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Expression of cytotoxic molecules and proinflammatory cytokines in the total population of circulating <t>CD4</t> + T cells and the CD28 + and CD28null subsets within ESRD patients before kidney transplantation. In (a), a typical flow cytometric example is depicted with respect to the dissection of CD4 + T cells into those expressing CD28 (CD4 + CD28 + ) and those lacking CD28 (CD4 + CD28null). Next, we determined the cytotoxic potential by analyzing percentages of perforin + (b) and Granzyme B + (c) CD4 + T cells as well as those expressing or lacking CD28 in 11 ESRD patients known with atherosclerotic disease (CVDpos, closed bars) before transplantation and compared that to 27 age- and sex-matched ESRD patients, not known with preexisting atherosclerotic disease (CVDneg, open bars). In addition, <t>PBMC</t> of 8 CVDpos ESRD patients (closed bars) and 12 age- and sex-matched CVDneg patients (open bars) were stimulated with PMA and ionomycin in order to be able to analyze percentages of the proinflammatory cytokines TNF- α + (d) as well as IFN- γ + (e) within CD4 + and CD4 + CD28 + and CD4 + CD28null T cells.
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1) Product Images from "Circulating CD4+CD28null T Cells May Increase the Risk of an Atherosclerotic Vascular Event Shortly after Kidney Transplantation"

Article Title: Circulating CD4+CD28null T Cells May Increase the Risk of an Atherosclerotic Vascular Event Shortly after Kidney Transplantation

Journal: Journal of Transplantation

doi: 10.1155/2013/841430

Expression of cytotoxic molecules and proinflammatory cytokines in the total population of circulating CD4 + T cells and the CD28 + and CD28null subsets within ESRD patients before kidney transplantation. In (a), a typical flow cytometric example is depicted with respect to the dissection of CD4 + T cells into those expressing CD28 (CD4 + CD28 + ) and those lacking CD28 (CD4 + CD28null). Next, we determined the cytotoxic potential by analyzing percentages of perforin + (b) and Granzyme B + (c) CD4 + T cells as well as those expressing or lacking CD28 in 11 ESRD patients known with atherosclerotic disease (CVDpos, closed bars) before transplantation and compared that to 27 age- and sex-matched ESRD patients, not known with preexisting atherosclerotic disease (CVDneg, open bars). In addition, PBMC of 8 CVDpos ESRD patients (closed bars) and 12 age- and sex-matched CVDneg patients (open bars) were stimulated with PMA and ionomycin in order to be able to analyze percentages of the proinflammatory cytokines TNF- α + (d) as well as IFN- γ + (e) within CD4 + and CD4 + CD28 + and CD4 + CD28null T cells.
Figure Legend Snippet: Expression of cytotoxic molecules and proinflammatory cytokines in the total population of circulating CD4 + T cells and the CD28 + and CD28null subsets within ESRD patients before kidney transplantation. In (a), a typical flow cytometric example is depicted with respect to the dissection of CD4 + T cells into those expressing CD28 (CD4 + CD28 + ) and those lacking CD28 (CD4 + CD28null). Next, we determined the cytotoxic potential by analyzing percentages of perforin + (b) and Granzyme B + (c) CD4 + T cells as well as those expressing or lacking CD28 in 11 ESRD patients known with atherosclerotic disease (CVDpos, closed bars) before transplantation and compared that to 27 age- and sex-matched ESRD patients, not known with preexisting atherosclerotic disease (CVDneg, open bars). In addition, PBMC of 8 CVDpos ESRD patients (closed bars) and 12 age- and sex-matched CVDneg patients (open bars) were stimulated with PMA and ionomycin in order to be able to analyze percentages of the proinflammatory cytokines TNF- α + (d) as well as IFN- γ + (e) within CD4 + and CD4 + CD28 + and CD4 + CD28null T cells.

Techniques Used: Expressing, Transplantation Assay, Flow Cytometry, Dissection

2) Product Images from "Expression and Activation by Epstein Barr Virus of Human Endogenous Retroviruses-W in Blood Cells and Astrocytes: Inference for Multiple Sclerosis"

Article Title: Expression and Activation by Epstein Barr Virus of Human Endogenous Retroviruses-W in Blood Cells and Astrocytes: Inference for Multiple Sclerosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0044991

Expression of MSRVenv and syncytin-1 by PBMC subsets exposed to EBVgp350 or proinflammatory cytokines. A. Levels of MSRV env and syncytin-1 mRNAs of PBMC from MSRV(+) HD treated overnight with 1–100 ng/ml of EBVgp350, either as such or separated in T, B, NK and monocyte subsets. B. Comparison of the MSRVenv and syncytin-1 mRNA levels of monocytes and MDM after overnight EBVgp350 treatment. A and B: Data are the means of three experiments run in duplicate, calculated by the 2 −ΔΔCt method; C. Expression of the HERV-Wenv protein on the plasma membrane, evaluated by flow cytometry as present env-specific positivity of PBMC treated for 24 h with TNFα (1 ng/ml), IFNγ (1000 IU/ml), or PMA (50 NM); the bars indicate standard deviations.
Figure Legend Snippet: Expression of MSRVenv and syncytin-1 by PBMC subsets exposed to EBVgp350 or proinflammatory cytokines. A. Levels of MSRV env and syncytin-1 mRNAs of PBMC from MSRV(+) HD treated overnight with 1–100 ng/ml of EBVgp350, either as such or separated in T, B, NK and monocyte subsets. B. Comparison of the MSRVenv and syncytin-1 mRNA levels of monocytes and MDM after overnight EBVgp350 treatment. A and B: Data are the means of three experiments run in duplicate, calculated by the 2 −ΔΔCt method; C. Expression of the HERV-Wenv protein on the plasma membrane, evaluated by flow cytometry as present env-specific positivity of PBMC treated for 24 h with TNFα (1 ng/ml), IFNγ (1000 IU/ml), or PMA (50 NM); the bars indicate standard deviations.

