controls were stimulated with LPS for 24 hours and PI for the last 5 hours. IL-10 in supernatants was detected by ELISA. Results shown are representative of at least three independent experiments.
Figure Legend Snippet: IL-10 production in Breg cells of SLE patients. (A) Real-time RT-PCR analysis of IL-10 mRNA expression in PBMCs from patients with active SLE (n = 16), patients with inactive SLE (n = 14), and control subject (n = 15). (B) Serum levels of IL-10 were detected in patients with active SLE (n = 16), patients with inactive SLE (n = 14), and control subject (n = 15) by ELISA. (C) PBMCs were isolated and stimulated with LPS for 24 hours and PIB for the final 5 hours. CD19 + IL-10 + cells were detected by flow cytometry analysis in a CD19 gate (left). Results of flow cytometric analysis of CD19 + IL-10 + cells (right, n = 6 for each group). (D) Sorted CD19 + CD5 + CD1d high B cells from SLE patients and healthy controls were stimulated with LPS for 24 hours and PIB for the last 5 hours. IL-10 mRNA expression was detected by real-time RT-PCR. Results shown are representative of at least three independent experiments. (E) Sorted CD19 + CD5 + CD1d high B cells from SLE patients and healthy controls were stimulated with LPS for 24 hours and PI for the last 5 hours. IL-10 in supernatants was detected by ELISA. Results shown are representative of at least three independent experiments.

Techniques Used: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Isolation, Flow Cytometry, Cytometry

Tfh cells are associated with expansion of Breg cells in SLE patients. (A) Human PBMCs were labeled with lymphocyte-specific antibodies (CD4, CXCR5 and PD-1). The percentage of CXCR5 + PD-1 + cells among CD4 + T cells was analyzed by flow cytometry (left). Results of flow cytometric analysis of Tfh cells (right) in patients with active SLE (n = 16), patients with inactive SLE (n = 14), and control subject (n = 15). (B) A positive correlation between the proportion of CXCR5 + PD-1 + cells among CD4 + T cells and the clinical severity of the flare as scored using the SLEDAI (n = 30) was noted. (C) A positive correlation between the proportion of CD4 + CXCR5 + PD-1 + T cells and CD19 + CD5 + CD1d high B cells in PBMCs from SLE patients (n = 30) was observed. (D) Serum IL-21 levels were detected in patients with active SLE (n = 16), patients with inactive SLE (n = 14), and control subject (n = 15) by ELISA. (E) A positive correlation between serum IL-10 levels and IL-21 levels in SLE patients (n = 30) was observed.
Figure Legend Snippet: Tfh cells are associated with expansion of Breg cells in SLE patients. (A) Human PBMCs were labeled with lymphocyte-specific antibodies (CD4, CXCR5 and PD-1). The percentage of CXCR5 + PD-1 + cells among CD4 + T cells was analyzed by flow cytometry (left). Results of flow cytometric analysis of Tfh cells (right) in patients with active SLE (n = 16), patients with inactive SLE (n = 14), and control subject (n = 15). (B) A positive correlation between the proportion of CXCR5 + PD-1 + cells among CD4 + T cells and the clinical severity of the flare as scored using the SLEDAI (n = 30) was noted. (C) A positive correlation between the proportion of CD4 + CXCR5 + PD-1 + T cells and CD19 + CD5 + CD1d high B cells in PBMCs from SLE patients (n = 30) was observed. (D) Serum IL-21 levels were detected in patients with active SLE (n = 16), patients with inactive SLE (n = 14), and control subject (n = 15) by ELISA. (E) A positive correlation between serum IL-10 levels and IL-21 levels in SLE patients (n = 30) was observed.

Techniques Used: Labeling, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

12) Product Images from "Human milk miRNAs primarily originate from the mammary gland resulting in unique miRNA profiles of fractionated milk"

Article Title: Human milk miRNAs primarily originate from the mammary gland resulting in unique miRNA profiles of fractionated milk

Journal: Scientific Reports

doi: 10.1038/srep20680

RNA enriched in miRNA in HM cells and fat, and maternal PBMCs and plasma, and associations with HM components. (A , B) RNA concentration of HM cells, PBMCs, HM fat, and plasma, obtained with NanoDrop 2000 (N) and the Bioanalyzer 2100 (B). (C,D) RNA integrity measured by the Bioanalyzer 2100, and RNA purity (260/280 ratio) using NanoDrop 2000 in all four sample groups. (E,F) Associations between total RNA eenriched in miRNA and HM cell content or maternal blood PBMC content using Bioanalyzer 2100 and Nanodrop 2000. (G) HM fat content (%) and RNA concentration of HM fat (ng). (H,I) Associations between HM volume or maternal blood volume with the total RNA enriched in miRNA. (J) Association between PBMC content of blood and HM cell content.
Figure Legend Snippet: RNA enriched in miRNA in HM cells and fat, and maternal PBMCs and plasma, and associations with HM components. (A , B) RNA concentration of HM cells, PBMCs, HM fat, and plasma, obtained with NanoDrop 2000 (N) and the Bioanalyzer 2100 (B). (C,D) RNA integrity measured by the Bioanalyzer 2100, and RNA purity (260/280 ratio) using NanoDrop 2000 in all four sample groups. (E,F) Associations between total RNA eenriched in miRNA and HM cell content or maternal blood PBMC content using Bioanalyzer 2100 and Nanodrop 2000. (G) HM fat content (%) and RNA concentration of HM fat (ng). (H,I) Associations between HM volume or maternal blood volume with the total RNA enriched in miRNA. (J) Association between PBMC content of blood and HM cell content.

Techniques Used: Concentration Assay

13) Product Images from "The role of Card9 overexpression in peripheral blood mononuclear cells from patients with aseptic acute pancreatitis"

Article Title: The role of Card9 overexpression in peripheral blood mononuclear cells from patients with aseptic acute pancreatitis

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.12738

Immunoprecipitation analysis and mRNA levels in PBMC s. ( A ) Card9m RNA expression; ( B ) Bcl10 mRNA expression; ( C ) immunoprecipitation of Card9‐Bcl10 complex (input: 1, 4, 7, 10, 13, 16; anti‐Bcl‐10 immunoprecipitated complex: 2, 5, 8; anti‐Card9 immunoprecipitated complex: 11, 14, 17; lank control: 3, 6, 9, 12, 15, 18). Immunoprecipitation analysis was representative from 20 healthy volunteers, 35 MAP and 17 SAP patients. PCR results were means ± S.D. of measurement on day 1, 3 and 5 (n controlled group = 20, n MAP patients = 17, n SAP patients = 35). a: control versus SAP . MAP , P
Figure Legend Snippet: Immunoprecipitation analysis and mRNA levels in PBMC s. ( A ) Card9m RNA expression; ( B ) Bcl10 mRNA expression; ( C ) immunoprecipitation of Card9‐Bcl10 complex (input: 1, 4, 7, 10, 13, 16; anti‐Bcl‐10 immunoprecipitated complex: 2, 5, 8; anti‐Card9 immunoprecipitated complex: 11, 14, 17; lank control: 3, 6, 9, 12, 15, 18). Immunoprecipitation analysis was representative from 20 healthy volunteers, 35 MAP and 17 SAP patients. PCR results were means ± S.D. of measurement on day 1, 3 and 5 (n controlled group = 20, n MAP patients = 17, n SAP patients = 35). a: control versus SAP . MAP , P

Techniques Used: Immunoprecipitation, RNA Expression, Expressing, Polymerase Chain Reaction

14) Product Images from "Fluctuations in Blood Marginal Zone B-Cell Frequencies May Reflect Migratory Patterns Associated with HIV-1 Disease Progression Status"

Article Title: Fluctuations in Blood Marginal Zone B-Cell Frequencies May Reflect Migratory Patterns Associated with HIV-1 Disease Progression Status

Journal: PLoS ONE

doi: 10.1371/journal.pone.0155868

Flow-Cytometry gating strategy for analysis of blood B-cell populations of HIV-1 infected individuals. Representative plot showing gating strategy on 105 live PBMCs. Total CD19+ B-cells were selected based on expression of CD27 and/or IgM, and levels of CD21. CD1c and CD10 expression were used for further characterization of blood MZ and TI B-cell populations. Quadrants were set based on the expression values obtained with fluorescence minus one (FMO) and isotype controls. Mature activated B-cells were defined as CD19+CD27+IgM-CD21loCD1c-CD10-, resting switched memory B-cells were CD19+CD27+IgM-CD21hiCD10-, precursor marginal-zone (MZ)-like B-cells were CD19+CD27+IgM+CD21loCD1c+CD10+, mature MZ-like B-cells were CD19+CD27+IgM+CD21hiCD1c+CD10- and transitional immature (TI) B-cells were CD19+CD27-IgM+CD21hiCD1c-CD10+. The mean events gated were: total B-cells (9320 ± 1750), mature activated (360 ± 67), resting switched memory (632 ± 301), precursor MZ-like (145 ± 36) mature MZ-like (327 ± 233) and TI (944 ± 174).
Figure Legend Snippet: Flow-Cytometry gating strategy for analysis of blood B-cell populations of HIV-1 infected individuals. Representative plot showing gating strategy on 105 live PBMCs. Total CD19+ B-cells were selected based on expression of CD27 and/or IgM, and levels of CD21. CD1c and CD10 expression were used for further characterization of blood MZ and TI B-cell populations. Quadrants were set based on the expression values obtained with fluorescence minus one (FMO) and isotype controls. Mature activated B-cells were defined as CD19+CD27+IgM-CD21loCD1c-CD10-, resting switched memory B-cells were CD19+CD27+IgM-CD21hiCD10-, precursor marginal-zone (MZ)-like B-cells were CD19+CD27+IgM+CD21loCD1c+CD10+, mature MZ-like B-cells were CD19+CD27+IgM+CD21hiCD1c+CD10- and transitional immature (TI) B-cells were CD19+CD27-IgM+CD21hiCD1c-CD10+. The mean events gated were: total B-cells (9320 ± 1750), mature activated (360 ± 67), resting switched memory (632 ± 301), precursor MZ-like (145 ± 36) mature MZ-like (327 ± 233) and TI (944 ± 174).

Techniques Used: Flow Cytometry, Cytometry, Infection, Expressing, Fluorescence

15) Product Images from "CGG Repeat-Induced FMR1 Silencing Depends on the Expansion Size in Human iPSCs and Neurons Carrying Unmethylated Full Mutations"

Article Title: CGG Repeat-Induced FMR1 Silencing Depends on the Expansion Size in Human iPSCs and Neurons Carrying Unmethylated Full Mutations

Journal: Stem Cell Reports

doi: 10.1016/j.stemcr.2016.10.004

Increased Silencing Threshold in iPSC Clones Derived from UFM Subjects (A) CGG repeat size in 5′ UTR of FMR1 analyzed by PCR in iPSCs derived from PBMCs from UFM1 and UFM2. Background color of a clone name indicates expression of FMR1 based on data in (B), expressing (blue), not expressing, or expressing below 5% of WT level (red). Circles under the clone names indicate mean percentage of DNA methylation across 22 CpGs of the FMR1 promoter based on data in Table S1 . Silent and fully methylated clones are observed in both UFM subjects. Yellow lines indicate the proposed approximate thresholds of CGG repeat numbers triggering FMR1 silencing in a given individual. The grey line corresponds to 200 CGG repeats. See also Figure S2 . (B) Expression of FMR1 mRNA in iPSC clones from PBMCs from UFM1 and UFM2 analyzed by TaqMan assay. WT iPSC clone 86-14 derived from a normal individual is included as a WT reference. Data are presented as a mean of three independent biological replicates. Error bars represent SD.
Figure Legend Snippet: Increased Silencing Threshold in iPSC Clones Derived from UFM Subjects (A) CGG repeat size in 5′ UTR of FMR1 analyzed by PCR in iPSCs derived from PBMCs from UFM1 and UFM2. Background color of a clone name indicates expression of FMR1 based on data in (B), expressing (blue), not expressing, or expressing below 5% of WT level (red). Circles under the clone names indicate mean percentage of DNA methylation across 22 CpGs of the FMR1 promoter based on data in Table S1 . Silent and fully methylated clones are observed in both UFM subjects. Yellow lines indicate the proposed approximate thresholds of CGG repeat numbers triggering FMR1 silencing in a given individual. The grey line corresponds to 200 CGG repeats. See also Figure S2 . (B) Expression of FMR1 mRNA in iPSC clones from PBMCs from UFM1 and UFM2 analyzed by TaqMan assay. WT iPSC clone 86-14 derived from a normal individual is included as a WT reference. Data are presented as a mean of three independent biological replicates. Error bars represent SD.

Techniques Used: Clone Assay, Derivative Assay, Polymerase Chain Reaction, Expressing, DNA Methylation Assay, Methylation, TaqMan Assay

16) Product Images from "Plasmodium vivax: Induction of CD4+CD25+FoxP3+ Regulatory T Cells during Infection Are Directly Associated with Level of Circulating Parasites"

Article Title: Plasmodium vivax: Induction of CD4+CD25+FoxP3+ Regulatory T Cells during Infection Are Directly Associated with Level of Circulating Parasites

Journal: PLoS ONE

doi: 10.1371/journal.pone.0009623

Indirect suppression elicited by CD4 + CD25 + T regulatory cells from P. vivax -infected individuals. (A) CFDA-SE Proliferation ratio of Pv-AMA-1- stimulated PBMCs (sPBMCs) and sPBMCs co-cultured with different proportions of autologous CD4 + CD25 + lymphocytes (1∶2, 1∶5 and 1∶10, CD4 + CD25 + cells: sPBMCs). Results are expressed for two malaria-infected donors who presented positive proliferative response after Pv-AMA-1 stimulation. (B) Representative FACS histogram plots for 1 out 2 donors with positive proliferative response after Pv-AMA-1 stimulation showing CFDA-SE staining after antigen stimulation and co-culturing with CD4 + CD25 + T cells. CFDA-SE Proliferation ratio for sPBMCs was calculated by proliferative response observed in Pv-AMA-1-stimulated PBMCs (indicated by positivity for CFDA-SE) divided by basal proliferative response of non-stimulated cells (PBMCs only). CFDA-SE Proliferation ratio for co-cultured cells were calculated by proliferative response observed in Pv-AMA-1-stimulated PBMCs with CD4 + CD25 + T cells divided by proliferative response observed in Pv-AMA-1-stimulated PBMCs only.
Figure Legend Snippet: Indirect suppression elicited by CD4 + CD25 + T regulatory cells from P. vivax -infected individuals. (A) CFDA-SE Proliferation ratio of Pv-AMA-1- stimulated PBMCs (sPBMCs) and sPBMCs co-cultured with different proportions of autologous CD4 + CD25 + lymphocytes (1∶2, 1∶5 and 1∶10, CD4 + CD25 + cells: sPBMCs). Results are expressed for two malaria-infected donors who presented positive proliferative response after Pv-AMA-1 stimulation. (B) Representative FACS histogram plots for 1 out 2 donors with positive proliferative response after Pv-AMA-1 stimulation showing CFDA-SE staining after antigen stimulation and co-culturing with CD4 + CD25 + T cells. CFDA-SE Proliferation ratio for sPBMCs was calculated by proliferative response observed in Pv-AMA-1-stimulated PBMCs (indicated by positivity for CFDA-SE) divided by basal proliferative response of non-stimulated cells (PBMCs only). CFDA-SE Proliferation ratio for co-cultured cells were calculated by proliferative response observed in Pv-AMA-1-stimulated PBMCs with CD4 + CD25 + T cells divided by proliferative response observed in Pv-AMA-1-stimulated PBMCs only.

Techniques Used: Infection, Cell Culture, FACS, Staining

17) Product Images from "Identification of the MicroRNA Repertoire in TLR-Ligand Challenged Bubaline PBMCs as a Model of Bacterial and Viral Infection"

Article Title: Identification of the MicroRNA Repertoire in TLR-Ligand Challenged Bubaline PBMCs as a Model of Bacterial and Viral Infection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0156598

Box-plot showing abundance of known and novel miRNAs across TLR ligand treated and control samples. LPS stimulated PBMCs (A), poly I:C stimulated PBMCs (B), CpG ODN stimulated PBMCs (C), non-LPS-stimulated control PBMCs (D), non-poly I:C-stimulated control PBMCs (E), non-CpG stimulated control PBMCs (F). The central lines inside the boxes indicate median values, box width indicates 25% and 75% quartile ranges around the median, “T” indicates the maximum and minimum values, and the dots represent outliers.
Figure Legend Snippet: Box-plot showing abundance of known and novel miRNAs across TLR ligand treated and control samples. LPS stimulated PBMCs (A), poly I:C stimulated PBMCs (B), CpG ODN stimulated PBMCs (C), non-LPS-stimulated control PBMCs (D), non-poly I:C-stimulated control PBMCs (E), non-CpG stimulated control PBMCs (F). The central lines inside the boxes indicate median values, box width indicates 25% and 75% quartile ranges around the median, “T” indicates the maximum and minimum values, and the dots represent outliers.

Techniques Used:

Heatmap of known miRNA expression across the TLR stimulated and control samples. LPS stimulated PBMCs (A), non-LPS-stimulated control PBMCs (B), poly I:C stimulated PBMCs (C), non-poly I:C-stimulated control PBMCs (D), CpG ODN stimulated PBMCs (E), non-CpG stimulated control PBMCs (F). The color scale bar (white-orange-red) indicate increasing expression levels (low-medium-high).
Figure Legend Snippet: Heatmap of known miRNA expression across the TLR stimulated and control samples. LPS stimulated PBMCs (A), non-LPS-stimulated control PBMCs (B), poly I:C stimulated PBMCs (C), non-poly I:C-stimulated control PBMCs (D), CpG ODN stimulated PBMCs (E), non-CpG stimulated control PBMCs (F). The color scale bar (white-orange-red) indicate increasing expression levels (low-medium-high).

Techniques Used: Expressing

The sample dendrogram and expression profiles of known and novel miRNAs across TLR stimulated and control samples. CpG ODN stimulated PBMCs (A), non-LPS-stimulated control PBMCs (B), non-CpG stimulated control PBMCs (C), poly I:C stimulated PBMCs (D), LPS stimulated PBMCs (E), non-poly I:C-stimulated control PBMCs (F).
Figure Legend Snippet: The sample dendrogram and expression profiles of known and novel miRNAs across TLR stimulated and control samples. CpG ODN stimulated PBMCs (A), non-LPS-stimulated control PBMCs (B), non-CpG stimulated control PBMCs (C), poly I:C stimulated PBMCs (D), LPS stimulated PBMCs (E), non-poly I:C-stimulated control PBMCs (F).

Techniques Used: Expressing

Heatmap of novel miRNA expression across the TLR stimulated and control samples. LPS stimulated PBMCs (A), non-LPS-stimulated control PBMCs (B), poly I:C stimulated PBMCs (C), non-poly I:C-stimulated control PBMCs (D), CpG ODN stimulated PBMCs (E), non-CpG stimulated control PBMCs (F). The color scale bar (white-orange-red) indicate increasing expression levels (low-medium-high).
Figure Legend Snippet: Heatmap of novel miRNA expression across the TLR stimulated and control samples. LPS stimulated PBMCs (A), non-LPS-stimulated control PBMCs (B), poly I:C stimulated PBMCs (C), non-poly I:C-stimulated control PBMCs (D), CpG ODN stimulated PBMCs (E), non-CpG stimulated control PBMCs (F). The color scale bar (white-orange-red) indicate increasing expression levels (low-medium-high).

Techniques Used: Expressing

18) Product Images from "Natural-Killer-Derived Extracellular Vesicles: Immune Sensors and Interactors"

Article Title: Natural-Killer-Derived Extracellular Vesicles: Immune Sensors and Interactors

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.00262

Effects of natural killer cell-derived extracellular vesicles (NKEVs) on monocytes. (A) Flow cytometry analysis of CD80–CD86 geo mean fluorescence intensity (gMFI) of gated CD14 + cells in peripheral blood mononuclear cells (PBMCs) cultured in the presence or absence of NK-cell-derived microvesicles (NKMV), NKExo, and/or lipopolysacharride (LPS) for 24 h. Upper panels: representative dot plots showing CD80–CD86 expression in the presence of NKMV and NKExo, lower panel: graphical summary of different healthy donors. (B) Flow cytometry of human leukocyte antigen DR isotype (HLA-DR) gMFI of CD14 + gated monocytes as in (A) . Results for different donors ( n = 3) are shown. (C) Effects of NKEVs on isolated monocytes, measured by flow cytometry after 24 h culture of CD14 + cells with NKMV or NKExo. Left panels: gMFI of CD80-86 and HLA-DR expression by monocytes of one healthy donor. Right panel: results from different healthy donors ( n = 3). (D) Stimulatory potential of monocytes preconditioned with NKMV and NKExo. Left panels: flow cytometry analysis of 72 h proliferation and CD25 expression by CD3, CD4, and CD8 T cells cultured in the presence of monocytes (medium), monocytes preconditioned with NKMV or NKExo. Right panels: graphical summary showing results from different healthy donors ( n = 3). Percentage of proliferation is indicated. Statistical significance was achieved by paired t test. Means ± SD are plotted, * p
Figure Legend Snippet: Effects of natural killer cell-derived extracellular vesicles (NKEVs) on monocytes. (A) Flow cytometry analysis of CD80–CD86 geo mean fluorescence intensity (gMFI) of gated CD14 + cells in peripheral blood mononuclear cells (PBMCs) cultured in the presence or absence of NK-cell-derived microvesicles (NKMV), NKExo, and/or lipopolysacharride (LPS) for 24 h. Upper panels: representative dot plots showing CD80–CD86 expression in the presence of NKMV and NKExo, lower panel: graphical summary of different healthy donors. (B) Flow cytometry of human leukocyte antigen DR isotype (HLA-DR) gMFI of CD14 + gated monocytes as in (A) . Results for different donors ( n = 3) are shown. (C) Effects of NKEVs on isolated monocytes, measured by flow cytometry after 24 h culture of CD14 + cells with NKMV or NKExo. Left panels: gMFI of CD80-86 and HLA-DR expression by monocytes of one healthy donor. Right panel: results from different healthy donors ( n = 3). (D) Stimulatory potential of monocytes preconditioned with NKMV and NKExo. Left panels: flow cytometry analysis of 72 h proliferation and CD25 expression by CD3, CD4, and CD8 T cells cultured in the presence of monocytes (medium), monocytes preconditioned with NKMV or NKExo. Right panels: graphical summary showing results from different healthy donors ( n = 3). Percentage of proliferation is indicated. Statistical significance was achieved by paired t test. Means ± SD are plotted, * p

Techniques Used: Derivative Assay, Flow Cytometry, Fluorescence, Cell Culture, Expressing, Isolation

Effects of natural killer cell-derived extracellular vesicles (NKEVs) on T cells. (A) Flow cytometry analysis of CD25 expression by CD3 + gated T cells in peripheral blood mononuclear cells (PBMCs) evaluated after 72 h of culture with NK-cell-derived microvesicle (NKMV) and NKExo. Left panels: representative dot plots of a healthy donor. Right panel: the graph shows the results obtained with PBMCs of different healthy donors ( n = 3), in the presence or absence of transforming growth factor beta (TGFβ)/interleukin (IL)-10 (10 ng/ml each). (B) Cytometric bead array (CBA)-measured cytokine production of 72 h PBMCs cultured as described in (A) . (C) Geo mean fluorescence intensity (gMFI) of CD25 expression of CD3 + gated T cells in CD3/CD28 activated PBMCs in the presence or absence of NKMV and NKExo and/or TGFβ/IL-10 (10 ng/ml each). (D) PD-1 gMFI on gated CD3 + T cells as in (C) . (E) Pearson correlation analysis of PD-1 gMFI and GRZB production. Results are shown for different healthy donors ( n = 3). Statistical significance was achieved by paired t -test. Means ± SD are plotted, * p
Figure Legend Snippet: Effects of natural killer cell-derived extracellular vesicles (NKEVs) on T cells. (A) Flow cytometry analysis of CD25 expression by CD3 + gated T cells in peripheral blood mononuclear cells (PBMCs) evaluated after 72 h of culture with NK-cell-derived microvesicle (NKMV) and NKExo. Left panels: representative dot plots of a healthy donor. Right panel: the graph shows the results obtained with PBMCs of different healthy donors ( n = 3), in the presence or absence of transforming growth factor beta (TGFβ)/interleukin (IL)-10 (10 ng/ml each). (B) Cytometric bead array (CBA)-measured cytokine production of 72 h PBMCs cultured as described in (A) . (C) Geo mean fluorescence intensity (gMFI) of CD25 expression of CD3 + gated T cells in CD3/CD28 activated PBMCs in the presence or absence of NKMV and NKExo and/or TGFβ/IL-10 (10 ng/ml each). (D) PD-1 gMFI on gated CD3 + T cells as in (C) . (E) Pearson correlation analysis of PD-1 gMFI and GRZB production. Results are shown for different healthy donors ( n = 3). Statistical significance was achieved by paired t -test. Means ± SD are plotted, * p

Techniques Used: Derivative Assay, Flow Cytometry, Expressing, Crocin Bleaching Assay, Cell Culture, Fluorescence

Effects of natural killer cell-derived extracellular vesicles (NKEVs) on NK cells. (A) Upper panels: representative dot plots showing CD56 + total NK cells in the CD3 negative lymphocyte gate after culturing PBMCs for 72 h in the presence or absence of NK-cell-derived microvesicle (NKMV) and NKExo and/or transforming growth factor beta (TGFβ)/interleukin (IL)-10 (10 ng/ml each). Lower panels: representative dot plots showing the percentage of NK cell subpopulations in the CD56 + gate for each culture condition as in upper panels: CD56 bright , CD56 dim CD16 + , and CD56 dim CD16 neg NK cells. (B) Graphs show the results obtained for total NK cells and each subpopulation shown in (A) for different healthy donors ( n = 3). (C) Graphs show total NK cells and NK subpopulations after 72 h culture of the CD14 negative fraction in the presence or absence of NKMV or NKExo and/or CD3/CD28 stimulation. Results were obtained with cells from different donors ( n = 3). Statistical significance was achieved by paired t test. Means ± SD are plotted, * p
Figure Legend Snippet: Effects of natural killer cell-derived extracellular vesicles (NKEVs) on NK cells. (A) Upper panels: representative dot plots showing CD56 + total NK cells in the CD3 negative lymphocyte gate after culturing PBMCs for 72 h in the presence or absence of NK-cell-derived microvesicle (NKMV) and NKExo and/or transforming growth factor beta (TGFβ)/interleukin (IL)-10 (10 ng/ml each). Lower panels: representative dot plots showing the percentage of NK cell subpopulations in the CD56 + gate for each culture condition as in upper panels: CD56 bright , CD56 dim CD16 + , and CD56 dim CD16 neg NK cells. (B) Graphs show the results obtained for total NK cells and each subpopulation shown in (A) for different healthy donors ( n = 3). (C) Graphs show total NK cells and NK subpopulations after 72 h culture of the CD14 negative fraction in the presence or absence of NKMV or NKExo and/or CD3/CD28 stimulation. Results were obtained with cells from different donors ( n = 3). Statistical significance was achieved by paired t test. Means ± SD are plotted, * p

Techniques Used: Derivative Assay

19) Product Images from "Human CD8 T cells are susceptible to TNF-mediated activation-induced cell death"

Article Title: Human CD8 T cells are susceptible to TNF-mediated activation-induced cell death

Journal: Theranostics

doi: 10.7150/thno.41646

TNFR2 stimulation on human CD8 T cells increases ROS production via mitochondrial hyperpolarization and causes DNA damage. PBMCs from healthy donors were activated with plate-bound anti-CD3 and soluble anti-CD28 mAbs for seven days in the presence or absence of blocking TNF agents. Flow cytometry assessment of intracellular ROS geometric mean fluorescence in FACs-gated CD8 T cells stimulated in the presence or absence of etanercept or infliximab (A) , or selective blocking antibodies against TNFR1 or TNFR2 (B) (n=8). C , Flow cytometry measurement of TMRM geometric mean fluorescence of FACs-gated CD8 T cells that were stimulated in the presence or absence of TNF blocking agents (n=8). DNA damage was measured by intracellular staining of phosphorylated gH2AX on FACs gated CD8 T cells in the presence or absence of etanercept (n=17) or infliximab (n=14) (D) , or selective blocking antibodies against TNFR1 or TNFR2 (E) (n=12). CD8 T cells were pre-gated on Live+/CD3+/CD4-. Data for A-C are mean ± s.e.m. and P values were calculated using one-way ANOVA with Tukey's multiple comparsisions test. In D and E, the samples were compared by a paired two-tailed t-test.
Figure Legend Snippet: TNFR2 stimulation on human CD8 T cells increases ROS production via mitochondrial hyperpolarization and causes DNA damage. PBMCs from healthy donors were activated with plate-bound anti-CD3 and soluble anti-CD28 mAbs for seven days in the presence or absence of blocking TNF agents. Flow cytometry assessment of intracellular ROS geometric mean fluorescence in FACs-gated CD8 T cells stimulated in the presence or absence of etanercept or infliximab (A) , or selective blocking antibodies against TNFR1 or TNFR2 (B) (n=8). C , Flow cytometry measurement of TMRM geometric mean fluorescence of FACs-gated CD8 T cells that were stimulated in the presence or absence of TNF blocking agents (n=8). DNA damage was measured by intracellular staining of phosphorylated gH2AX on FACs gated CD8 T cells in the presence or absence of etanercept (n=17) or infliximab (n=14) (D) , or selective blocking antibodies against TNFR1 or TNFR2 (E) (n=12). CD8 T cells were pre-gated on Live+/CD3+/CD4-. Data for A-C are mean ± s.e.m. and P values were calculated using one-way ANOVA with Tukey's multiple comparsisions test. In D and E, the samples were compared by a paired two-tailed t-test.

Techniques Used: Blocking Assay, Flow Cytometry, Fluorescence, FACS, Staining, Two Tailed Test

20) Product Images from "Expression of miR‐18a and miR‐34c in circulating monocytes associated with vulnerability to type 2 diabetes mellitus and insulin resistance"

Article Title: Expression of miR‐18a and miR‐34c in circulating monocytes associated with vulnerability to type 2 diabetes mellitus and insulin resistance

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.13240

Comparison of micro RNA expression in PBMC s among T2 DM , IFG and control groups.
Figure Legend Snippet: Comparison of micro RNA expression in PBMC s among T2 DM , IFG and control groups.

Techniques Used: RNA Expression

21) Product Images from "Synovial fluid CD69+CD8+ T cells with tissue‐resident phenotype mediate perforin‐dependent citrullination in rheumatoid arthritis"

Article Title: Synovial fluid CD69+CD8+ T cells with tissue‐resident phenotype mediate perforin‐dependent citrullination in rheumatoid arthritis

Journal: Clinical & Translational Immunology

doi: 10.1002/cti2.1140

CD69 + CD103 + /‐ CD45RA ‐ CD8 + T cells are increased in the synovial fluid of RA patients. ( a ) The frequency of CD8 + T cells within the lymphocyte population in peripheral mononuclear cells (PBMCs) from healthy controls (HCs; n = 9) and rheumatoid arthritis (RA) patients ( n = 13) and in synovial fluid mononuclear cells (SFMCs) from RA patients ( n = 60). ( b ) Left: Representative histograms of CD45RA expression in CD8 + T cells among PBMCs from HCs and RA patients and SFMCs from RA patients. Right: Dot plot graph depicting the frequency of CD45RA ‐ CD8 + T cells among PBMCs from HCs ( n = 9) and RA patients ( n = 13) and SFMCs from RA patients ( n = 60). ( c ) Left: Representative flow cytometry plots for CD69 and CD103 expression in CD45RA ‐ CD8 + T cells among PBMCs from HCs or RA patients and SFMCs from RA patients. Right: The frequencies of CD69 + CD103 ‐ CD45RA ‐ and CD69 + CD103 + CD45RA ‐ CD8 + T cells among PBMCs from HCs ( n = 9) and RA patients ( n = 13) and SFMCs from RA patients ( n = 60). ( d ) The proportions of CD45RA + CCR7 + (naive), CD45RA ‐ CCR7 + (central memory), CD45RA ‐ CCR7 ‐ (effector memory) and CD45RA + CCR7 ‐ (CD45RA + effector memory) cells among CD69 ‐ CD103 ‐ , CD69 + CD103 ‐ and CD69 + CD103 + CD8 + T cells from the synovial fluid in patients with RA. ( e ) Representative images of the immunohistochemical staining in synovial tissues from patients with RA and osteoarthritis (OA); red‐coloured cells represent CD8, white represent CD69, and green represent CD103. White arrows indicate CD69 + CD103 + CD8 + cells, and red arrows indicate CD69 + CD103 ‐ CD8 + cells. Scale bars = 20μm. Statistical test: one‐way ANOVA with Tukey’s multiple comparisons test; ** P ‐value
Figure Legend Snippet: CD69 + CD103 + /‐ CD45RA ‐ CD8 + T cells are increased in the synovial fluid of RA patients. ( a ) The frequency of CD8 + T cells within the lymphocyte population in peripheral mononuclear cells (PBMCs) from healthy controls (HCs; n = 9) and rheumatoid arthritis (RA) patients ( n = 13) and in synovial fluid mononuclear cells (SFMCs) from RA patients ( n = 60). ( b ) Left: Representative histograms of CD45RA expression in CD8 + T cells among PBMCs from HCs and RA patients and SFMCs from RA patients. Right: Dot plot graph depicting the frequency of CD45RA ‐ CD8 + T cells among PBMCs from HCs ( n = 9) and RA patients ( n = 13) and SFMCs from RA patients ( n = 60). ( c ) Left: Representative flow cytometry plots for CD69 and CD103 expression in CD45RA ‐ CD8 + T cells among PBMCs from HCs or RA patients and SFMCs from RA patients. Right: The frequencies of CD69 + CD103 ‐ CD45RA ‐ and CD69 + CD103 + CD45RA ‐ CD8 + T cells among PBMCs from HCs ( n = 9) and RA patients ( n = 13) and SFMCs from RA patients ( n = 60). ( d ) The proportions of CD45RA + CCR7 + (naive), CD45RA ‐ CCR7 + (central memory), CD45RA ‐ CCR7 ‐ (effector memory) and CD45RA + CCR7 ‐ (CD45RA + effector memory) cells among CD69 ‐ CD103 ‐ , CD69 + CD103 ‐ and CD69 + CD103 + CD8 + T cells from the synovial fluid in patients with RA. ( e ) Representative images of the immunohistochemical staining in synovial tissues from patients with RA and osteoarthritis (OA); red‐coloured cells represent CD8, white represent CD69, and green represent CD103. White arrows indicate CD69 + CD103 + CD8 + cells, and red arrows indicate CD69 + CD103 ‐ CD8 + cells. Scale bars = 20μm. Statistical test: one‐way ANOVA with Tukey’s multiple comparisons test; ** P ‐value