Techniques Used: Expressing, Flow Cytometry, Cytometry

3) Product Images from "Dysregulated CD25 and Cytokine Expression by γδ T Cells of Systemic Sclerosis Patients Stimulated With Cardiolipin and Zoledronate"

Article Title: Dysregulated CD25 and Cytokine Expression by γδ T Cells of Systemic Sclerosis Patients Stimulated With Cardiolipin and Zoledronate

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00753

Cytokine production by systemic sclerosis (SSc) patients and healthy controls (HCs) T cells. Peripheral blood mononuclear cells from seven SSc patients (patients 13–19; Table 1 ) and five HC were cultured overnight in medium alone (FM), cardiolipin (CL), zoledronate (zol), or both (FM + zl or CL + zol). Subsequently, cell surface membranes were stained with monoclonal antibody (mAb) to CD3, Vγ9, or Vδ1, and cells were stained intracellularly with mAb to IL-4 (upper panel) or IFNγ. Bars indicate the mean ± 1 SEM of %cytokine + cells for the subsets as indicated. * p
Figure Legend Snippet: Cytokine production by systemic sclerosis (SSc) patients and healthy controls (HCs) T cells. Peripheral blood mononuclear cells from seven SSc patients (patients 13–19; Table 1 ) and five HC were cultured overnight in medium alone (FM), cardiolipin (CL), zoledronate (zol), or both (FM + zl or CL + zol). Subsequently, cell surface membranes were stained with monoclonal antibody (mAb) to CD3, Vγ9, or Vδ1, and cells were stained intracellularly with mAb to IL-4 (upper panel) or IFNγ. Bars indicate the mean ± 1 SEM of %cytokine + cells for the subsets as indicated. * p

Techniques Used: Cell Culture, Staining

4) Product Images from "Euglena gracilis paramylon activates human lymphocytes by upregulating pro‐inflammatory factors"

Article Title: Euglena gracilis paramylon activates human lymphocytes by upregulating pro‐inflammatory factors

Journal: Food Science & Nutrition

doi: 10.1002/fsn3.383

Nitric oxide production by peripheral blood mononuclear cell stimulated with glucans and lipopolysaccharide after 4 and 24 h, expressed as percent nitrite concentration versus control. ** = P
Figure Legend Snippet: Nitric oxide production by peripheral blood mononuclear cell stimulated with glucans and lipopolysaccharide after 4 and 24 h, expressed as percent nitrite concentration versus control. ** = P

Techniques Used: Concentration Assay

Relative expression of interleukin‐6, tumor necrosis factor alpha, cyclooxygenase 2, and inducible nitric oxide synthase in peripheral blood mononuclear cell after 24 h stimulation with glucans and lipopolysaccharide. Results were expressed as fold‐increase respect to control and plotted as the mean ± SD. ** = P
Figure Legend Snippet: Relative expression of interleukin‐6, tumor necrosis factor alpha, cyclooxygenase 2, and inducible nitric oxide synthase in peripheral blood mononuclear cell after 24 h stimulation with glucans and lipopolysaccharide. Results were expressed as fold‐increase respect to control and plotted as the mean ± SD. ** = P

Techniques Used: Expressing

5) Product Images from "Design, Expression, and Processing of Epitomized Hepatitis C Virus-Encoded CTL Epitopes"

Article Title: Design, Expression, and Processing of Epitomized Hepatitis C Virus-Encoded CTL Epitopes

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

doi:

T cell reactivity against a 406 ten-mer peptide set covering the top 95% sequence diversity: CTL responses to 406 ten-mer peptides were assessed by IFN- γ ELISPOT using a peptide matrix approach and PBMC after a 2-wk Ag-specific in vitro expansion. Responses are shown as either ( A ) number of individual responses against the entire set of 406 test peptides (i.e., counting each 10-mer and any of its reactive variants as individual responses) or ( B ) by counting all responses to 10-mer peptides covering the same 10-aaa long stretch as one response (i.e., responses to variant 10-mer peptides with an identical starting and ending position were counted only once, even if multiple variants scored positive in the same individual). Response rates in 40 HCV-infected subjects are compared with responses seen in 6 HCV-negative individuals. Only responses seen in HCV-infected individuals are graphed in C , showing the distribution of responses when responses to 10-mer peptides and their variant(s) are only scored once (filled histograms) and compared with the cumulative Shannon entropy for each 10-mer region (gray histograms; i.e., Shannon entropy was calculated for every residue, based on 165 sequences and cumulative values of each residues appearing within a 10-mer region were plotted). A statistically significant inverse correlation between the cumulative entropy of each 10-mer peptide and its frequency of recognition was observed ( p = 0.0011; two-sided Spearman rank test).
Figure Legend Snippet: T cell reactivity against a 406 ten-mer peptide set covering the top 95% sequence diversity: CTL responses to 406 ten-mer peptides were assessed by IFN- γ ELISPOT using a peptide matrix approach and PBMC after a 2-wk Ag-specific in vitro expansion. Responses are shown as either ( A ) number of individual responses against the entire set of 406 test peptides (i.e., counting each 10-mer and any of its reactive variants as individual responses) or ( B ) by counting all responses to 10-mer peptides covering the same 10-aaa long stretch as one response (i.e., responses to variant 10-mer peptides with an identical starting and ending position were counted only once, even if multiple variants scored positive in the same individual). Response rates in 40 HCV-infected subjects are compared with responses seen in 6 HCV-negative individuals. Only responses seen in HCV-infected individuals are graphed in C , showing the distribution of responses when responses to 10-mer peptides and their variant(s) are only scored once (filled histograms) and compared with the cumulative Shannon entropy for each 10-mer region (gray histograms; i.e., Shannon entropy was calculated for every residue, based on 165 sequences and cumulative values of each residues appearing within a 10-mer region were plotted). A statistically significant inverse correlation between the cumulative entropy of each 10-mer peptide and its frequency of recognition was observed ( p = 0.0011; two-sided Spearman rank test).

Techniques Used: Sequencing, CTL Assay, Enzyme-linked Immunospot, In Vitro, Variant Assay, Infection

6) Product Images from "Defective T-cell control of Epstein–Barr virus infection in multiple sclerosis"

Article Title: Defective T-cell control of Epstein–Barr virus infection in multiple sclerosis

Journal: Clinical & Translational Immunology

doi: 10.1038/cti.2016.87

Decreased CD8 + T-cell response to EBV lytic phase antigens throughout the course of MS. ( a ) Median percentages of CD8 + T cells of different phenotypes producing IFN-γ in response to a pool of HLA-class-I-restricted EBV lytic peptides in the PBMC in EBV-seropositive healthy controls (HC), the total group of MS patients (All MS), patients with clinically isolated syndrome (CIS), patients with relapsing–remitting (RR), secondary progressive (SP) and primary progressive (PP) MS, as well as patients during a clinical attack (Attack) and during remission (Remission). As expected, there was no CD4 + T-cell response to these HLA-class-I-restricted peptides. ( b ) Percentages of EBV-lytic-specific CD8 + T cells in the PBMC in the total group of patients (MS) compared with healthy controls (HC) (medians with interquartile ranges indicated by black horizontal lines), with bracketed P values determined by the Mann–Whitney test. ( c ) Percentages of EBV-lytic-specific CD8 + T cells in the PBMC in HLA-A*02 + patients (A2+ MS) compared with HLA-A*02 + healthy controls (A2+ HC) (medians with interquartile ranges indicated by black horizontal lines), with bracketed P values determined by the Mann–Whitney test. ( d ) Percentages of CD8 + T cells of different phenotypes producing IFN-γ in response to a pool of HLA-class-I-restricted CMV peptides in the PBMC in CMV-seropositive HC, and CMV-seropositive MS patients (MS) (medians with interquartile ranges indicated by black horizontal lines), with bracketed P -values determined by the Mann–Whitney test. There was no CD4 + T-cell response.
Figure Legend Snippet: Decreased CD8 + T-cell response to EBV lytic phase antigens throughout the course of MS. ( a ) Median percentages of CD8 + T cells of different phenotypes producing IFN-γ in response to a pool of HLA-class-I-restricted EBV lytic peptides in the PBMC in EBV-seropositive healthy controls (HC), the total group of MS patients (All MS), patients with clinically isolated syndrome (CIS), patients with relapsing–remitting (RR), secondary progressive (SP) and primary progressive (PP) MS, as well as patients during a clinical attack (Attack) and during remission (Remission). As expected, there was no CD4 + T-cell response to these HLA-class-I-restricted peptides. ( b ) Percentages of EBV-lytic-specific CD8 + T cells in the PBMC in the total group of patients (MS) compared with healthy controls (HC) (medians with interquartile ranges indicated by black horizontal lines), with bracketed P values determined by the Mann–Whitney test. ( c ) Percentages of EBV-lytic-specific CD8 + T cells in the PBMC in HLA-A*02 + patients (A2+ MS) compared with HLA-A*02 + healthy controls (A2+ HC) (medians with interquartile ranges indicated by black horizontal lines), with bracketed P values determined by the Mann–Whitney test. ( d ) Percentages of CD8 + T cells of different phenotypes producing IFN-γ in response to a pool of HLA-class-I-restricted CMV peptides in the PBMC in CMV-seropositive HC, and CMV-seropositive MS patients (MS) (medians with interquartile ranges indicated by black horizontal lines), with bracketed P -values determined by the Mann–Whitney test. There was no CD4 + T-cell response.

Techniques Used: Mass Spectrometry, Isolation, MANN-WHITNEY

Frequencies of T cells producing IFN-γ in response to autologous EBV-infected LCL. ( a ) Median percentages of T cells of different phenotypes producing IFN-γ in response to autologous EBV-infected LCL in the PBMC in EBV-seropositive healthy controls (HC), patients with clinically isolated syndrome (CIS), patients with relapsing–remitting (RR), secondary progressive (SP) and primary progressive (PP) MS, as well as patients during a clinical attack (Attack) and during remission (Remission). ( b ) Percentages of LCL-specific T cells in the PBMC in patients not having a clinical attack (Remission+SP+PP) compared with HC (medians with interquartile ranges indicated by black horizontal lines), with bracketed P values determined by the Mann–Whitney test. ( c ) Percentage of LCL-specific CD8 + EM/EMRA T cells in the PBMC plotted against the percentage of total CD8 + EM/EMRA T cells in the PBMC in healthy subjects (HC) and the total group of MS patients (MS). ( d ) Percentages of LCL-specific cells within the CD3 + , CD4 + , CD8 + , CD4 + EM, CD4 + EMRA + , CD4 + CM, CD4 + naive, CD8 + EM, CD8 + EMRA, CD8 + CM and CD8 + naive T-cell populations in patients during a clinical attack (Attack) compared with patients not having an attack (Remission+SP+PP) (medians with interquartile ranges indicated by black horizontal lines), with bracketed P values determined by the Mann–Whitney test. ( e ) The frequency of LCL-specific T cells within the CD8 + population strongly correlated with the frequency of LCL-specific T cells within the CD4 + population in MS patients whereas this correlation was weaker in healthy subjects. On multiple linear regression analysis, the slope of the regression line in the MS patients was significantly greater than that in healthy subjects ( P= 0.006).
Figure Legend Snippet: Frequencies of T cells producing IFN-γ in response to autologous EBV-infected LCL. ( a ) Median percentages of T cells of different phenotypes producing IFN-γ in response to autologous EBV-infected LCL in the PBMC in EBV-seropositive healthy controls (HC), patients with clinically isolated syndrome (CIS), patients with relapsing–remitting (RR), secondary progressive (SP) and primary progressive (PP) MS, as well as patients during a clinical attack (Attack) and during remission (Remission). ( b ) Percentages of LCL-specific T cells in the PBMC in patients not having a clinical attack (Remission+SP+PP) compared with HC (medians with interquartile ranges indicated by black horizontal lines), with bracketed P values determined by the Mann–Whitney test. ( c ) Percentage of LCL-specific CD8 + EM/EMRA T cells in the PBMC plotted against the percentage of total CD8 + EM/EMRA T cells in the PBMC in healthy subjects (HC) and the total group of MS patients (MS). ( d ) Percentages of LCL-specific cells within the CD3 + , CD4 + , CD8 + , CD4 + EM, CD4 + EMRA + , CD4 + CM, CD4 + naive, CD8 + EM, CD8 + EMRA, CD8 + CM and CD8 + naive T-cell populations in patients during a clinical attack (Attack) compared with patients not having an attack (Remission+SP+PP) (medians with interquartile ranges indicated by black horizontal lines), with bracketed P values determined by the Mann–Whitney test. ( e ) The frequency of LCL-specific T cells within the CD8 + population strongly correlated with the frequency of LCL-specific T cells within the CD4 + population in MS patients whereas this correlation was weaker in healthy subjects. On multiple linear regression analysis, the slope of the regression line in the MS patients was significantly greater than that in healthy subjects ( P= 0.006).