Techniques Used: Expressing, Flow Cytometry, Immunohistochemistry, Staining

22) Product Images from "Dissecting the mechanisms involved in anti-human T-lymphocyte immunoglobulin (ATG)-induced tolerance in the setting of allogeneic stem cell transplantation - potential implications for graft versus host disease"

Article Title: Dissecting the mechanisms involved in anti-human T-lymphocyte immunoglobulin (ATG)-induced tolerance in the setting of allogeneic stem cell transplantation - potential implications for graft versus host disease

Journal: Oncotarget

doi: 10.18632/oncotarget.21797

Pre-treatment with SB431542 or CSA interferes with ATG-mediated Treg induction PBMCs were incubated during 48 hours with either IgG or ATG (60 µg/ml), in the absence or presence of SB431542 (20 µM) or CSA (1 µg/ml ). A. Expression of Treg markers was evaluated by flow cytometry. Percentage of CD4+ cells expressing CD25+/Foxp3, CD95, PD-1 and GITR are presented as mean of triplicates ±STDEV, ** p
Figure Legend Snippet: Pre-treatment with SB431542 or CSA interferes with ATG-mediated Treg induction PBMCs were incubated during 48 hours with either IgG or ATG (60 µg/ml), in the absence or presence of SB431542 (20 µM) or CSA (1 µg/ml ). A. Expression of Treg markers was evaluated by flow cytometry. Percentage of CD4+ cells expressing CD25+/Foxp3, CD95, PD-1 and GITR are presented as mean of triplicates ±STDEV, ** p

Techniques Used: Incubation, Expressing, Flow Cytometry, Cytometry

ATG-mediated induction of Treg phenotype is reversible PBMCs were incubated for 48 hours or 96 hours with ATG (60 µg/ml) or rabbit IgG. Alternatively, PBMCs were exposed to ATG for 48 hours, then ATG was removed, cells were washed with PBSx1 three times and re-plated in fresh medium without ATG for additional 48 hours. Cells were analyzed by flow cytometry for CD4, CD25, CD127 and FoxP3 expression. A. Representative plots demonstrating the frequency of CD4+CD25+CD127low and CD4+CD25+FoxP3+ Treg cells following the treatments. B. Percentage of CD4-positive cells co-expressing high CD25 and low CD127 (left panel), or high CD25 and high FoxP3 (right panel) presented as mean of triplicates ±STDEV (** p
Figure Legend Snippet: ATG-mediated induction of Treg phenotype is reversible PBMCs were incubated for 48 hours or 96 hours with ATG (60 µg/ml) or rabbit IgG. Alternatively, PBMCs were exposed to ATG for 48 hours, then ATG was removed, cells were washed with PBSx1 three times and re-plated in fresh medium without ATG for additional 48 hours. Cells were analyzed by flow cytometry for CD4, CD25, CD127 and FoxP3 expression. A. Representative plots demonstrating the frequency of CD4+CD25+CD127low and CD4+CD25+FoxP3+ Treg cells following the treatments. B. Percentage of CD4-positive cells co-expressing high CD25 and low CD127 (left panel), or high CD25 and high FoxP3 (right panel) presented as mean of triplicates ±STDEV (** p

Techniques Used: Incubation, Flow Cytometry, Cytometry, Expressing

Proliferative response of different T cell subsets PBMCs were stimulated with anti-CD3/anti-CD28 antibodies, in the presence of autologous ATG- or rabbit IgG-pretreated cells (at 1:1 ratio), for 5 days of incubation. Proliferation of CD8-positive, CD4-positvie or CD4/CD8-negativie cells was determined by Ki67 expression. Representative plots showing Ki67 expression in specific cell sub-populations. Suppressive potential of ATG-induced Treg cells on different sub-populations, percentage of proliferated cells is presented as mean of triplicates ±STDEV at the lower panels (* p
Figure Legend Snippet: Proliferative response of different T cell subsets PBMCs were stimulated with anti-CD3/anti-CD28 antibodies, in the presence of autologous ATG- or rabbit IgG-pretreated cells (at 1:1 ratio), for 5 days of incubation. Proliferation of CD8-positive, CD4-positvie or CD4/CD8-negativie cells was determined by Ki67 expression. Representative plots showing Ki67 expression in specific cell sub-populations. Suppressive potential of ATG-induced Treg cells on different sub-populations, percentage of proliferated cells is presented as mean of triplicates ±STDEV at the lower panels (* p

Techniques Used: Incubation, Expressing

Effect of sorted iTreg cells on T cell proliferation A. PBMCs were incubated during 48 hours with ATG (60 µg/ml), stained for CD4, CD25 and CD127, and separated into two fractions: CD4+CD25+CD127- cells (enriched with viable Treg cells) and negative fraction. Treg phenotype was confirmed by FoxP3 staining. B. Sorted enriched Treg cells and negative fraction were applied to the stimulated autologous PBMCs and proliferation was measured by CFSE staining. Percentage of proliferated cells is presented by bars as mean of triplicates ±STDEV (** p
Figure Legend Snippet: Effect of sorted iTreg cells on T cell proliferation A. PBMCs were incubated during 48 hours with ATG (60 µg/ml), stained for CD4, CD25 and CD127, and separated into two fractions: CD4+CD25+CD127- cells (enriched with viable Treg cells) and negative fraction. Treg phenotype was confirmed by FoxP3 staining. B. Sorted enriched Treg cells and negative fraction were applied to the stimulated autologous PBMCs and proliferation was measured by CFSE staining. Percentage of proliferated cells is presented by bars as mean of triplicates ±STDEV (** p

Techniques Used: Incubation, Staining

ATG treatment induces the expression of Treg markers and complement inhibitory receptors Normal PBMCs were incubated during 48 hours with ATG (60 µg/ml) or rabbit IgG. Cell surface staining for CD4, CD25, CD127, CD95, GITR, ICOS, PD-1, CD55, CD58, CD59 and intra-cellular staining for FoxP3 was performed. A. Representative plots showing CD25 and CD127 expression on gated CD4+ cells. CD4+CD25+CD127-low subset of Treg cells is enumerated. B. Representative histograms showing the expression of Treg markers and complement inhibitory receptors in the CD4+CD25+ cell subset. C. Mean fluorescence intensity (MFI) and percentage of CD4+ CD25+ cells expressing the specific markers are presented as mean of triplicates (** p
Figure Legend Snippet: ATG treatment induces the expression of Treg markers and complement inhibitory receptors Normal PBMCs were incubated during 48 hours with ATG (60 µg/ml) or rabbit IgG. Cell surface staining for CD4, CD25, CD127, CD95, GITR, ICOS, PD-1, CD55, CD58, CD59 and intra-cellular staining for FoxP3 was performed. A. Representative plots showing CD25 and CD127 expression on gated CD4+ cells. CD4+CD25+CD127-low subset of Treg cells is enumerated. B. Representative histograms showing the expression of Treg markers and complement inhibitory receptors in the CD4+CD25+ cell subset. C. Mean fluorescence intensity (MFI) and percentage of CD4+ CD25+ cells expressing the specific markers are presented as mean of triplicates (** p

Techniques Used: Expressing, Incubation, Staining, Fluorescence

23) Product Images from "Increased Complement 3a Receptor is Associated with Behcet’s disease and Vogt-Koyanagi-Harada disease"

Article Title: Increased Complement 3a Receptor is Associated with Behcet’s disease and Vogt-Koyanagi-Harada disease

Journal: Scientific Reports

doi: 10.1038/s41598-017-15740-8

Expression of C3aR and C5aR in PBMCs and monocytes from AAU patients and normal controls. The expression of C3aR and C5aR was detected by FACS in PBMCs ( A , B ) and monocytes ( C , D ) from acute anterior uveitis (AAU) (n = 12) patients and normal controls (n = 17). The Independent-sample test or Mann-Whitney U test was used to compare the difference between AAU patients and normal controls. Data are expressed as mean ± s.e.m.
Figure Legend Snippet: Expression of C3aR and C5aR in PBMCs and monocytes from AAU patients and normal controls. The expression of C3aR and C5aR was detected by FACS in PBMCs ( A , B ) and monocytes ( C , D ) from acute anterior uveitis (AAU) (n = 12) patients and normal controls (n = 17). The Independent-sample test or Mann-Whitney U test was used to compare the difference between AAU patients and normal controls. Data are expressed as mean ± s.e.m.

Techniques Used: Expressing, FACS, MANN-WHITNEY

Expression of C3aR in PBMCs from inactive BD patients and inactive VKH patients. ( A and C ) The expression of C3aR was detected by FACS in PBMCs from inactive BD patients (n = 8) and normal controls (n = 8). ( B and D ). The expression of C3aR was detected by FACS in PBMCs from inactive VKH patients (n = 8) and normal controls (n = 8).The Independent-sample test or Mann-Whitney U test was used to compare the difference between patients with inactive BD or inactive VKH and normal controls. Data are expressed as mean ± s.e.m.
Figure Legend Snippet: Expression of C3aR in PBMCs from inactive BD patients and inactive VKH patients. ( A and C ) The expression of C3aR was detected by FACS in PBMCs from inactive BD patients (n = 8) and normal controls (n = 8). ( B and D ). The expression of C3aR was detected by FACS in PBMCs from inactive VKH patients (n = 8) and normal controls (n = 8).The Independent-sample test or Mann-Whitney U test was used to compare the difference between patients with inactive BD or inactive VKH and normal controls. Data are expressed as mean ± s.e.m.

Techniques Used: Expressing, FACS, MANN-WHITNEY

Effect of serum from patients with aBD or aVKH on C3aR and C5aR expression in PBMCs. ( A , B ) PBMCs were cultured in RPMI 1640 supplemented with 10% serum that from patients with aBD (n = 7) and normal controls (n = 11) in the presence of anti-CD3/CD28 for 3 days. ( C , D ) PBMCs were cultured in RPMI 1640 supplemented with 10% serum that from patients with aVKH patients (n = 13) and normal controls (n = 18) in the presence of anti-CD3/CD28 for 3 days. The expression of C3aR and C5aR in the cultured PBMCs was detected by FACS. The Independent-sample test or Mann-Whitney U test was used to compare the difference between patients with aBD or aVKH and normal controls. Data are expressed as mean ± s.e.m.
Figure Legend Snippet: Effect of serum from patients with aBD or aVKH on C3aR and C5aR expression in PBMCs. ( A , B ) PBMCs were cultured in RPMI 1640 supplemented with 10% serum that from patients with aBD (n = 7) and normal controls (n = 11) in the presence of anti-CD3/CD28 for 3 days. ( C , D ) PBMCs were cultured in RPMI 1640 supplemented with 10% serum that from patients with aVKH patients (n = 13) and normal controls (n = 18) in the presence of anti-CD3/CD28 for 3 days. The expression of C3aR and C5aR in the cultured PBMCs was detected by FACS. The Independent-sample test or Mann-Whitney U test was used to compare the difference between patients with aBD or aVKH and normal controls. Data are expressed as mean ± s.e.m.

Techniques Used: Expressing, Cell Culture, FACS, MANN-WHITNEY

Expression of C3aR and C5aR in PBMCs and monocytes from patients with aBD and aVKH. ( A and D ) The expression of C3aR and C5aR was detected by FACS in PBMCs from patients with aBD (n = 5) and normal controls (n = 8). ( B , C and E , F ) The expression of of C3aR and C5aR was detected by FACS in PBMCs and monocytes from patients with aVKH (n = 18) and normal controls (n = 23). ( G , H ) Representative figures of the flow cytometric analyses of C3aR between aBD or aVKH and normal controls. Results are expressed as percentage difference compared with isotypic control. The Independent-sample test or Mann-Whitney U test was used to assess the difference of C3aR and C5aR level between patients with aBD or aVKH and normal controls. Data are expressed as mean ± s.e.m.
Figure Legend Snippet: Expression of C3aR and C5aR in PBMCs and monocytes from patients with aBD and aVKH. ( A and D ) The expression of C3aR and C5aR was detected by FACS in PBMCs from patients with aBD (n = 5) and normal controls (n = 8). ( B , C and E , F ) The expression of of C3aR and C5aR was detected by FACS in PBMCs and monocytes from patients with aVKH (n = 18) and normal controls (n = 23). ( G , H ) Representative figures of the flow cytometric analyses of C3aR between aBD or aVKH and normal controls. Results are expressed as percentage difference compared with isotypic control. The Independent-sample test or Mann-Whitney U test was used to assess the difference of C3aR and C5aR level between patients with aBD or aVKH and normal controls. Data are expressed as mean ± s.e.m.

Techniques Used: Expressing, FACS, Flow Cytometry, MANN-WHITNEY

24) Product Images from "Reprogramming of Adult Peripheral Blood Cells into Human Induced Pluripotent Stem Cells as a Safe and Accessible Source of Endothelial Cells"

Article Title: Reprogramming of Adult Peripheral Blood Cells into Human Induced Pluripotent Stem Cells as a Safe and Accessible Source of Endothelial Cells

Journal: Stem Cells and Development

doi: 10.1089/scd.2017.0132

Detection of Ig and TCR gene recombinations in hiPSC-PB. Genomic DNA from PBMCs is provided as a positive control, while fibroblast line (HDFn) serves as a negative control. Sixty-three primers were used in seven multiplex PCR tubes. (A) Complete VH-JH rearrangement of IGH gene. Three tubes were used with valid sizes of amplicon (1) 310–360 bp, (2) 250–295 bp, and (3) 100–170 bp. (B) Complete Vβ-Jβ rearrangement of TRB gene. Two tubes were used with valid sizes (4) 240–285 bp and (5) 240–285 bp. (C) TRG gene rearrangements. Two tubes were used with valid sizes (6) 145–255 bp and (7) 80–220 bp. MwM, molecular weight marker. HDFn, human dermal neonatal fibroblast; hiPSC-PB, hiPSC derived from PBMCs; Ig, immunoglobulin; PCR, polymerase chain reaction; TCR, T-cell receptor.
Figure Legend Snippet: Detection of Ig and TCR gene recombinations in hiPSC-PB. Genomic DNA from PBMCs is provided as a positive control, while fibroblast line (HDFn) serves as a negative control. Sixty-three primers were used in seven multiplex PCR tubes. (A) Complete VH-JH rearrangement of IGH gene. Three tubes were used with valid sizes of amplicon (1) 310–360 bp, (2) 250–295 bp, and (3) 100–170 bp. (B) Complete Vβ-Jβ rearrangement of TRB gene. Two tubes were used with valid sizes (4) 240–285 bp and (5) 240–285 bp. (C) TRG gene rearrangements. Two tubes were used with valid sizes (6) 145–255 bp and (7) 80–220 bp. MwM, molecular weight marker. HDFn, human dermal neonatal fibroblast; hiPSC-PB, hiPSC derived from PBMCs; Ig, immunoglobulin; PCR, polymerase chain reaction; TCR, T-cell receptor.