Techniques Used: Infection, Isolation, Mass Spectrometry, MANN-WHITNEY

The relationships of EBV genome load and anti-EBV antibody titres with the frequency of EBV-specific CD8 + T cells in MS. ( a ) EBV DNA copy number in the PBMC in healthy EBV-seropositive subjects (healthy controls (HC)) and patients with MS (MS) (medians with interquartile ranges indicated by black horizontal lines), with P- value determined by the Mann–Whitney test. ( b ) Relationship between the EBV genome load and the LCL-specific CD8 + T-cell frequency in MS patients. ( c ) Titres of anti-EBNA1 IgG and anti-VCA IgG in the sera of healthy EBV-seropositive subjects (HC) and patients with MS (MS) (medians with interquartile ranges indicated by black horizontal lines), with bracketed P- values determined by the Mann–Whitney test. ( d ) Relationship between the anti-EBNA1 IgG titre and the LCL-specific CD8 + EMRA T-cell frequency in the PBMC in MS patients. ( e ) Relationship between the anti-VCA IgG titre and the LCL-specific CD8 + EMRA T-cell frequency in the PBMC in MS patients ( f ) Relationship between the anti-VCA IgG titre and the EBV genome load in HC and patients with MS (MS). ( g ) Relationship between the anti-VCA IgG titre and the EBV genome load in the combined groups of HC and patients with MS. ( h ) Relationship between the anti-EBNA1 IgG titre and the EBV genome load in HC and patients with MS.
Figure Legend Snippet: The relationships of EBV genome load and anti-EBV antibody titres with the frequency of EBV-specific CD8 + T cells in MS. ( a ) EBV DNA copy number in the PBMC in healthy EBV-seropositive subjects (healthy controls (HC)) and patients with MS (MS) (medians with interquartile ranges indicated by black horizontal lines), with P- value determined by the Mann–Whitney test. ( b ) Relationship between the EBV genome load and the LCL-specific CD8 + T-cell frequency in MS patients. ( c ) Titres of anti-EBNA1 IgG and anti-VCA IgG in the sera of healthy EBV-seropositive subjects (HC) and patients with MS (MS) (medians with interquartile ranges indicated by black horizontal lines), with bracketed P- values determined by the Mann–Whitney test. ( d ) Relationship between the anti-EBNA1 IgG titre and the LCL-specific CD8 + EMRA T-cell frequency in the PBMC in MS patients. ( e ) Relationship between the anti-VCA IgG titre and the LCL-specific CD8 + EMRA T-cell frequency in the PBMC in MS patients ( f ) Relationship between the anti-VCA IgG titre and the EBV genome load in HC and patients with MS (MS). ( g ) Relationship between the anti-VCA IgG titre and the EBV genome load in the combined groups of HC and patients with MS. ( h ) Relationship between the anti-EBNA1 IgG titre and the EBV genome load in HC and patients with MS.

Techniques Used: Mass Spectrometry, MANN-WHITNEY

7) Product Images from "Heterologous prime-boost-boost immunisation of Chinese cynomolgus macaques using DNA and recombinant poxvirus vectors expressing HIV-1 virus-like particles"

Article Title: Heterologous prime-boost-boost immunisation of Chinese cynomolgus macaques using DNA and recombinant poxvirus vectors expressing HIV-1 virus-like particles

Journal: Virology Journal

doi: 10.1186/1743-422X-8-429

HIV-specific T cell responses by IFN-γ ELISpots at week 9 . (A) Pre-immunised and post-immunised Env-specific peripheral blood mononuclear cell (PBMC) responses; (B) pre-immunised and post-immunised Gag-specific PBMC responses; (C) Env-specific splenocyte responses; (D) Gag-specific splenocyte responses; (E) Env-specific axillary and inguinal lymph node responses and (F) Gag-specific axillary and inguinal lymph node responses. Data is duplicated and presented as the mean number of spot-forming units/10 6 cells ± SEM.
Figure Legend Snippet: HIV-specific T cell responses by IFN-γ ELISpots at week 9 . (A) Pre-immunised and post-immunised Env-specific peripheral blood mononuclear cell (PBMC) responses; (B) pre-immunised and post-immunised Gag-specific PBMC responses; (C) Env-specific splenocyte responses; (D) Gag-specific splenocyte responses; (E) Env-specific axillary and inguinal lymph node responses and (F) Gag-specific axillary and inguinal lymph node responses. Data is duplicated and presented as the mean number of spot-forming units/10 6 cells ± SEM.

Techniques Used:

8) Product Images from "Induction of Potent Immune Responses by Cationic Microparticles with Adsorbed Human Immunodeficiency Virus DNA Vaccines"

Article Title: Induction of Potent Immune Responses by Cationic Microparticles with Adsorbed Human Immunodeficiency Virus DNA Vaccines

Journal: Journal of Virology

doi: 10.1128/JVI.75.19.9037-9043.2001

Gag-specific cytolytic activity of PBMC from individual rhesus macaques given two doses of PLG/CTAB-formulated pCMV-gag DNA (A) or unformulated pCMV-gag DNA (B). Two weeks after the second DNA immunization, PBMC were stimulated with a pool of overlapping Gag peptides and cultured in the presence of IL-2 for 8 days. Serial dilutions of cultures (1:15, 1:45, 1:135, and 1:405) were added to 51 Cr-labeled rVVgagpol SF2 -infected autologous B-LCL (●) or rVVgp160env SF162 -infected autologous B-LCL (○). 51 Cr release was determined 4 h after addition of cultured PBMC to B-LCL.
Figure Legend Snippet: Gag-specific cytolytic activity of PBMC from individual rhesus macaques given two doses of PLG/CTAB-formulated pCMV-gag DNA (A) or unformulated pCMV-gag DNA (B). Two weeks after the second DNA immunization, PBMC were stimulated with a pool of overlapping Gag peptides and cultured in the presence of IL-2 for 8 days. Serial dilutions of cultures (1:15, 1:45, 1:135, and 1:405) were added to 51 Cr-labeled rVVgagpol SF2 -infected autologous B-LCL (●) or rVVgp160env SF162 -infected autologous B-LCL (○). 51 Cr release was determined 4 h after addition of cultured PBMC to B-LCL.

Techniques Used: Activity Assay, Cell Culture, Labeling, Infection

9) Product Images from "Salmonella Flagellin Induces Tumor Necrosis Factor Alpha in a Human Promonocytic Cell Line"

Article Title: Salmonella Flagellin Induces Tumor Necrosis Factor Alpha in a Human Promonocytic Cell Line

Journal: Infection and Immunity

doi:

Effect of purified flagella on TNF-α expression in U38 cells and LPS-tolerant PBMC. Flagellar structures were isolated from Salmonella strain CD5 and tested on 3 × 10 6 U38 cells or PBMC at various concentrations. The curves shown are from two representative experiments. Control TNF-α levels were
Figure Legend Snippet: Effect of purified flagella on TNF-α expression in U38 cells and LPS-tolerant PBMC. Flagellar structures were isolated from Salmonella strain CD5 and tested on 3 × 10 6 U38 cells or PBMC at various concentrations. The curves shown are from two representative experiments. Control TNF-α levels were

Techniques Used: Purification, Expressing, Isolation

10) Product Images from "Generation of Th1 T cell responses directed to a HLA Class II restricted epitope from the Aspergillus f16 allergen"

Article Title: Generation of Th1 T cell responses directed to a HLA Class II restricted epitope from the Aspergillus f16 allergen

Journal: Clinical and Experimental Immunology

doi: 10.1111/j.1365-2249.2005.02699.x

Proliferative response to Asp f16 protein-derived overlapping pentadecapeptides. (a) PBMC from five different donors were stimulated with soluble Asp f16-PPC (not presented on autologous DC) in the presence or absence of IL-2 added at day 3 of a 6-day
Figure Legend Snippet: Proliferative response to Asp f16 protein-derived overlapping pentadecapeptides. (a) PBMC from five different donors were stimulated with soluble Asp f16-PPC (not presented on autologous DC) in the presence or absence of IL-2 added at day 3 of a 6-day

Techniques Used: Derivative Assay

11) Product Images from "A blood dendritic cell vaccine for acute myeloid leukemia expands anti-tumor T cell responses at remission"

Article Title: A blood dendritic cell vaccine for acute myeloid leukemia expands anti-tumor T cell responses at remission