Techniques Used: Positive Control, Negative Control, Multiplex Assay, Polymerase Chain Reaction, Amplification, Molecular Weight, Marker, Derivative Assay

hiPSC reprogramming and characterization. (A) Experimental timeline for the reprogramming of PBMCs and ECs into hiPSCs. Cell morphology observed during the reprogramming of PBMCs at days 4, 9, and 17 (scale bar = 100 μm). (B) Immunofluorescence staining for the pluripotency markers Oct3/4, Sox2, and Nanog in hiPSCs (scale bar = 200 μm). (C) Flow cytometry-based detection of the pluripotency markers SSEA-4, Tra-1-60, and Tra-1-81 in hiPSCs. (D) Teratoma formation in immunodeficient mice after transplantation of hiPSCs; teratomas contained tissues from all three germ layers. ECs, endothelial cells; hiPSC, human induced pluripotent stem cell; PBMCs, peripheral blood mononuclear cells. Color images are available online at www.libeberpub.com/scd
Figure Legend Snippet: hiPSC reprogramming and characterization. (A) Experimental timeline for the reprogramming of PBMCs and ECs into hiPSCs. Cell morphology observed during the reprogramming of PBMCs at days 4, 9, and 17 (scale bar = 100 μm). (B) Immunofluorescence staining for the pluripotency markers Oct3/4, Sox2, and Nanog in hiPSCs (scale bar = 200 μm). (C) Flow cytometry-based detection of the pluripotency markers SSEA-4, Tra-1-60, and Tra-1-81 in hiPSCs. (D) Teratoma formation in immunodeficient mice after transplantation of hiPSCs; teratomas contained tissues from all three germ layers. ECs, endothelial cells; hiPSC, human induced pluripotent stem cell; PBMCs, peripheral blood mononuclear cells. Color images are available online at www.libeberpub.com/scd

Techniques Used: Immunofluorescence, Staining, Flow Cytometry, Cytometry, Mouse Assay, Transplantation Assay

25) Product Images from "Toll-like receptor 2 induced cytotoxic T-lymphocyte-associated protein 4 regulates Aspergillus-induced regulatory T-cells with pro-inflammatory characteristics"

Article Title: Toll-like receptor 2 induced cytotoxic T-lymphocyte-associated protein 4 regulates Aspergillus-induced regulatory T-cells with pro-inflammatory characteristics

Journal: Scientific Reports

doi: 10.1038/s41598-017-11738-4

CTLA4 regulates Aspergillus -induced regulatory T-cells with aT H 17-like phenotype. ( A ) Percentage of CTLA4 expressing CD4 + T-cells and Mean fluorescence intensity (MFI) of CTLA4 staining following stimulation of human PBMCs (n = 13 donors) with heat-inactivated A. fumigatus conidia (1 × 10 7 /mL), 10 µg/mL P3C or a combination of both. ( B ) IL-17A concentration in culture supernatants of human PBMCs (n = 6 donors) stimulated with heat-inactivated A. fumigatus conidia (1 × 10 7 /mL) after 1 hour pre-incubation with 24 µg/mL Abatacept, or CoIgG. ( C ) Regulatory T-cell (CD25 + FoxP3 + ) induction after 7 days in human PBMCs (n = 7 donors) stimulated with heat-inactivated A. fumigatus conidia (1 × 10 7 /mL) after 1 hour pre-incubation with 24 µg/mL Abatacept, or CoIgG. T H 17 cell (IL-17A + ) induction after 7 days in human PBMCs stimulated with heat- inactivated A. fumigatus conidia (1 × 10 7 /mL) after 1 hour pre-incubation with 24 µg/mL Abatacept, or CoIgG. IL-17A + regulatory T-cell induction after 7 days in human PBMCs stimulated with heat- inactivated A. fumigatus conidia (1 × 10 7 /mL) after 1 hour pre-incubation with 24 µg/mL Abatacept, or CoIgG. Data are represented as mean ± SEM. Abbreviations: P3C = Pam3Cys-SKKKK; Af = A spergillus fumigatus ; CoIgG = Control immunoglobulin G. *p-value ≤ 0.05, **p-value ≤ 0.01,***p-value ≤ 0.001.
Figure Legend Snippet: CTLA4 regulates Aspergillus -induced regulatory T-cells with aT H 17-like phenotype. ( A ) Percentage of CTLA4 expressing CD4 + T-cells and Mean fluorescence intensity (MFI) of CTLA4 staining following stimulation of human PBMCs (n = 13 donors) with heat-inactivated A. fumigatus conidia (1 × 10 7 /mL), 10 µg/mL P3C or a combination of both. ( B ) IL-17A concentration in culture supernatants of human PBMCs (n = 6 donors) stimulated with heat-inactivated A. fumigatus conidia (1 × 10 7 /mL) after 1 hour pre-incubation with 24 µg/mL Abatacept, or CoIgG. ( C ) Regulatory T-cell (CD25 + FoxP3 + ) induction after 7 days in human PBMCs (n = 7 donors) stimulated with heat-inactivated A. fumigatus conidia (1 × 10 7 /mL) after 1 hour pre-incubation with 24 µg/mL Abatacept, or CoIgG. T H 17 cell (IL-17A + ) induction after 7 days in human PBMCs stimulated with heat- inactivated A. fumigatus conidia (1 × 10 7 /mL) after 1 hour pre-incubation with 24 µg/mL Abatacept, or CoIgG. IL-17A + regulatory T-cell induction after 7 days in human PBMCs stimulated with heat- inactivated A. fumigatus conidia (1 × 10 7 /mL) after 1 hour pre-incubation with 24 µg/mL Abatacept, or CoIgG. Data are represented as mean ± SEM. Abbreviations: P3C = Pam3Cys-SKKKK; Af = A spergillus fumigatus ; CoIgG = Control immunoglobulin G. *p-value ≤ 0.05, **p-value ≤ 0.01,***p-value ≤ 0.001.

Techniques Used: Expressing, Fluorescence, Staining, Concentration Assay, Incubation

26) Product Images from "B-Cell Responses to Intramuscular Administration of a Bivalent Virus-Like Particle Human Norovirus Vaccine"

Article Title: B-Cell Responses to Intramuscular Administration of a Bivalent Virus-Like Particle Human Norovirus Vaccine

Journal: Clinical and Vaccine Immunology : CVI

doi: 10.1128/CVI.00571-16

Correlation of IgA and IgG ASC responses obtained using fresh and cryopreserved PBMCs. ASC responses at days 7 and 35 (7 days after each vaccine dose) were compared. A strong correlation between the results obtained using fresh and cryopreserved PBMCs was seen, as indicated by the Spearman correlation coefficient given in each panel. Results for ASC assays using fresh PBMCs were published previously ( 19 ).
Figure Legend Snippet: Correlation of IgA and IgG ASC responses obtained using fresh and cryopreserved PBMCs. ASC responses at days 7 and 35 (7 days after each vaccine dose) were compared. A strong correlation between the results obtained using fresh and cryopreserved PBMCs was seen, as indicated by the Spearman correlation coefficient given in each panel. Results for ASC assays using fresh PBMCs were published previously ( 19 ).

Techniques Used:

27) Product Images from "Cytomegalovirus Infection Leads to Development of High Frequencies of Cytotoxic Virus-Specific CD4+ T Cells Targeted to Vascular Endothelium"

Article Title: Cytomegalovirus Infection Leads to Development of High Frequencies of Cytotoxic Virus-Specific CD4+ T Cells Targeted to Vascular Endothelium

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005832

CMV-specific CD4+ T cells are highly cytotoxic directly ex vivo . PBMCs were stained with LIVE/DEAD fixable dye, before staining with HLA class II tetramer followed by anti-CD3, CD4 and CX3CR1. After fixation and permeabilisation Granzyme B and Perforin were stained intracellularly. (A) Pie charts represent the proportion of CD4+ EM T cells (in CMV seronegative individuals) or the TM+ population expressing the indicated markers and combinations thereof. To compare pie charts permutation analysis was performed in SPICE. (B) Expression of Granzyme B, perforin and CX3CR1 on pp65-specific (on the left) and gB-specific (on the right) T cells in relation to donor age. Spearman‘s rank correlation was used to analyse the strength of associations between variables. (C) Percentage killing of peptide-loaded target cells (HLA-matched LCLs) mediated by ex vivo separated CD4+TM+ or CD4+EM T cells following over night co-culture at effector:target (E:T) ratios indicated. All conditions were performed in triplicate; bars represent means with SEM. (D) Proportion of cells expressing NKG2D within the CD4+ EM or the CMV-specific T cell population in young, middle aged or older adults. Error bars represent medians and IQR. p-values: ** p = 0.001–0.01, *** p = 0.001–0.0001. Kruskal-Wallis test was performed with Dunn‘s multiple comparison in GraphPad Prism5 to derive p-values.
Figure Legend Snippet: CMV-specific CD4+ T cells are highly cytotoxic directly ex vivo . PBMCs were stained with LIVE/DEAD fixable dye, before staining with HLA class II tetramer followed by anti-CD3, CD4 and CX3CR1. After fixation and permeabilisation Granzyme B and Perforin were stained intracellularly. (A) Pie charts represent the proportion of CD4+ EM T cells (in CMV seronegative individuals) or the TM+ population expressing the indicated markers and combinations thereof. To compare pie charts permutation analysis was performed in SPICE. (B) Expression of Granzyme B, perforin and CX3CR1 on pp65-specific (on the left) and gB-specific (on the right) T cells in relation to donor age. Spearman‘s rank correlation was used to analyse the strength of associations between variables. (C) Percentage killing of peptide-loaded target cells (HLA-matched LCLs) mediated by ex vivo separated CD4+TM+ or CD4+EM T cells following over night co-culture at effector:target (E:T) ratios indicated. All conditions were performed in triplicate; bars represent means with SEM. (D) Proportion of cells expressing NKG2D within the CD4+ EM or the CMV-specific T cell population in young, middle aged or older adults. Error bars represent medians and IQR. p-values: ** p = 0.001–0.01, *** p = 0.001–0.0001. Kruskal-Wallis test was performed with Dunn‘s multiple comparison in GraphPad Prism5 to derive p-values.

Techniques Used: Ex Vivo, Staining, Expressing, Co-Culture Assay

Analysis of inhibitory molecule PD-1 on CMV-specific T cells. PBMCs were stained with LIVE/DEAD fixeable dye, followed by HLA class II tetramer, before staining to detect surface molecules. (A) Percentage of PD-1+ cells within T cell memory subsets defined by co-expression of CCR7 and CD45RA for the total CD4+ population (n = 53). (B) Percentage of PD-1+ cells within those subsets for the TM+ cells (n = 53). Columns represent medians with IQR. (C) The frequency of PD-1+ cells within the CD4+EM T cell subset, LLQ-, AGI- and DYS-specific T cell populations. (D) MFI of PD-1 expression on CD4+EM T cells as well as LLQ-, AGI- and DYS-specific T cells. Error bars indicate medians and IQR. p-values: * p = 0.01–0.05, ** p = 0.001–0.01. Kruskal-Wallis test was performed with Dunn‘s multiple comparison in GraphPad Prism5 to derive p-values (C and D). (E) The proportion of TM+ cells and CD4+EM cells expressing PD-1 in relation to donor age. (F) Median fluorescence intensity (MFI) of PD-1 expression on TM+ cells and CD4+EM cells correlated with donor age. Spearman‘s rank correlation was used to analyse the strength of associations between variables (D and E).
Figure Legend Snippet: Analysis of inhibitory molecule PD-1 on CMV-specific T cells. PBMCs were stained with LIVE/DEAD fixeable dye, followed by HLA class II tetramer, before staining to detect surface molecules. (A) Percentage of PD-1+ cells within T cell memory subsets defined by co-expression of CCR7 and CD45RA for the total CD4+ population (n = 53). (B) Percentage of PD-1+ cells within those subsets for the TM+ cells (n = 53). Columns represent medians with IQR. (C) The frequency of PD-1+ cells within the CD4+EM T cell subset, LLQ-, AGI- and DYS-specific T cell populations. (D) MFI of PD-1 expression on CD4+EM T cells as well as LLQ-, AGI- and DYS-specific T cells. Error bars indicate medians and IQR. p-values: * p = 0.01–0.05, ** p = 0.001–0.01. Kruskal-Wallis test was performed with Dunn‘s multiple comparison in GraphPad Prism5 to derive p-values (C and D). (E) The proportion of TM+ cells and CD4+EM cells expressing PD-1 in relation to donor age. (F) Median fluorescence intensity (MFI) of PD-1 expression on TM+ cells and CD4+EM cells correlated with donor age. Spearman‘s rank correlation was used to analyse the strength of associations between variables (D and E).

Techniques Used: Staining, Expressing, Fluorescence

28) Product Images from "M1-like monocytes are a major immunological determinant of severity in previously healthy adults with life-threatening influenza"

Article Title: M1-like monocytes are a major immunological determinant of severity in previously healthy adults with life-threatening influenza

Journal: JCI Insight

doi: 10.1172/jci.insight.91868

Monocytes in severe NRF patients are M1 like. ( A – C ) Classical, inflammatory, and patrolling monocytes in blood of mild and severe NRF and WRF patients. ( D ) Pie chart representation of classical, inflammatory, and patrolling monocyte frequencies in blood of NRF or WRF patients and healthy controls (HC) observed in A–C . Values refer to % of total monocytes. ( E ) Ex vivo expression of genes associated with M1 and M2 macrophage differentiation in CD14 + monocytes isolated from n = 10 NRF and n = 5 WRF patients with severe disease. Each gene is normalized to β-actin in the sample and then compared with the mean of the gene/β-actin of healthy controls ( n = 9) (Mann-Whitney test with Bonferroni correction for multiple testing; ** adjusted P = 0.01). ( F ) TNF-α gene expression normalized to CD14 gene expression for each of the severe NRF and WRF patients and healthy controls. ( E and F ) Asterisks refer to statistically different genes comparing NRF severe and WRF sever. ( G ) TNF-α/IL-10 gene expression. ( H ) Ratio of TNF-α/IL-10 protein expression by intracellular cytokine staining. TNF-α and IL-10 expression (as cytokine-positive cells, as proportion of CD14 + monocytes) was measured following 6 hours of LPS stimulation of PBMCs. ( I and J ) Expression of M1 (CCR7 surface staining) and M2 (CD163 surface staining) markers on monocytes, measured by flow cytometry. P values were calculated using Kruskal-Wallis test and Dunn’s multiple comparison test for F – G and Student’s t test if data were normally distributed and Mann-Whitney test if not for A – D and H – J . * P
Figure Legend Snippet: Monocytes in severe NRF patients are M1 like. ( A – C ) Classical, inflammatory, and patrolling monocytes in blood of mild and severe NRF and WRF patients. ( D ) Pie chart representation of classical, inflammatory, and patrolling monocyte frequencies in blood of NRF or WRF patients and healthy controls (HC) observed in A–C . Values refer to % of total monocytes. ( E ) Ex vivo expression of genes associated with M1 and M2 macrophage differentiation in CD14 + monocytes isolated from n = 10 NRF and n = 5 WRF patients with severe disease. Each gene is normalized to β-actin in the sample and then compared with the mean of the gene/β-actin of healthy controls ( n = 9) (Mann-Whitney test with Bonferroni correction for multiple testing; ** adjusted P = 0.01). ( F ) TNF-α gene expression normalized to CD14 gene expression for each of the severe NRF and WRF patients and healthy controls. ( E and F ) Asterisks refer to statistically different genes comparing NRF severe and WRF sever. ( G ) TNF-α/IL-10 gene expression. ( H ) Ratio of TNF-α/IL-10 protein expression by intracellular cytokine staining. TNF-α and IL-10 expression (as cytokine-positive cells, as proportion of CD14 + monocytes) was measured following 6 hours of LPS stimulation of PBMCs. ( I and J ) Expression of M1 (CCR7 surface staining) and M2 (CD163 surface staining) markers on monocytes, measured by flow cytometry. P values were calculated using Kruskal-Wallis test and Dunn’s multiple comparison test for F – G and Student’s t test if data were normally distributed and Mann-Whitney test if not for A – D and H – J . * P

Techniques Used: Ex Vivo, Expressing, Isolation, MANN-WHITNEY, Staining, Flow Cytometry, Cytometry

29) Product Images from "Annexin A1 in blood mononuclear cells from patients with coronary artery disease: Its association with inflammatory status and glucocorticoid sensitivity"

Article Title: Annexin A1 in blood mononuclear cells from patients with coronary artery disease: Its association with inflammatory status and glucocorticoid sensitivity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0174177

Correlation between surface AnxA1 expression on monocytes and glucocorticoid sensitivity. The figure shows the correlation between log transformed values of MFI values of surface and glucocorticoid sensitivity ex vivo, assessed by dexamethasone (M-7) suppression of LPS-stimulated IL-6 secretion from PBMCs (% of LPS-stimulated levels), r = 0.340, p = 0.002.
Figure Legend Snippet: Correlation between surface AnxA1 expression on monocytes and glucocorticoid sensitivity. The figure shows the correlation between log transformed values of MFI values of surface and glucocorticoid sensitivity ex vivo, assessed by dexamethasone (M-7) suppression of LPS-stimulated IL-6 secretion from PBMCs (% of LPS-stimulated levels), r = 0.340, p = 0.002.