Journal: Oncoimmunology

doi: 10.1080/2162402X.2017.1419114

T Cell Subset Distribution and Function in AML Patients is Affected by Prior Therapy. (A) Total T cells (CD3 + ), CD4 + T cells (CD3 + CD4 + CD8 − ) and CD8 + T cells (CD3 + CD4 − CD8 + ) were enumerated in AML patients in CR following chemotherapy with NFR regimens (n = 7) or FR regimens (FR, n = 4) or > 12 months post-allogeneic stem cell transplant (ATx, n = 7), compared to age-matched HD (n = 8). PBMC were thawed and analysed by flow cytometry. Enumeration was estimated based on cell subset frequency and the lymphocyte count recorded at the time of sample collection. (B) CD8 + T cell subsets (naïve CD28 + CD45RO − CD57 − , memory CD28 + CD45RO + CD57 +/− , effector/effector memory CD28 − CD45RO + CD57 + and terminally differentiated CD28 − CD45RO − CD57 + ) were detected in the experiment shown in (A). (C) Thawed PBMC were rested overnight then stimulated with 50ng/mL PMA and 1ug/ml ionomycin or (D) anti-CD2/CD3/CD28 beads (1 bead: 2 PBMC) for 5 hours, with brefeldin A and monensin added for the final 4 hours. IFN-gamma production by CD4 + and CD8 + T cells was evaluated by flow cytometry. AICD was calculated as % loss of viable CD3 + T cells (determined by Fixable Live/Dead Violet staining) in the PBMC samples in stimulated versus unstimulated conditions. Each data set was tested for normality by the Shapiro-Wilk normality test and analysed by standard one-way ANOVA with Dunnett's multiple comparison test or Kruskal-Wallis Test with Dunn's multiple comparison test as appropriate. # p
Figure Legend Snippet: T Cell Subset Distribution and Function in AML Patients is Affected by Prior Therapy. (A) Total T cells (CD3 + ), CD4 + T cells (CD3 + CD4 + CD8 − ) and CD8 + T cells (CD3 + CD4 − CD8 + ) were enumerated in AML patients in CR following chemotherapy with NFR regimens (n = 7) or FR regimens (FR, n = 4) or > 12 months post-allogeneic stem cell transplant (ATx, n = 7), compared to age-matched HD (n = 8). PBMC were thawed and analysed by flow cytometry. Enumeration was estimated based on cell subset frequency and the lymphocyte count recorded at the time of sample collection. (B) CD8 + T cell subsets (naïve CD28 + CD45RO − CD57 − , memory CD28 + CD45RO + CD57 +/− , effector/effector memory CD28 − CD45RO + CD57 + and terminally differentiated CD28 − CD45RO − CD57 + ) were detected in the experiment shown in (A). (C) Thawed PBMC were rested overnight then stimulated with 50ng/mL PMA and 1ug/ml ionomycin or (D) anti-CD2/CD3/CD28 beads (1 bead: 2 PBMC) for 5 hours, with brefeldin A and monensin added for the final 4 hours. IFN-gamma production by CD4 + and CD8 + T cells was evaluated by flow cytometry. AICD was calculated as % loss of viable CD3 + T cells (determined by Fixable Live/Dead Violet staining) in the PBMC samples in stimulated versus unstimulated conditions. Each data set was tested for normality by the Shapiro-Wilk normality test and analysed by standard one-way ANOVA with Dunnett's multiple comparison test or Kruskal-Wallis Test with Dunn's multiple comparison test as appropriate. # p

Techniques Used: Flow Cytometry, Cytometry, Staining

12) Product Images from "Rheumatoid arthritis fibroblast‐like synoviocytes co‐cultured with PBMC increased peripheral CD4+CXCR5+ICOS+ T cell numbers"

Article Title: Rheumatoid arthritis fibroblast‐like synoviocytes co‐cultured with PBMC increased peripheral CD4+CXCR5+ICOS+ T cell numbers

Journal: Clinical and Experimental Immunology

doi: 10.1111/cei.13025

Rheumatoid arthritis fibroblast‐like synoviocytes (RA‐FLS), co‐cultured with activated peripheral blood mononuclear cells (PBMC), increased the number of peripheral CD4 + chemokine receptor 5 (CXCR5) + ‐inducible co‐stimulatory molecule (ICOS) + T cells. (a) Anti‐CD3/CD28‐stimulated PBMC was cultured with or without RA‐FLS for 72 h in cell‐to‐cell contact and the Transwell system; peripheral CD4 + CXCR5 + ICOS + T cell frequency in RA patients was assayed by flow cytometric analysis ( n = 10). (b) Interleukin (IL)‐21 level in co‐cultured supernatants was detected by enzyme‐linked immunosorbent assay (ELISA) ( n = 10); (c) mRNA expressions of IL‐21 and B cell lymphoma (Bcl)‐6 in PBMC were tested by reverse transcription–polymerase chain reaction (RT–PCR) ( n = 5). * P
Figure Legend Snippet: Rheumatoid arthritis fibroblast‐like synoviocytes (RA‐FLS), co‐cultured with activated peripheral blood mononuclear cells (PBMC), increased the number of peripheral CD4 + chemokine receptor 5 (CXCR5) + ‐inducible co‐stimulatory molecule (ICOS) + T cells. (a) Anti‐CD3/CD28‐stimulated PBMC was cultured with or without RA‐FLS for 72 h in cell‐to‐cell contact and the Transwell system; peripheral CD4 + CXCR5 + ICOS + T cell frequency in RA patients was assayed by flow cytometric analysis ( n = 10). (b) Interleukin (IL)‐21 level in co‐cultured supernatants was detected by enzyme‐linked immunosorbent assay (ELISA) ( n = 10); (c) mRNA expressions of IL‐21 and B cell lymphoma (Bcl)‐6 in PBMC were tested by reverse transcription–polymerase chain reaction (RT–PCR) ( n = 5). * P