Techniques Used: Expressing, Transformation Assay, Ex Vivo

Expression of AnxA1 mRNA in isolated PBMCs from ACS patients, SCAD patients and controls. AnxA1 mRNA expression was analyzed in PBMCs collected immediately after density gradient isolation from 31 healthy controls, 49 SCAD patients and 10 ACS patients. The AnxA1 mRNA expression is presented as arbitrary units and was calculated using the ΔΔCT method. Each sample was run in duplicates. Box-and-whisker plots show median and interquartile range. Kruskal-Wallis test was used to assess differences between all groups, p
Figure Legend Snippet: Expression of AnxA1 mRNA in isolated PBMCs from ACS patients, SCAD patients and controls. AnxA1 mRNA expression was analyzed in PBMCs collected immediately after density gradient isolation from 31 healthy controls, 49 SCAD patients and 10 ACS patients. The AnxA1 mRNA expression is presented as arbitrary units and was calculated using the ΔΔCT method. Each sample was run in duplicates. Box-and-whisker plots show median and interquartile range. Kruskal-Wallis test was used to assess differences between all groups, p

Techniques Used: Expressing, Isolation, Whisker Assay

30) Product Images from "Oviduct epithelium induces interferon-tau in bovine Day-4 embryos, which generates an anti-inflammatory response in immune cells"

Article Title: Oviduct epithelium induces interferon-tau in bovine Day-4 embryos, which generates an anti-inflammatory response in immune cells

Journal: Scientific Reports

doi: 10.1038/s41598-018-26224-8

( a ) Relative mRNA expression of candidate genes in PBMCs cultured in embryo-BOEC co-cultures medium or in BOEC cultures medium (cont). ( b ) Relative mRNA expression of candidate genes in PBMCs cultured in medium from embryos alone cultures or in culture medium without embryos (cont). Data are presented as mean ± SEM of six independent experiments performed in duplicate. * P
Figure Legend Snippet: ( a ) Relative mRNA expression of candidate genes in PBMCs cultured in embryo-BOEC co-cultures medium or in BOEC cultures medium (cont). ( b ) Relative mRNA expression of candidate genes in PBMCs cultured in medium from embryos alone cultures or in culture medium without embryos (cont). Data are presented as mean ± SEM of six independent experiments performed in duplicate. * P

Techniques Used: Expressing, Cell Culture

( a ) Relative mRNA expression of candidate genes in PBMCs cultured in IFNT-treated (50 pg/ml) BOEC medium or in BOEC medium (cont). ( b ) Relative mRNA expression of candidate genes in PBMCs cultured in fresh medium supplemented with IFNT (50 pg/ml) or in fresh medium without IFNT (cont). Data are presented as mean ± SEM of three independent experiments performed in triplicate. * P
Figure Legend Snippet: ( a ) Relative mRNA expression of candidate genes in PBMCs cultured in IFNT-treated (50 pg/ml) BOEC medium or in BOEC medium (cont). ( b ) Relative mRNA expression of candidate genes in PBMCs cultured in fresh medium supplemented with IFNT (50 pg/ml) or in fresh medium without IFNT (cont). Data are presented as mean ± SEM of three independent experiments performed in triplicate. * P

Techniques Used: Expressing, Cell Culture

( a ) Relative mRNA expression of target genes in PBMCs cultured in embryo-BOEC co-culture medium after antibody neutralization of IFNT. ( b ) Relative mRNA expression of target genes in PBMCs after antibody neutralization of IFNT (50 pg/ml). Data are presented as mean ± SEM of three independent experiments performed in triplicate. Different letters (a,b,c) above the bars for each gene denote significant differences, when compared to the control (cont).
Figure Legend Snippet: ( a ) Relative mRNA expression of target genes in PBMCs cultured in embryo-BOEC co-culture medium after antibody neutralization of IFNT. ( b ) Relative mRNA expression of target genes in PBMCs after antibody neutralization of IFNT (50 pg/ml). Data are presented as mean ± SEM of three independent experiments performed in triplicate. Different letters (a,b,c) above the bars for each gene denote significant differences, when compared to the control (cont).

Techniques Used: Expressing, Cell Culture, Co-Culture Assay, Neutralization

31) Product Images from "Characterization of the Myeloid Cell Populations’ Resident in the Porcine Palatine Tonsil"

Article Title: Characterization of the Myeloid Cell Populations’ Resident in the Porcine Palatine Tonsil

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01800

Evaluation of antigen processing, T cell stimulatory capacity and cytokine secretion profile following TLR stimulation (A) . Differential endocytosis between cell populations was evaluated with OVA-DQ-FITC by culture of lineage depleted myeloid cells for 1.5 h at 37°C (and 4°C), and FITC fluorescence was assessed by flow cytometry. Histogram shows the mean percentage of cells taking up DQ-OVA from eight pigs (each tested in triplicate), following subtraction of non-specific fluorescence (uptake at 4°C) for each cell population from three independent experiments. CD4 T cells were also assessed as a negative control. (B) Myeloid cells (APC) were sorted and peripheral blood mononuclear cells from allogeneic animals were stained with Violet CellTrace and mixed at a APC:T cell ratio of 1:10 before being culture for 5 days at 37°C. Proliferation of CD4 + CD8α − (CD4 T cells), CD4 − CD8α + (CD8 T cells), and CD4 + CD8α + (memory T cells) was evaluated by flow cytometry. A value of 100 was assigned to the population with the highest proliferation value and all other populations were compared to this value (and repeated for each pig). Data are from three separate experiments and a minimum of four different animals for each cell type. A one-way ANOVA was performed and statistical significance is described by **** p
Figure Legend Snippet: Evaluation of antigen processing, T cell stimulatory capacity and cytokine secretion profile following TLR stimulation (A) . Differential endocytosis between cell populations was evaluated with OVA-DQ-FITC by culture of lineage depleted myeloid cells for 1.5 h at 37°C (and 4°C), and FITC fluorescence was assessed by flow cytometry. Histogram shows the mean percentage of cells taking up DQ-OVA from eight pigs (each tested in triplicate), following subtraction of non-specific fluorescence (uptake at 4°C) for each cell population from three independent experiments. CD4 T cells were also assessed as a negative control. (B) Myeloid cells (APC) were sorted and peripheral blood mononuclear cells from allogeneic animals were stained with Violet CellTrace and mixed at a APC:T cell ratio of 1:10 before being culture for 5 days at 37°C. Proliferation of CD4 + CD8α − (CD4 T cells), CD4 − CD8α + (CD8 T cells), and CD4 + CD8α + (memory T cells) was evaluated by flow cytometry. A value of 100 was assigned to the population with the highest proliferation value and all other populations were compared to this value (and repeated for each pig). Data are from three separate experiments and a minimum of four different animals for each cell type. A one-way ANOVA was performed and statistical significance is described by **** p

Techniques Used: Fluorescence, Flow Cytometry, Cytometry, Negative Control, Staining

32) Product Images from "Differential Inhibition of Human Immunodeficiency Virus Type 1 in Peripheral Blood Mononuclear Cells and TZM-bl Cells by Endotoxin-Mediated Chemokine and Gamma Interferon Production"

Article Title: Differential Inhibition of Human Immunodeficiency Virus Type 1 in Peripheral Blood Mononuclear Cells and TZM-bl Cells by Endotoxin-Mediated Chemokine and Gamma Interferon Production

Journal: AIDS Research and Human Retroviruses

doi: 10.1089/aid.2009.0186

Prolonged stimulation of PBMCs with PHA-P diminishes the antiviral effect of LPS against HIV-1 NL-LucR.T2A-BaL.ecto ( A ) and the amount of cytokines released ( B ). PBMCs from two different donors were stimulated with PHA-P either overnight (filled symbols)
Figure Legend Snippet: Prolonged stimulation of PBMCs with PHA-P diminishes the antiviral effect of LPS against HIV-1 NL-LucR.T2A-BaL.ecto ( A ) and the amount of cytokines released ( B ). PBMCs from two different donors were stimulated with PHA-P either overnight (filled symbols)

Techniques Used:

33) Product Images from "Human T cell responses to Japanese encephalitis virus in health and disease"

Article Title: Human T cell responses to Japanese encephalitis virus in health and disease

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20151517

Breadth of the T cell response to peptide pools of JEV in JEV-exposed healthy donors. (A) Relative frequency of ex vivo IFN-γ responses were measured by ELISPOT or ICS in JEV-exposed healthy donors ( n = 35, 29 for ELISPOT, and 6 for ICS). Peptide pools are shown grouped by viral proteins. For a subset of five subjects, ICS and ELISPOT were performed at least three times with consistent results. C, core. E, envelope. (B) Spot-forming cells (SFCs) per million PBMCs were measured by ELISPOT in 13 healthy JEV-exposed donors (18 responses, black circles) and three DENV-exposed subjects (four responses, red triangles). (C) Proliferative responses were measured by CFSE dilution and flow cytometry in healthy JEV-exposed donors once per subject. Data are relative frequency ( n = 24) for CD4 + and CD8 + T cells. (D) Based on data from ICS assays, the proportion of the total IFN-γ response produced by CD8 + T cells in each healthy JEV-exposed donor was calculated. The bar depicts the median. n = 11.
Figure Legend Snippet: Breadth of the T cell response to peptide pools of JEV in JEV-exposed healthy donors. (A) Relative frequency of ex vivo IFN-γ responses were measured by ELISPOT or ICS in JEV-exposed healthy donors ( n = 35, 29 for ELISPOT, and 6 for ICS). Peptide pools are shown grouped by viral proteins. For a subset of five subjects, ICS and ELISPOT were performed at least three times with consistent results. C, core. E, envelope. (B) Spot-forming cells (SFCs) per million PBMCs were measured by ELISPOT in 13 healthy JEV-exposed donors (18 responses, black circles) and three DENV-exposed subjects (four responses, red triangles). (C) Proliferative responses were measured by CFSE dilution and flow cytometry in healthy JEV-exposed donors once per subject. Data are relative frequency ( n = 24) for CD4 + and CD8 + T cells. (D) Based on data from ICS assays, the proportion of the total IFN-γ response produced by CD8 + T cells in each healthy JEV-exposed donor was calculated. The bar depicts the median. n = 11.

Techniques Used: Ex Vivo, Enzyme-linked Immunospot, Flow Cytometry, Cytometry, Produced

34) Product Images from "Cord-Blood-Derived Mesenchymal Stromal Cells Downmodulate CD4+ T-Cell Activation by Inducing IL-10-Producing Th1 Cells"

Article Title: Cord-Blood-Derived Mesenchymal Stromal Cells Downmodulate CD4+ T-Cell Activation by Inducing IL-10-Producing Th1 Cells

Journal: Stem Cells and Development

doi: 10.1089/scd.2012.0315

Mesenchymal stromal cells (MSCs) inhibit CD4 + T-cell proliferation, but do not induce a classical Treg phenotype. Total peripheral blood mononuclear cells (PBMCs) were tested upon stimulation with allogeneic PBMCs in the presence and absence of MSCs. (A) CD4 + T-cell proliferation, analyzed by CFSE dilution, was inhibited by the presence of MSCs. (B) CD4 + T-cells were also analyzed for the presence of Tregs (CD4 + CD25 + CD127 − FoxP3 + cells). (C) CD69 expression was analyzed on CD4 + FoxP3 − CD25 − cells. n =4. * p =0.05, ** p =0.01, and *** p =0.0005 (paired t -test). CFSE, 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester.
Figure Legend Snippet: Mesenchymal stromal cells (MSCs) inhibit CD4 + T-cell proliferation, but do not induce a classical Treg phenotype. Total peripheral blood mononuclear cells (PBMCs) were tested upon stimulation with allogeneic PBMCs in the presence and absence of MSCs. (A) CD4 + T-cell proliferation, analyzed by CFSE dilution, was inhibited by the presence of MSCs. (B) CD4 + T-cells were also analyzed for the presence of Tregs (CD4 + CD25 + CD127 − FoxP3 + cells). (C) CD69 expression was analyzed on CD4 + FoxP3 − CD25 − cells. n =4. * p =0.05, ** p =0.01, and *** p =0.0005 (paired t -test). CFSE, 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester.

Techniques Used: Expressing

35) Product Images from "Allogeneic Periodontal Ligament Stem Cell Therapy for Periodontitis in Swine"

Article Title: Allogeneic Periodontal Ligament Stem Cell Therapy for Periodontitis in Swine

Journal: Stem Cells (Dayton, Ohio)

doi: 10.1002/stem.512

Prostaglandin E2 (PGE2) is the key inhibitor of T-cell proliferation mediated by PDLSCs. (A): T-cell proliferation was significantly inhibited when cocultured with human PDLSCs (hPDLSCs) in the Transwell culture system and in cell-cell contact culture. (B): TGF-β1 levels were similar between hPDLSCs alone and the coculture of hPDLSCs and PHA-induced T-cells. (C): The concentration of PGE2 significantly increased in the coculture of hPDLSCs and PHA-induced T cells. (D): The neutralizing monoclonal antibody against TGF-β failed to restore T-cell proliferation, while the PGE2 inhibitor indomethacine restored T-cell proliferation. (E, F): The percentage of Foxp3 + T cells in coculture of PBMCs and PHA (E) was similar to that in coculture of PBMCs, PHA, and hPDLSCs (F) . (G): The expression of COX2 was significantly higher in regenerated periodontal tissues of the allogeneic PDLSCs group than in the autologous PDLSCs group 2 weeks after transplantation, whereas TGF-β1, HGF, and IL-10 levels did not change ( n = 4). (H, I): T-cell apoptosis rates were similar between PHA-stimulated T cells ( I ) and a coculture group with hPDLSCs (H) . (J): Inhibition of T cell proliferation mediated by hPDLSCs was restored when T cells were restimulated by PHA or IL-2. Data are presented as mean ± SD of triplicates of six independent experiments. *, p
Figure Legend Snippet: Prostaglandin E2 (PGE2) is the key inhibitor of T-cell proliferation mediated by PDLSCs. (A): T-cell proliferation was significantly inhibited when cocultured with human PDLSCs (hPDLSCs) in the Transwell culture system and in cell-cell contact culture. (B): TGF-β1 levels were similar between hPDLSCs alone and the coculture of hPDLSCs and PHA-induced T-cells. (C): The concentration of PGE2 significantly increased in the coculture of hPDLSCs and PHA-induced T cells. (D): The neutralizing monoclonal antibody against TGF-β failed to restore T-cell proliferation, while the PGE2 inhibitor indomethacine restored T-cell proliferation. (E, F): The percentage of Foxp3 + T cells in coculture of PBMCs and PHA (E) was similar to that in coculture of PBMCs, PHA, and hPDLSCs (F) . (G): The expression of COX2 was significantly higher in regenerated periodontal tissues of the allogeneic PDLSCs group than in the autologous PDLSCs group 2 weeks after transplantation, whereas TGF-β1, HGF, and IL-10 levels did not change ( n = 4). (H, I): T-cell apoptosis rates were similar between PHA-stimulated T cells ( I ) and a coculture group with hPDLSCs (H) . (J): Inhibition of T cell proliferation mediated by hPDLSCs was restored when T cells were restimulated by PHA or IL-2. Data are presented as mean ± SD of triplicates of six independent experiments. *, p

Techniques Used: Concentration Assay, Expressing, Transplantation Assay, Inhibition

36) Product Images from "Immunization with live attenuated simian immunodeficiency virus induces strong type 1 T helper responses and ?-chemokine production"

Article Title: Immunization with live attenuated simian immunodeficiency virus induces strong type 1 T helper responses and ?-chemokine production

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

doi:

Intracellular cytokine analysis of CD4 + T cells after stimulation with SIV p55. ( A ) PBMCs from an SIVmac239Δnef-immunized macaque were stimulated with SIV p55- or VSV-N-pulsed autologous adherent cells for 24 h, and then intracellular expression of IL-2 and IFN-γ in CD4 + T lymphocytes was evaluated by flow cytometry. The numbers shown in the upper right-hand corner of each panel in A correspond to the percentages of CD4 + T cells expressing the given cytokine. ( B ) Intracellular cytokine expression by SIV p55-specific CD4 + T cells from macaques infected with live attenuated SIV strains.
Figure Legend Snippet: Intracellular cytokine analysis of CD4 + T cells after stimulation with SIV p55. ( A ) PBMCs from an SIVmac239Δnef-immunized macaque were stimulated with SIV p55- or VSV-N-pulsed autologous adherent cells for 24 h, and then intracellular expression of IL-2 and IFN-γ in CD4 + T lymphocytes was evaluated by flow cytometry. The numbers shown in the upper right-hand corner of each panel in A correspond to the percentages of CD4 + T cells expressing the given cytokine. ( B ) Intracellular cytokine expression by SIV p55-specific CD4 + T cells from macaques infected with live attenuated SIV strains.

Techniques Used: Expressing, Flow Cytometry, Cytometry, Infection

ELISPOT analysis of SIV-specific CD4 + T cells from vaccinated macaques. CD4 + T cells from animals immunized with SIVmac239nef ( A ) or SIVmac239Δ3 ( B ) were stimulated with autologous antigen-presenting cells pulsed with SIV p55 and IFN-γ-producing cells identified by using ELISPOT analysis. ( C ) Correlation of SIV p55-CD4 + T cells as assessed by ELISPOT with proliferative responses by using PBMCs isolated from the Δnef- or Δ3-immunized macaques.
Figure Legend Snippet: ELISPOT analysis of SIV-specific CD4 + T cells from vaccinated macaques. CD4 + T cells from animals immunized with SIVmac239nef ( A ) or SIVmac239Δ3 ( B ) were stimulated with autologous antigen-presenting cells pulsed with SIV p55 and IFN-γ-producing cells identified by using ELISPOT analysis. ( C ) Correlation of SIV p55-CD4 + T cells as assessed by ELISPOT with proliferative responses by using PBMCs isolated from the Δnef- or Δ3-immunized macaques.

Techniques Used: Enzyme-linked Immunospot, Isolation

37) Product Images from "Safety of AAV Factor IX Peripheral Transvenular Gene Delivery to Muscle in Hemophilia B Dogs"

Article Title: Safety of AAV Factor IX Peripheral Transvenular Gene Delivery to Muscle in Hemophilia B Dogs

Journal: Molecular Therapy

doi: 10.1038/mt.2010.73

Time course of IL-10 ELISpot responses to canine FIX (cFIX) in dogs receiving AAV-2-cFIX vectors by ATVRX. Secretion of IL-10 from PBMCs after stimulation with peptides spanning the cFIX sequence organized in 19 pools. Samples were collected and analyzed at baseline, during IS and after IS discontinuation. Responses were considered positive when the number of spot-forming units (SFU) per million PBMCs were > 50 and at least threefold higher than the media control (Med). All test conditions were assayed in triplicate. ( a ) Group A (1 × 10 12 vg/kg with IS); ( b ) Group B (3 × 10 12 vg/kg with IS); ( c ) Group C (8.5 × 10 12 vg/kg with IS); ( d ) Group D (3 × 10 12 vg/kg without IS). Baseline sample for J62 was not available; a sample obtained 1 week after vector delivery was analyzed instead. ATVRX, afferent transvenular retrograde extravasation; IL, interleukin; IS, immunosuppression; PBMC, peripheral blood mononuclear cell.
Figure Legend Snippet: Time course of IL-10 ELISpot responses to canine FIX (cFIX) in dogs receiving AAV-2-cFIX vectors by ATVRX. Secretion of IL-10 from PBMCs after stimulation with peptides spanning the cFIX sequence organized in 19 pools. Samples were collected and analyzed at baseline, during IS and after IS discontinuation. Responses were considered positive when the number of spot-forming units (SFU) per million PBMCs were > 50 and at least threefold higher than the media control (Med). All test conditions were assayed in triplicate. ( a ) Group A (1 × 10 12 vg/kg with IS); ( b ) Group B (3 × 10 12 vg/kg with IS); ( c ) Group C (8.5 × 10 12 vg/kg with IS); ( d ) Group D (3 × 10 12 vg/kg without IS). Baseline sample for J62 was not available; a sample obtained 1 week after vector delivery was analyzed instead. ATVRX, afferent transvenular retrograde extravasation; IL, interleukin; IS, immunosuppression; PBMC, peripheral blood mononuclear cell.

Techniques Used: Enzyme-linked Immunospot, Sequencing, Plasmid Preparation

Analysis of canine FIX (cFIX) peptide pools which showed reactivity in the IL-10 ELISpots. ( a ) A peptide library of 89 peptides spanning the sequence of cFIX was organized into a matrix in which each peptide was represented in two orthogonal pools. Highlighted in grey are the pools that scored positive in most of the dogs analyzed. Positivity in two orthogonal pools identified a single stimulatory peptide, peptide 68. Peptide 68 amino acid sequence is indicated. ( b ) Peptides contained in pool 4 were rearranged into a smaller orthogonal matrix, allowing the identification of another reactive peptide that spanned the Chapel Hill mutation (peptide 84). *Chapel Hill mutation (glycin→glutamic acid) within peptide 84. ( c ) IL-10 secretion detected by ELISpot in PBMCs obtained form H24 (group C, ATVRX, 8.5 × 10 12 vg/kg with IS) stimulated with the matrix represented in b . ELISpot, enzyme-linked immunosorbent assay; IL, interleukin; PBMC, peripheral blood mononuclear cell.
Figure Legend Snippet: Analysis of canine FIX (cFIX) peptide pools which showed reactivity in the IL-10 ELISpots. ( a ) A peptide library of 89 peptides spanning the sequence of cFIX was organized into a matrix in which each peptide was represented in two orthogonal pools. Highlighted in grey are the pools that scored positive in most of the dogs analyzed. Positivity in two orthogonal pools identified a single stimulatory peptide, peptide 68. Peptide 68 amino acid sequence is indicated. ( b ) Peptides contained in pool 4 were rearranged into a smaller orthogonal matrix, allowing the identification of another reactive peptide that spanned the Chapel Hill mutation (peptide 84). *Chapel Hill mutation (glycin→glutamic acid) within peptide 84. ( c ) IL-10 secretion detected by ELISpot in PBMCs obtained form H24 (group C, ATVRX, 8.5 × 10 12 vg/kg with IS) stimulated with the matrix represented in b . ELISpot, enzyme-linked immunosorbent assay; IL, interleukin; PBMC, peripheral blood mononuclear cell.

Techniques Used: Sequencing, Mutagenesis, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay

38) Product Images from "Soluble Rhesus Lymphocryptovirus gp350 Protects against Infection and Reduces Viral Loads in Animals that Become Infected with Virus after Challenge"

Article Title: Soluble Rhesus Lymphocryptovirus gp350 Protects against Infection and Reduces Viral Loads in Animals that Become Infected with Virus after Challenge

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002308

Detection of rhesus LCV EBER1 in the blood of monkeys immunized with soluble gp350, VRP-gp350, a combination of VRP-350, VRP-EBNA-3A, and VRP-EBNA-3B, or PBS. RNA was isolated from PBMCs and reverse transcription was performed followed by real time PCR with a probe that detects EBER1 DNA.
Figure Legend Snippet: Detection of rhesus LCV EBER1 in the blood of monkeys immunized with soluble gp350, VRP-gp350, a combination of VRP-350, VRP-EBNA-3A, and VRP-EBNA-3B, or PBS. RNA was isolated from PBMCs and reverse transcription was performed followed by real time PCR with a probe that detects EBER1 DNA.

Techniques Used: Isolation, Real-time Polymerase Chain Reaction

39) Product Images from "Full-Length GB Virus C (Hepatitis G Virus) RNA Transcripts Are Infectious in Primary CD4-Positive T Cells"

Article Title: Full-Length GB Virus C (Hepatitis G Virus) RNA Transcripts Are Infectious in Primary CD4-Positive T Cells

Journal: Journal of Virology

doi:

GBV-C E2 expression in PBMCs. PBMCs were infected with supernatant from the passage 4 cell culture supernatant fluid, and, 2 (A) and 5 days (B) postinfection, the cells were fixed and processed as described in Materials and Methods. GBV-C E2 expression was detected using a murine monoclonal anti-GBV-C E2 antibody as described in Materials and Methods. The same cells (2 days postinfection) did not show specific cytoplasmic fluorescence when evaluated with an isotypic control antibody (C), nor did mock-infected PBMCs evaluated with the GBV-C anti-E2 antibody as for panel A (D).
Figure Legend Snippet: GBV-C E2 expression in PBMCs. PBMCs were infected with supernatant from the passage 4 cell culture supernatant fluid, and, 2 (A) and 5 days (B) postinfection, the cells were fixed and processed as described in Materials and Methods. GBV-C E2 expression was detected using a murine monoclonal anti-GBV-C E2 antibody as described in Materials and Methods. The same cells (2 days postinfection) did not show specific cytoplasmic fluorescence when evaluated with an isotypic control antibody (C), nor did mock-infected PBMCs evaluated with the GBV-C anti-E2 antibody as for panel A (D).

Techniques Used: Expressing, Infection, Cell Culture, Fluorescence

40) Product Images from "Human CD8+ T Cells Clear Cryptosporidium parvum from Infected Intestinal Epithelial Cells"

Article Title: Human CD8+ T Cells Clear Cryptosporidium parvum from Infected Intestinal Epithelial Cells

Journal: The American Journal of Tropical Medicine and Hygiene

doi: 10.4269/ajtmh.2010.09-0590

Parasite clearance is blocked by Concanamycin A or antibody to Fas ligand. CD8 + T cells were isolated from gp15 antigen expanded peripheral blood mononuclear cells (PBMCs) from sensitized Donor 1 and used as effector cells in co-cultures with Cryptosporidium
Figure Legend Snippet: Parasite clearance is blocked by Concanamycin A or antibody to Fas ligand. CD8 + T cells were isolated from gp15 antigen expanded peripheral blood mononuclear cells (PBMCs) from sensitized Donor 1 and used as effector cells in co-cultures with Cryptosporidium

Techniques Used: Isolation

Parasite clearance is blocked by antibody to HLA-A or HLA-B/C. CD8 + T cells were isolated from gp15 antigen expanded peripheral blood mononuclear cells (PBMCs) from sensitized Donor 1 (HLA-A and HLA-B matched with the CaCo-2 cells) and used as effector
Figure Legend Snippet: Parasite clearance is blocked by antibody to HLA-A or HLA-B/C. CD8 + T cells were isolated from gp15 antigen expanded peripheral blood mononuclear cells (PBMCs) from sensitized Donor 1 (HLA-A and HLA-B matched with the CaCo-2 cells) and used as effector

Techniques Used: Isolation

Related Articles

Transfection:

Article Title: Polymorphism of rs1836882 in NOX4 Gene Modifies Associations between Dietary Caloric Intake and ROS Levels in Peripheral Blood Mononuclear Cells
Article Snippet: .. Transfection of HNF3γ plasmids for PBMC or HEK293 cells HNF3γ plasmid transfection was performed using Lipofectamine 2000 (Invitrogen). .. On reaching 60% confluence, PBMC or HEK293 cells were incubated with the HNF3γ plasmid-Lipofectamine 2000 complexes at 37°C for 4 h, followed by recovery in growth RPMI-1640 medium for PBMC or MEM medium for HEK293.

Concentration Assay:

Article Title: Effects of anticancer agents on cell viability, proliferative activity and cytokine production of peripheral blood mononuclear cells
Article Snippet: .. PBMCs were then stimulated with PHA (5 µg/ml) and subsequently dispersed at a concentration of 2 × 105 cells/well/200 µl into a 96-well plate and incubated for 48 h. Cytokine concentrations in PBMC culture supernatant were determined by cytokine-specific solid phase sandwich enzyme-linked immuosorbent assay (ELISA) kits (eBioscience, San Diego, CA and R & D systems, Minneapolis, MN) according to the manufacturer’s instructions. .. Flow cytometric analysis PBMCs were incubated with 10 µM GEM for 2 h prior to stimulation with PHA (5 µg/ml).

Isolation:

Article Title: Targeted Delivery of an Antigenic Peptide to the Endoplasmic Reticulum: Application for Development of a Peptide Therapy for Ankylosing Spondylitis
Article Snippet: .. Stimulation of PBMCs with THUC PBMCs isolated from HLA-B27-positive AS patients were resuspended in RPMI 1640 medium (Invitrogen) with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. .. PBMCs (5 × 106 cells) in 1 ml medium were treated with 5 μM THUC or THUB and incubated at 37°C with 5% CO2 .

Article Title: New insights into the role and mechanism of macrophage migration inhibitory factor in steroid-resistant patients with systemic lupus erythematosus
Article Snippet: .. Real-time RT-PCR Total RNA from PBMCs was isolated with the TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to product recommendations. .. RNA was then reverse transcribed using the Reverse Transcription Kit (Takara Bio Inc., Otsu, Japan).

Cytometry:

Article Title: Overexpression of Programmed Death Ligands in Naturally Occurring Postweaning Multisystemic Wasting Syndrome
Article Snippet: .. Proliferation of PBMCs was evaluated via dilution of CFSE on an Applied Biosystems Attune Acoustic Focusing Cytometer (Applied Biosystems). .. Data from 1×104 cells were analyzed using software version 1.2.5.

Quantitative RT-PCR:

Article Title: New insights into the role and mechanism of macrophage migration inhibitory factor in steroid-resistant patients with systemic lupus erythematosus
Article Snippet: .. Real-time RT-PCR Total RNA from PBMCs was isolated with the TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to product recommendations. .. RNA was then reverse transcribed using the Reverse Transcription Kit (Takara Bio Inc., Otsu, Japan).

Enzyme-linked Immunosorbent Assay:

Article Title: Effects of anticancer agents on cell viability, proliferative activity and cytokine production of peripheral blood mononuclear cells
Article Snippet: .. PBMCs were then stimulated with PHA (5 µg/ml) and subsequently dispersed at a concentration of 2 × 105 cells/well/200 µl into a 96-well plate and incubated for 48 h. Cytokine concentrations in PBMC culture supernatant were determined by cytokine-specific solid phase sandwich enzyme-linked immuosorbent assay (ELISA) kits (eBioscience, San Diego, CA and R & D systems, Minneapolis, MN) according to the manufacturer’s instructions. .. Flow cytometric analysis PBMCs were incubated with 10 µM GEM for 2 h prior to stimulation with PHA (5 µg/ml).

Activation Assay:

Article Title: Zika Virus Infection Preferentially Counterbalances Human Peripheral Monocyte and/or NK Cell Activity
Article Snippet: .. Cytokine levels in supernatant obtained from mock- and ZIKV-infected PBMCs were measured simultaneously using the ProcartaPlex immunoassay (Thermo Fisher Scientific) detecting 45 secreted cytokines, chemokines, and growth factors, including brain-derived neurotropic factor (BDNF); eotaxin/CCL11; epidermal growth factor (EGF); fibroblast growth factor 2 (FGF-2); granulocyte-macrophage colony-stimulating factor (GM-CSF); growth-related oncogene alpha (GROα)/CXCL1; hepatocyte growth factor (HGF); nerve growth factor (NGF) beta ( ); leukemia inhibitory factor ( ); alpha interferon (IFN-α); IFN-γ; interleukin-1β (IL-1β); IL-1α; IL-1RA; IL-2; IL-4; IL-5; IL-6; IL-7; IL-8/CXCL8; IL-9; IL-10; IL-12p70; IL-13; IL-15; IL-17A; IL-18; IL-21; IL-22; IL-23; IL-27; IL-31; gamma interferon-induced protein 10 (IP-10)/CXCL10; monocyte chemoattractant protein (MCP-1/CCL2); macrophage inflammatory protein 1α (MIP-1α)/CCL3; MIP-1β/CCL4; regulated on activation, normal T cell expressed and secreted (RANTES)/CCL5; stromal cell-derived factor 1α (SDF-1α)/CXCL12; tumor necrosis factor alpha (TNF-α); TNF-β/lymphotoxin alpha (LTA); platelet-derived growth factor (PDGF)-BB; placental growth factor (PLGF); stem cell factor (SCF); vascular endothelial growth factor A (VEGF-A); and VEGF-D. .. Successful RNA depletion was verified using a Qubit (Thermo Fisher Scientific) and an Agilent 2100 Bioanalyzer (Agilent), and all of the depleted RNA was used as input material for the ScriptSeq v2 RNA-seq library preparation protocol.

Incubation:

Article Title: Effects of anticancer agents on cell viability, proliferative activity and cytokine production of peripheral blood mononuclear cells
Article Snippet: .. PBMCs were then stimulated with PHA (5 µg/ml) and subsequently dispersed at a concentration of 2 × 105 cells/well/200 µl into a 96-well plate and incubated for 48 h. Cytokine concentrations in PBMC culture supernatant were determined by cytokine-specific solid phase sandwich enzyme-linked immuosorbent assay (ELISA) kits (eBioscience, San Diego, CA and R & D systems, Minneapolis, MN) according to the manufacturer’s instructions. .. Flow cytometric analysis PBMCs were incubated with 10 µM GEM for 2 h prior to stimulation with PHA (5 µg/ml).

Staining:

Article Title: Age-dependent changes in innate immune phenotype and function in rhesus macaques (Macaca mulatta)
Article Snippet: .. For the analysis of innate immune cell cytokine production, PBMC were stained with the innate surface panel described above, with the addition of Aqua LIVE/DEAD stain (Invitrogen) to exclude dead cells. .. Following surface staining, cells were fixed and permeabilized according to the manufacturer's instructions (Biolegend) and stained for TNFα, IL-6 (eBioscience) and IFNα (PBL InterferonSource, Piscataway, NJ).

Plasmid Preparation:

Article Title: Polymorphism of rs1836882 in NOX4 Gene Modifies Associations between Dietary Caloric Intake and ROS Levels in Peripheral Blood Mononuclear Cells
Article Snippet: .. Transfection of HNF3γ plasmids for PBMC or HEK293 cells HNF3γ plasmid transfection was performed using Lipofectamine 2000 (Invitrogen). .. On reaching 60% confluence, PBMC or HEK293 cells were incubated with the HNF3γ plasmid-Lipofectamine 2000 complexes at 37°C for 4 h, followed by recovery in growth RPMI-1640 medium for PBMC or MEM medium for HEK293.

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    Thermo Fisher frozen thawed pbmcs
    More Complex acDC Cytokine Cocktails Induce APCs of Similar Phenotype (A) <t>CBMCs</t> and <t>PBMCs</t> (2 × 10 6 per well in 48-well plates) were cultured using the indicated acDC cytokine cocktails followed by TNF-α, PGE2, IL-1β, and low-dose IL-7 after 24 h. At the end of this 48-h culture, the phenotype of adherent CD3 − CD19 − CD11c + cells was assessed by flow cytometry using the indicated cell surface markers. A representative staining obtained from one CBMC sample (left) and one PBMC sample (right) is shown. (B) Cumulative data obtained from 3 CBMC donors (top) and 3 PBMC donors (bottom), displayed as the relative representation of CD14 + and CD14 − cells among adherent CD3 − CD19 − CD11c + cells expressing 0 to 3 different markers among HLA-DR, CD80, and CD86.
    Frozen Thawed Pbmcs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pbmcs
    Immune profiling of <t>ZIKV-infected</t> <t>PBMCs.</t> (A) Immune mediators in the culture supernatant of ZIKV-infected PBMCs and CD14-depleted PBMCs were quantified with a 45-plex microbead assay. Concentrations were scaled between 0 and 1. (B) Bar charts of three cytokines, levels of which were significantly affected by both the depletion of CD14 + monocytes and ZIKV infection. (C) Stimulatory capacity of the culture supernatants was further evaluated with freshly isolated PBMCs. Culture supernatant was added in a ratio of 1:10, and cells were harvested at 36 h poststimulation. Compiled percentages of CD107a-, IFN-γ-, and NKG2D-positive CD94 + CD56 + NK cells are shown as normalized to the respective mock sample. Data displayed were derived from seven donors. Lineage markers CD3, CD19, CD20, and CD14 have been included to rule out the presence of non-NK cells. All data are presented as means ± standard deviations. *, P
    Pbmcs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 288 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    More Complex acDC Cytokine Cocktails Induce APCs of Similar Phenotype (A) CBMCs and PBMCs (2 × 10 6 per well in 48-well plates) were cultured using the indicated acDC cytokine cocktails followed by TNF-α, PGE2, IL-1β, and low-dose IL-7 after 24 h. At the end of this 48-h culture, the phenotype of adherent CD3 − CD19 − CD11c + cells was assessed by flow cytometry using the indicated cell surface markers. A representative staining obtained from one CBMC sample (left) and one PBMC sample (right) is shown. (B) Cumulative data obtained from 3 CBMC donors (top) and 3 PBMC donors (bottom), displayed as the relative representation of CD14 + and CD14 − cells among adherent CD3 − CD19 − CD11c + cells expressing 0 to 3 different markers among HLA-DR, CD80, and CD86.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: In Vitro Expansion of Anti-viral T Cells from Cord Blood by Accelerated Co-cultured Dendritic Cells

    doi: 10.1016/j.omtm.2018.12.010

    Figure Lengend Snippet: More Complex acDC Cytokine Cocktails Induce APCs of Similar Phenotype (A) CBMCs and PBMCs (2 × 10 6 per well in 48-well plates) were cultured using the indicated acDC cytokine cocktails followed by TNF-α, PGE2, IL-1β, and low-dose IL-7 after 24 h. At the end of this 48-h culture, the phenotype of adherent CD3 − CD19 − CD11c + cells was assessed by flow cytometry using the indicated cell surface markers. A representative staining obtained from one CBMC sample (left) and one PBMC sample (right) is shown. (B) Cumulative data obtained from 3 CBMC donors (top) and 3 PBMC donors (bottom), displayed as the relative representation of CD14 + and CD14 − cells among adherent CD3 − CD19 − CD11c + cells expressing 0 to 3 different markers among HLA-DR, CD80, and CD86.

    Article Snippet: acDC Cytokine Treatment of PBMCs and CBMCs We adapted a methodology that we previously developed to generate Ag-reactive T cells from PBMCs., , , , Frozen-thawed PBMCs or CBMCs were plated at the following densities in AIM-V medium (Thermo Fisher Scientific): 2 × 106 cells per 500 μL per well in 48-well plates; 5 × 106 cells per 1 mL per well in 24-well plates; 10 × 106 cells per 2 mL per well in 12-well plates; and 10 × 106 cells per 20 mL per flask in G-Rex10 flasks (Wilson Wolf).

    Techniques: Cell Culture, Flow Cytometry, Cytometry, Staining, Expressing

    More Complex acDC Cytokine Cocktails Induce Ag-Reactive CD8 + TC in Similar Numbers but Produce More Cytokines (A) PBMCs and CBMCs (3 donors each, same donors as in Figure 3 ; 2 × 10 6 CBMCs per well in 48-well plates) were cultured using the indicated acDC cytokine cocktails followed by TNF-α, PGE2, IL-1β, and low-dose IL-7 along with MelanA 26–35 peptide after 24 h and by IL-2, IL-15, and IL-7 from day 2 of culture. Absolute numbers of MelanA 26–-35 -reactive CD8 + T cells per million PBMCs (green) or CBMCs (black) obtained at the end of the 10-day culture are plotted, as determined with MelanA 26–35 -loaded HLA-A2 MMrs and CountBright beads. (B) The same cultures were tested at day 13 during a 6-h recall assay in the presence of HLA-A2 + LCL cells pulsed with MelanA 26–35 or no peptide. The graph displays absolute numbers of CD8 + T cells from PBMCs (green) or CBMCs (black) producing at least one cytokine among IFN-γ, TNF-α, IL-2, and MIP-1β in response to MelanA 26–35 -pulsed LCL cells after background subtraction, i.e., the number of cytokine-positive CD8 + T cells detected in response to unpulsed LCL cells. Results are expressed as mean ± SD (*p

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: In Vitro Expansion of Anti-viral T Cells from Cord Blood by Accelerated Co-cultured Dendritic Cells

    doi: 10.1016/j.omtm.2018.12.010

    Figure Lengend Snippet: More Complex acDC Cytokine Cocktails Induce Ag-Reactive CD8 + TC in Similar Numbers but Produce More Cytokines (A) PBMCs and CBMCs (3 donors each, same donors as in Figure 3 ; 2 × 10 6 CBMCs per well in 48-well plates) were cultured using the indicated acDC cytokine cocktails followed by TNF-α, PGE2, IL-1β, and low-dose IL-7 along with MelanA 26–35 peptide after 24 h and by IL-2, IL-15, and IL-7 from day 2 of culture. Absolute numbers of MelanA 26–-35 -reactive CD8 + T cells per million PBMCs (green) or CBMCs (black) obtained at the end of the 10-day culture are plotted, as determined with MelanA 26–35 -loaded HLA-A2 MMrs and CountBright beads. (B) The same cultures were tested at day 13 during a 6-h recall assay in the presence of HLA-A2 + LCL cells pulsed with MelanA 26–35 or no peptide. The graph displays absolute numbers of CD8 + T cells from PBMCs (green) or CBMCs (black) producing at least one cytokine among IFN-γ, TNF-α, IL-2, and MIP-1β in response to MelanA 26–35 -pulsed LCL cells after background subtraction, i.e., the number of cytokine-positive CD8 + T cells detected in response to unpulsed LCL cells. Results are expressed as mean ± SD (*p

    Article Snippet: acDC Cytokine Treatment of PBMCs and CBMCs We adapted a methodology that we previously developed to generate Ag-reactive T cells from PBMCs., , , , Frozen-thawed PBMCs or CBMCs were plated at the following densities in AIM-V medium (Thermo Fisher Scientific): 2 × 106 cells per 500 μL per well in 48-well plates; 5 × 106 cells per 1 mL per well in 24-well plates; 10 × 106 cells per 2 mL per well in 12-well plates; and 10 × 106 cells per 20 mL per flask in G-Rex10 flasks (Wilson Wolf).

    Techniques: Cell Culture

    acDC Cytokine Cocktails Induce Equivalent APC Populations in CB and PB PBMCs or CBMCs (2 × 10 6 cells per well in 48-well plates) were cultured for 48 h with the indicated cytokine cocktails, namely, GM-CSF/IL-4, IL-1β, Flt3L, or no cytokines during 24 h followed by the addition of TNF-α, PGE2, IL-1β, and low-dose IL-7 for another 24 h. At the end of this 48-h culture, the phenotype of CD3 − CD19 − CD11c + cells was assessed by flow cytometry using the indicated cell surface markers. (A) Representative staining obtained from one PBMC (white profiles) and one CBMC sample (gray profiles), as compared to isotype control (dotted line indicates the mean fluorescence intensity; MFI). (B) Cumulative data obtained from 3 CBMC and 3 PBMC donors, represented as relative MFI ± SD for each of the indicated markers, normalized to the MFI registered for PBMC samples in the absence of cytokines.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: In Vitro Expansion of Anti-viral T Cells from Cord Blood by Accelerated Co-cultured Dendritic Cells

    doi: 10.1016/j.omtm.2018.12.010

    Figure Lengend Snippet: acDC Cytokine Cocktails Induce Equivalent APC Populations in CB and PB PBMCs or CBMCs (2 × 10 6 cells per well in 48-well plates) were cultured for 48 h with the indicated cytokine cocktails, namely, GM-CSF/IL-4, IL-1β, Flt3L, or no cytokines during 24 h followed by the addition of TNF-α, PGE2, IL-1β, and low-dose IL-7 for another 24 h. At the end of this 48-h culture, the phenotype of CD3 − CD19 − CD11c + cells was assessed by flow cytometry using the indicated cell surface markers. (A) Representative staining obtained from one PBMC (white profiles) and one CBMC sample (gray profiles), as compared to isotype control (dotted line indicates the mean fluorescence intensity; MFI). (B) Cumulative data obtained from 3 CBMC and 3 PBMC donors, represented as relative MFI ± SD for each of the indicated markers, normalized to the MFI registered for PBMC samples in the absence of cytokines.

    Article Snippet: acDC Cytokine Treatment of PBMCs and CBMCs We adapted a methodology that we previously developed to generate Ag-reactive T cells from PBMCs., , , , Frozen-thawed PBMCs or CBMCs were plated at the following densities in AIM-V medium (Thermo Fisher Scientific): 2 × 106 cells per 500 μL per well in 48-well plates; 5 × 106 cells per 1 mL per well in 24-well plates; 10 × 106 cells per 2 mL per well in 12-well plates; and 10 × 106 cells per 20 mL per flask in G-Rex10 flasks (Wilson Wolf).

    Techniques: Cell Culture, Flow Cytometry, Cytometry, Staining, Fluorescence

    Immune profiling of ZIKV-infected PBMCs. (A) Immune mediators in the culture supernatant of ZIKV-infected PBMCs and CD14-depleted PBMCs were quantified with a 45-plex microbead assay. Concentrations were scaled between 0 and 1. (B) Bar charts of three cytokines, levels of which were significantly affected by both the depletion of CD14 + monocytes and ZIKV infection. (C) Stimulatory capacity of the culture supernatants was further evaluated with freshly isolated PBMCs. Culture supernatant was added in a ratio of 1:10, and cells were harvested at 36 h poststimulation. Compiled percentages of CD107a-, IFN-γ-, and NKG2D-positive CD94 + CD56 + NK cells are shown as normalized to the respective mock sample. Data displayed were derived from seven donors. Lineage markers CD3, CD19, CD20, and CD14 have been included to rule out the presence of non-NK cells. All data are presented as means ± standard deviations. *, P

    Journal: mSphere

    Article Title: Zika Virus Infection Preferentially Counterbalances Human Peripheral Monocyte and/or NK Cell Activity

    doi: 10.1128/mSphereDirect.00120-18

    Figure Lengend Snippet: Immune profiling of ZIKV-infected PBMCs. (A) Immune mediators in the culture supernatant of ZIKV-infected PBMCs and CD14-depleted PBMCs were quantified with a 45-plex microbead assay. Concentrations were scaled between 0 and 1. (B) Bar charts of three cytokines, levels of which were significantly affected by both the depletion of CD14 + monocytes and ZIKV infection. (C) Stimulatory capacity of the culture supernatants was further evaluated with freshly isolated PBMCs. Culture supernatant was added in a ratio of 1:10, and cells were harvested at 36 h poststimulation. Compiled percentages of CD107a-, IFN-γ-, and NKG2D-positive CD94 + CD56 + NK cells are shown as normalized to the respective mock sample. Data displayed were derived from seven donors. Lineage markers CD3, CD19, CD20, and CD14 have been included to rule out the presence of non-NK cells. All data are presented as means ± standard deviations. *, P

    Article Snippet: Cytokine levels in supernatant obtained from mock- and ZIKV-infected PBMCs were measured simultaneously using the ProcartaPlex immunoassay (Thermo Fisher Scientific) detecting 45 secreted cytokines, chemokines, and growth factors, including brain-derived neurotropic factor (BDNF); eotaxin/CCL11; epidermal growth factor (EGF); fibroblast growth factor 2 (FGF-2); granulocyte-macrophage colony-stimulating factor (GM-CSF); growth-related oncogene alpha (GROα)/CXCL1; hepatocyte growth factor (HGF); nerve growth factor (NGF) beta ( ); leukemia inhibitory factor ( ); alpha interferon (IFN-α); IFN-γ; interleukin-1β (IL-1β); IL-1α; IL-1RA; IL-2; IL-4; IL-5; IL-6; IL-7; IL-8/CXCL8; IL-9; IL-10; IL-12p70; IL-13; IL-15; IL-17A; IL-18; IL-21; IL-22; IL-23; IL-27; IL-31; gamma interferon-induced protein 10 (IP-10)/CXCL10; monocyte chemoattractant protein (MCP-1/CCL2); macrophage inflammatory protein 1α (MIP-1α)/CCL3; MIP-1β/CCL4; regulated on activation, normal T cell expressed and secreted (RANTES)/CCL5; stromal cell-derived factor 1α (SDF-1α)/CXCL12; tumor necrosis factor alpha (TNF-α); TNF-β/lymphotoxin alpha (LTA); platelet-derived growth factor (PDGF)-BB; placental growth factor (PLGF); stem cell factor (SCF); vascular endothelial growth factor A (VEGF-A); and VEGF-D.

    Techniques: Infection, Isolation, Derivative Assay

    Role of monocytes in NK cell activity. Full PBMCs and CD14-depleted PBMCs (2 × 10 6 cells per infection) were infected with Zika virus (ZIKV) at an MOI of 10 and harvested at 36 hpi. (A) Gating strategy of CD94 + CD56 + Lineage − NK cells and their expression of CD69, CD107a, and IFN-γ. Plots from one representative donor are shown. The red circle indicates the presence or absence of CD14 + monocytes. (B) Compiled percentages of CD107a- and IFN-γ-positive NK cells (depicted in panel A) as normalized to the respective mock sample. (C) Viral load in the infected cells. Data shown were derived from seven donors. Lineage markers CD3, CD19, CD20, and CD14 have been included to rule out the presence of non-NK cells. All data are presented as means ± standard deviations. *, P

    Journal: mSphere

    Article Title: Zika Virus Infection Preferentially Counterbalances Human Peripheral Monocyte and/or NK Cell Activity

    doi: 10.1128/mSphereDirect.00120-18

    Figure Lengend Snippet: Role of monocytes in NK cell activity. Full PBMCs and CD14-depleted PBMCs (2 × 10 6 cells per infection) were infected with Zika virus (ZIKV) at an MOI of 10 and harvested at 36 hpi. (A) Gating strategy of CD94 + CD56 + Lineage − NK cells and their expression of CD69, CD107a, and IFN-γ. Plots from one representative donor are shown. The red circle indicates the presence or absence of CD14 + monocytes. (B) Compiled percentages of CD107a- and IFN-γ-positive NK cells (depicted in panel A) as normalized to the respective mock sample. (C) Viral load in the infected cells. Data shown were derived from seven donors. Lineage markers CD3, CD19, CD20, and CD14 have been included to rule out the presence of non-NK cells. All data are presented as means ± standard deviations. *, P

    Article Snippet: Cytokine levels in supernatant obtained from mock- and ZIKV-infected PBMCs were measured simultaneously using the ProcartaPlex immunoassay (Thermo Fisher Scientific) detecting 45 secreted cytokines, chemokines, and growth factors, including brain-derived neurotropic factor (BDNF); eotaxin/CCL11; epidermal growth factor (EGF); fibroblast growth factor 2 (FGF-2); granulocyte-macrophage colony-stimulating factor (GM-CSF); growth-related oncogene alpha (GROα)/CXCL1; hepatocyte growth factor (HGF); nerve growth factor (NGF) beta ( ); leukemia inhibitory factor ( ); alpha interferon (IFN-α); IFN-γ; interleukin-1β (IL-1β); IL-1α; IL-1RA; IL-2; IL-4; IL-5; IL-6; IL-7; IL-8/CXCL8; IL-9; IL-10; IL-12p70; IL-13; IL-15; IL-17A; IL-18; IL-21; IL-22; IL-23; IL-27; IL-31; gamma interferon-induced protein 10 (IP-10)/CXCL10; monocyte chemoattractant protein (MCP-1/CCL2); macrophage inflammatory protein 1α (MIP-1α)/CCL3; MIP-1β/CCL4; regulated on activation, normal T cell expressed and secreted (RANTES)/CCL5; stromal cell-derived factor 1α (SDF-1α)/CXCL12; tumor necrosis factor alpha (TNF-α); TNF-β/lymphotoxin alpha (LTA); platelet-derived growth factor (PDGF)-BB; placental growth factor (PLGF); stem cell factor (SCF); vascular endothelial growth factor A (VEGF-A); and VEGF-D.

    Techniques: Activity Assay, Infection, Expressing, Derivative Assay

    Effects of natural killer cell-derived extracellular vesicles (NKEVs) on monocytes. (A) Flow cytometry analysis of CD80–CD86 geo mean fluorescence intensity (gMFI) of gated CD14 + cells in peripheral blood mononuclear cells (PBMCs) cultured in the presence or absence of NK-cell-derived microvesicles (NKMV), NKExo, and/or lipopolysacharride (LPS) for 24 h. Upper panels: representative dot plots showing CD80–CD86 expression in the presence of NKMV and NKExo, lower panel: graphical summary of different healthy donors. (B) Flow cytometry of human leukocyte antigen DR isotype (HLA-DR) gMFI of CD14 + gated monocytes as in (A) . Results for different donors ( n = 3) are shown. (C) Effects of NKEVs on isolated monocytes, measured by flow cytometry after 24 h culture of CD14 + cells with NKMV or NKExo. Left panels: gMFI of CD80-86 and HLA-DR expression by monocytes of one healthy donor. Right panel: results from different healthy donors ( n = 3). (D) Stimulatory potential of monocytes preconditioned with NKMV and NKExo. Left panels: flow cytometry analysis of 72 h proliferation and CD25 expression by CD3, CD4, and CD8 T cells cultured in the presence of monocytes (medium), monocytes preconditioned with NKMV or NKExo. Right panels: graphical summary showing results from different healthy donors ( n = 3). Percentage of proliferation is indicated. Statistical significance was achieved by paired t test. Means ± SD are plotted, * p

    Journal: Frontiers in Immunology

    Article Title: Natural-Killer-Derived Extracellular Vesicles: Immune Sensors and Interactors

    doi: 10.3389/fimmu.2020.00262

    Figure Lengend Snippet: Effects of natural killer cell-derived extracellular vesicles (NKEVs) on monocytes. (A) Flow cytometry analysis of CD80–CD86 geo mean fluorescence intensity (gMFI) of gated CD14 + cells in peripheral blood mononuclear cells (PBMCs) cultured in the presence or absence of NK-cell-derived microvesicles (NKMV), NKExo, and/or lipopolysacharride (LPS) for 24 h. Upper panels: representative dot plots showing CD80–CD86 expression in the presence of NKMV and NKExo, lower panel: graphical summary of different healthy donors. (B) Flow cytometry of human leukocyte antigen DR isotype (HLA-DR) gMFI of CD14 + gated monocytes as in (A) . Results for different donors ( n = 3) are shown. (C) Effects of NKEVs on isolated monocytes, measured by flow cytometry after 24 h culture of CD14 + cells with NKMV or NKExo. Left panels: gMFI of CD80-86 and HLA-DR expression by monocytes of one healthy donor. Right panel: results from different healthy donors ( n = 3). (D) Stimulatory potential of monocytes preconditioned with NKMV and NKExo. Left panels: flow cytometry analysis of 72 h proliferation and CD25 expression by CD3, CD4, and CD8 T cells cultured in the presence of monocytes (medium), monocytes preconditioned with NKMV or NKExo. Right panels: graphical summary showing results from different healthy donors ( n = 3). Percentage of proliferation is indicated. Statistical significance was achieved by paired t test. Means ± SD are plotted, * p

    Article Snippet: The following antibodies were used for cell labeling of peripheral blood lymphocytes (PBL), NK cells and PBMCs harvested from CD3/CD28-stimulated or unstimulated NKEV-PBMC cocultures, in the presence or absence of TGFβ/IL-10: CFSE (CellTrace, Thermo Fisher Scientific), CD3-ECD (Beckman Coulter), CD4-Alexa700 (Beckman Coulter), CD8-BV605 (Biolegend), CD56-BV510 (Becton Dickinson), CD16-BV650 (Biolegend), PD-1-PC7 (Beckman Coulter), CD25-PercpCy5.5 (Becton Dickinson), HLA-DR-APC (Beckman Coulter), and CD14-Alexa750 (Beckman Coulter).

    Techniques: Derivative Assay, Flow Cytometry, Fluorescence, Cell Culture, Expressing, Isolation

    Effects of natural killer cell-derived extracellular vesicles (NKEVs) on T cells. (A) Flow cytometry analysis of CD25 expression by CD3 + gated T cells in peripheral blood mononuclear cells (PBMCs) evaluated after 72 h of culture with NK-cell-derived microvesicle (NKMV) and NKExo. Left panels: representative dot plots of a healthy donor. Right panel: the graph shows the results obtained with PBMCs of different healthy donors ( n = 3), in the presence or absence of transforming growth factor beta (TGFβ)/interleukin (IL)-10 (10 ng/ml each). (B) Cytometric bead array (CBA)-measured cytokine production of 72 h PBMCs cultured as described in (A) . (C) Geo mean fluorescence intensity (gMFI) of CD25 expression of CD3 + gated T cells in CD3/CD28 activated PBMCs in the presence or absence of NKMV and NKExo and/or TGFβ/IL-10 (10 ng/ml each). (D) PD-1 gMFI on gated CD3 + T cells as in (C) . (E) Pearson correlation analysis of PD-1 gMFI and GRZB production. Results are shown for different healthy donors ( n = 3). Statistical significance was achieved by paired t -test. Means ± SD are plotted, * p

    Journal: Frontiers in Immunology

    Article Title: Natural-Killer-Derived Extracellular Vesicles: Immune Sensors and Interactors

    doi: 10.3389/fimmu.2020.00262

    Figure Lengend Snippet: Effects of natural killer cell-derived extracellular vesicles (NKEVs) on T cells. (A) Flow cytometry analysis of CD25 expression by CD3 + gated T cells in peripheral blood mononuclear cells (PBMCs) evaluated after 72 h of culture with NK-cell-derived microvesicle (NKMV) and NKExo. Left panels: representative dot plots of a healthy donor. Right panel: the graph shows the results obtained with PBMCs of different healthy donors ( n = 3), in the presence or absence of transforming growth factor beta (TGFβ)/interleukin (IL)-10 (10 ng/ml each). (B) Cytometric bead array (CBA)-measured cytokine production of 72 h PBMCs cultured as described in (A) . (C) Geo mean fluorescence intensity (gMFI) of CD25 expression of CD3 + gated T cells in CD3/CD28 activated PBMCs in the presence or absence of NKMV and NKExo and/or TGFβ/IL-10 (10 ng/ml each). (D) PD-1 gMFI on gated CD3 + T cells as in (C) . (E) Pearson correlation analysis of PD-1 gMFI and GRZB production. Results are shown for different healthy donors ( n = 3). Statistical significance was achieved by paired t -test. Means ± SD are plotted, * p

    Article Snippet: The following antibodies were used for cell labeling of peripheral blood lymphocytes (PBL), NK cells and PBMCs harvested from CD3/CD28-stimulated or unstimulated NKEV-PBMC cocultures, in the presence or absence of TGFβ/IL-10: CFSE (CellTrace, Thermo Fisher Scientific), CD3-ECD (Beckman Coulter), CD4-Alexa700 (Beckman Coulter), CD8-BV605 (Biolegend), CD56-BV510 (Becton Dickinson), CD16-BV650 (Biolegend), PD-1-PC7 (Beckman Coulter), CD25-PercpCy5.5 (Becton Dickinson), HLA-DR-APC (Beckman Coulter), and CD14-Alexa750 (Beckman Coulter).

    Techniques: Derivative Assay, Flow Cytometry, Expressing, Crocin Bleaching Assay, Cell Culture, Fluorescence

    Effects of natural killer cell-derived extracellular vesicles (NKEVs) on NK cells. (A) Upper panels: representative dot plots showing CD56 + total NK cells in the CD3 negative lymphocyte gate after culturing PBMCs for 72 h in the presence or absence of NK-cell-derived microvesicle (NKMV) and NKExo and/or transforming growth factor beta (TGFβ)/interleukin (IL)-10 (10 ng/ml each). Lower panels: representative dot plots showing the percentage of NK cell subpopulations in the CD56 + gate for each culture condition as in upper panels: CD56 bright , CD56 dim CD16 + , and CD56 dim CD16 neg NK cells. (B) Graphs show the results obtained for total NK cells and each subpopulation shown in (A) for different healthy donors ( n = 3). (C) Graphs show total NK cells and NK subpopulations after 72 h culture of the CD14 negative fraction in the presence or absence of NKMV or NKExo and/or CD3/CD28 stimulation. Results were obtained with cells from different donors ( n = 3). Statistical significance was achieved by paired t test. Means ± SD are plotted, * p

    Journal: Frontiers in Immunology

    Article Title: Natural-Killer-Derived Extracellular Vesicles: Immune Sensors and Interactors

    doi: 10.3389/fimmu.2020.00262

    Figure Lengend Snippet: Effects of natural killer cell-derived extracellular vesicles (NKEVs) on NK cells. (A) Upper panels: representative dot plots showing CD56 + total NK cells in the CD3 negative lymphocyte gate after culturing PBMCs for 72 h in the presence or absence of NK-cell-derived microvesicle (NKMV) and NKExo and/or transforming growth factor beta (TGFβ)/interleukin (IL)-10 (10 ng/ml each). Lower panels: representative dot plots showing the percentage of NK cell subpopulations in the CD56 + gate for each culture condition as in upper panels: CD56 bright , CD56 dim CD16 + , and CD56 dim CD16 neg NK cells. (B) Graphs show the results obtained for total NK cells and each subpopulation shown in (A) for different healthy donors ( n = 3). (C) Graphs show total NK cells and NK subpopulations after 72 h culture of the CD14 negative fraction in the presence or absence of NKMV or NKExo and/or CD3/CD28 stimulation. Results were obtained with cells from different donors ( n = 3). Statistical significance was achieved by paired t test. Means ± SD are plotted, * p

    Article Snippet: The following antibodies were used for cell labeling of peripheral blood lymphocytes (PBL), NK cells and PBMCs harvested from CD3/CD28-stimulated or unstimulated NKEV-PBMC cocultures, in the presence or absence of TGFβ/IL-10: CFSE (CellTrace, Thermo Fisher Scientific), CD3-ECD (Beckman Coulter), CD4-Alexa700 (Beckman Coulter), CD8-BV605 (Biolegend), CD56-BV510 (Becton Dickinson), CD16-BV650 (Biolegend), PD-1-PC7 (Beckman Coulter), CD25-PercpCy5.5 (Becton Dickinson), HLA-DR-APC (Beckman Coulter), and CD14-Alexa750 (Beckman Coulter).

    Techniques: Derivative Assay

    TNFR2 stimulation on human CD8 T cells increases ROS production via mitochondrial hyperpolarization and causes DNA damage. PBMCs from healthy donors were activated with plate-bound anti-CD3 and soluble anti-CD28 mAbs for seven days in the presence or absence of blocking TNF agents. Flow cytometry assessment of intracellular ROS geometric mean fluorescence in FACs-gated CD8 T cells stimulated in the presence or absence of etanercept or infliximab (A) , or selective blocking antibodies against TNFR1 or TNFR2 (B) (n=8). C , Flow cytometry measurement of TMRM geometric mean fluorescence of FACs-gated CD8 T cells that were stimulated in the presence or absence of TNF blocking agents (n=8). DNA damage was measured by intracellular staining of phosphorylated gH2AX on FACs gated CD8 T cells in the presence or absence of etanercept (n=17) or infliximab (n=14) (D) , or selective blocking antibodies against TNFR1 or TNFR2 (E) (n=12). CD8 T cells were pre-gated on Live+/CD3+/CD4-. Data for A-C are mean ± s.e.m. and P values were calculated using one-way ANOVA with Tukey's multiple comparsisions test. In D and E, the samples were compared by a paired two-tailed t-test.

    Journal: Theranostics

    Article Title: Human CD8 T cells are susceptible to TNF-mediated activation-induced cell death

    doi: 10.7150/thno.41646

    Figure Lengend Snippet: TNFR2 stimulation on human CD8 T cells increases ROS production via mitochondrial hyperpolarization and causes DNA damage. PBMCs from healthy donors were activated with plate-bound anti-CD3 and soluble anti-CD28 mAbs for seven days in the presence or absence of blocking TNF agents. Flow cytometry assessment of intracellular ROS geometric mean fluorescence in FACs-gated CD8 T cells stimulated in the presence or absence of etanercept or infliximab (A) , or selective blocking antibodies against TNFR1 or TNFR2 (B) (n=8). C , Flow cytometry measurement of TMRM geometric mean fluorescence of FACs-gated CD8 T cells that were stimulated in the presence or absence of TNF blocking agents (n=8). DNA damage was measured by intracellular staining of phosphorylated gH2AX on FACs gated CD8 T cells in the presence or absence of etanercept (n=17) or infliximab (n=14) (D) , or selective blocking antibodies against TNFR1 or TNFR2 (E) (n=12). CD8 T cells were pre-gated on Live+/CD3+/CD4-. Data for A-C are mean ± s.e.m. and P values were calculated using one-way ANOVA with Tukey's multiple comparsisions test. In D and E, the samples were compared by a paired two-tailed t-test.

    Article Snippet: Intracellular ROS and TMRM measurement For analysis of intracellular ROS, PBMCs were incubated with 5 μM of CellROS Deep Red reagent (ThermoFisher) for 30 min at 37°C.

    Techniques: Blocking Assay, Flow Cytometry, Fluorescence, FACS, Staining, Two Tailed Test