Techniques Used: Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

Increased reactive oxygen species (ROS) generation in co‐cultured peripheral blood mononuclear cells (PBMC) and the rheumatoid arthritis fibroblast‐like synoviocytes (RA‐FLS) system caused higher levels of interleukin (IL)‐6 production. (a) RA‐FLS was cultured with or without anti‐CD3/CD28‐stimulated PBMC for 72 h, ROS levels of RA‐FLS were detected by flow cytometric analysis ( n = 10) and cell staining ( n = 5); (b) anti‐CD3/CD28‐stimulated PBMC was co‐cultured with or without RA‐FLS for 72 h, ROS levels of PBMC were tested by flow cytometric analysis ( n = 10) and cell staining ( n = 5); (c) N‐acetyl‐L‐cysteine (NAC) was added into the co‐culture system of RA‐FLS and activated PBMC for 72 h and the IL‐6 level in supernatants was detected by enzyme‐linked immunosorbent assay (ELISA) ( n = 10); (d) the percentage of CD4 + chemokine receptor 5 (CXCR5) + inducible co‐stimulatory molecule (ICOS) + T cells of PBMC was tested by flow cytometric analysis ( n = 10); (e) IL‐21 level in the supernatants was measured by ELISA ( n = 10) and the mRNA expressions of IL‐21 and B cell lymphoma (Bcl)‐6 in cultured PBMC were assessed by reverse transcription–polymerase chain reaction (RT–PCR) ( n = 5). * P
Figure Legend Snippet: Increased reactive oxygen species (ROS) generation in co‐cultured peripheral blood mononuclear cells (PBMC) and the rheumatoid arthritis fibroblast‐like synoviocytes (RA‐FLS) system caused higher levels of interleukin (IL)‐6 production. (a) RA‐FLS was cultured with or without anti‐CD3/CD28‐stimulated PBMC for 72 h, ROS levels of RA‐FLS were detected by flow cytometric analysis ( n = 10) and cell staining ( n = 5); (b) anti‐CD3/CD28‐stimulated PBMC was co‐cultured with or without RA‐FLS for 72 h, ROS levels of PBMC were tested by flow cytometric analysis ( n = 10) and cell staining ( n = 5); (c) N‐acetyl‐L‐cysteine (NAC) was added into the co‐culture system of RA‐FLS and activated PBMC for 72 h and the IL‐6 level in supernatants was detected by enzyme‐linked immunosorbent assay (ELISA) ( n = 10); (d) the percentage of CD4 + chemokine receptor 5 (CXCR5) + inducible co‐stimulatory molecule (ICOS) + T cells of PBMC was tested by flow cytometric analysis ( n = 10); (e) IL‐21 level in the supernatants was measured by ELISA ( n = 10) and the mRNA expressions of IL‐21 and B cell lymphoma (Bcl)‐6 in cultured PBMC were assessed by reverse transcription–polymerase chain reaction (RT–PCR) ( n = 5). * P

Techniques Used: Cell Culture, Flow Cytometry, Staining, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

Rheumatoid arthritis fibroblast‐like synoviocytes (RA‐FLS) co‐cultured with stimulated peripheral blood mononuclear cells (PBMC) up‐regulated CD4 + chemokine receptor 5 (CXCR5) + inducible co‐stimulatory molecule (ICOS) + T cells possibly through increased interleukin (IL)‐6 secretion. (a) Anti‐CD3/CD28‐stimulated PBMC was cultured with or without RA‐FLS for 72 h, and then 33 cytokine levels in supernatants were assayed by human cytokine antibody array AAH‐TH17‐G1; (b) IL‐6 and IL‐12 levels in co‐cultured supernatants were detected by enzyme‐linked immunosorbent assay (ELISA) ( n = 10); (c) after anti‐IL‐6R antibody was added into the co‐culture system for 72 h, the percentage of CD4 + CXCR5 + ICOS + T cells was analysed by flow cytometric analysis ( n = 10). The IL‐21 level in supernatants was detected by ELISA ( n = 10) and the mRNA expressions of IL‐21 and B cell lymphoma (Bcl)‐6 in PBMC were measured by reverse transcription–polymerase chain reaction (RT–PCR) ( n = 5); (d) after anti‐IL‐12R antibody was added into the co‐culture system for 72 h, CD4 + CXCR5 + ICOS + T cell frequency was assayed by flow cytometric analysis ( n = 10). The IL‐21 level in supernatants was measured by ELISA ( n = 10) and the mRNA expressions of IL‐21 and Bcl‐6 in PBMC were tested by RT–PCR ( n = 5). * P
Figure Legend Snippet: Rheumatoid arthritis fibroblast‐like synoviocytes (RA‐FLS) co‐cultured with stimulated peripheral blood mononuclear cells (PBMC) up‐regulated CD4 + chemokine receptor 5 (CXCR5) + inducible co‐stimulatory molecule (ICOS) + T cells possibly through increased interleukin (IL)‐6 secretion. (a) Anti‐CD3/CD28‐stimulated PBMC was cultured with or without RA‐FLS for 72 h, and then 33 cytokine levels in supernatants were assayed by human cytokine antibody array AAH‐TH17‐G1; (b) IL‐6 and IL‐12 levels in co‐cultured supernatants were detected by enzyme‐linked immunosorbent assay (ELISA) ( n = 10); (c) after anti‐IL‐6R antibody was added into the co‐culture system for 72 h, the percentage of CD4 + CXCR5 + ICOS + T cells was analysed by flow cytometric analysis ( n = 10). The IL‐21 level in supernatants was detected by ELISA ( n = 10) and the mRNA expressions of IL‐21 and B cell lymphoma (Bcl)‐6 in PBMC were measured by reverse transcription–polymerase chain reaction (RT–PCR) ( n = 5); (d) after anti‐IL‐12R antibody was added into the co‐culture system for 72 h, CD4 + CXCR5 + ICOS + T cell frequency was assayed by flow cytometric analysis ( n = 10). The IL‐21 level in supernatants was measured by ELISA ( n = 10) and the mRNA expressions of IL‐21 and Bcl‐6 in PBMC were tested by RT–PCR ( n = 5). * P

Techniques Used: Cell Culture, Ab Array, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction

Anti‐interleukin (IL)‐1βR antibody down‐regulated CD4 + chemokine receptor 5 (CXCR5) + ‐inducible co‐stimulatory molecule (ICOS) + T cells in the co‐culture system. (a) Co‐cultured supernatant of IL‐1β level was detected by enzyme‐linked immunosorbent assay (ELISA) ( n = 10); (b) after anti‐IL‐1βR antibody was added into the co‐culture system for 72 h, reactive oxygen species (ROS) levels of rheumatoid arthritis fibroblast‐like synoviocytes (RA‐FLS) cultured with or without stimulated peripheral blood mononuclear cells (PBMC) were detected by flow cytometric analysis ( n = 10) and cell staining ( n = 5); (c) ROS levels of anti‐CD3/CD28‐stimulated PBMC co‐cultured with or without RA‐FLS were tested by flow cytometric analysis ( n = 10) and cell staining ( n = 5); (d) the IL‐6 level in supernatants was tested by ELISA ( n = 10); (e) CD4 + CXCR5 + ICOS + T cell frequency was assayed by flow cytometric analysis ( n = 10); IL‐21 level in supernatants was detected by ELISA ( n = 10) and the mRNA expressions of IL‐21 and B cell lymphoma (Bcl‐6) were tested by reverse transcription–polymerase chain reaction (RT–PCR) ( n = 5). * P
Figure Legend Snippet: Anti‐interleukin (IL)‐1βR antibody down‐regulated CD4 + chemokine receptor 5 (CXCR5) + ‐inducible co‐stimulatory molecule (ICOS) + T cells in the co‐culture system. (a) Co‐cultured supernatant of IL‐1β level was detected by enzyme‐linked immunosorbent assay (ELISA) ( n = 10); (b) after anti‐IL‐1βR antibody was added into the co‐culture system for 72 h, reactive oxygen species (ROS) levels of rheumatoid arthritis fibroblast‐like synoviocytes (RA‐FLS) cultured with or without stimulated peripheral blood mononuclear cells (PBMC) were detected by flow cytometric analysis ( n = 10) and cell staining ( n = 5); (c) ROS levels of anti‐CD3/CD28‐stimulated PBMC co‐cultured with or without RA‐FLS were tested by flow cytometric analysis ( n = 10) and cell staining ( n = 5); (d) the IL‐6 level in supernatants was tested by ELISA ( n = 10); (e) CD4 + CXCR5 + ICOS + T cell frequency was assayed by flow cytometric analysis ( n = 10); IL‐21 level in supernatants was detected by ELISA ( n = 10) and the mRNA expressions of IL‐21 and B cell lymphoma (Bcl‐6) were tested by reverse transcription–polymerase chain reaction (RT–PCR) ( n = 5). * P

Techniques Used: Co-Culture Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining, Reverse Transcription Polymerase Chain Reaction

Anti‐tumour necrosis factor receptor 2 (TNF‐R2) antibody down‐regulated CD4 + chemokine receptor 5 (CXCR5) + ‐inducible co‐stimulatory molecule (ICOS) + T cells in the co‐culture system. (a) Rheumatoid arthritis fibroblast‐like synoviocytes (RA‐FLS) and anti‐CD3/CD28‐stimulated peripheral blood mononuclear cells (PBMC) were co‐cultured for 72 h and the TNF‐α level in supernatants was detected by enzyme‐linked immunosorbent assay (ELISA) ( n = 10); (b) after anti‐TNF‐R2 antibody was added into the co‐culture system for 72 h, ROS levels of RA‐FLS cultured with or without stimulated PBMC were detected by flow cytometric analysis ( n = 10) and cell staining ( n = 5); (c) ROS levels of anti‐CD3/CD28‐stimulated PBMC co‐cultured with or without RA‐FLS were tested by flow cytometric analysis ( n = 10) and cell staining ( n = 5); (d) interleukin (IL)‐6 level in supernatants was tested by ELISA ( n = 10); (e) CD4 + CXCR5 + ICOS + T cell frequency was assayed by flow cytometric analysis ( n = 10); the IL‐21 level in supernatants was detected by ELISA ( n = 10) and the mRNA expressions of IL‐21 and B cell lymphoma (Bcl)‐6 were tested by reverse transcription–polymerase chain reaction (RT–PCR) ( n = 5). * P
Figure Legend Snippet: Anti‐tumour necrosis factor receptor 2 (TNF‐R2) antibody down‐regulated CD4 + chemokine receptor 5 (CXCR5) + ‐inducible co‐stimulatory molecule (ICOS) + T cells in the co‐culture system. (a) Rheumatoid arthritis fibroblast‐like synoviocytes (RA‐FLS) and anti‐CD3/CD28‐stimulated peripheral blood mononuclear cells (PBMC) were co‐cultured for 72 h and the TNF‐α level in supernatants was detected by enzyme‐linked immunosorbent assay (ELISA) ( n = 10); (b) after anti‐TNF‐R2 antibody was added into the co‐culture system for 72 h, ROS levels of RA‐FLS cultured with or without stimulated PBMC were detected by flow cytometric analysis ( n = 10) and cell staining ( n = 5); (c) ROS levels of anti‐CD3/CD28‐stimulated PBMC co‐cultured with or without RA‐FLS were tested by flow cytometric analysis ( n = 10) and cell staining ( n = 5); (d) interleukin (IL)‐6 level in supernatants was tested by ELISA ( n = 10); (e) CD4 + CXCR5 + ICOS + T cell frequency was assayed by flow cytometric analysis ( n = 10); the IL‐21 level in supernatants was detected by ELISA ( n = 10) and the mRNA expressions of IL‐21 and B cell lymphoma (Bcl)‐6 were tested by reverse transcription–polymerase chain reaction (RT–PCR) ( n = 5). * P

Techniques Used: Co-Culture Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining, Reverse Transcription Polymerase Chain Reaction

13) Product Images from "Characterization and Functional Properties of Gastric Tissue-Resident Memory T Cells from Children, Adults, and the Elderly"

Article Title: Characterization and Functional Properties of Gastric Tissue-Resident Memory T Cells from Children, Adults, and the Elderly

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2014.00294

Activation of CD8 + and CD4 + tissue-resident memory T (T RM ) cells in gastric LPMC . Representative plot of the activation of gastric LPMC CD8 + (A) or CD4 + (B) T RM (CD62L − CD45RA − CD69 + CD103 + ) by two stimulants: (1) staphylococcal enterotoxin B (SEB; 10 μg/ml) and (2) anti-CD3/CD28 beads (α-CD3 α-CD28) to produce IL-2, IFN-γ, MIP-1β, TNF-α, IL-17A, and up-regulation of the expression of CD107a. Cells left unstimulated were used as negative control (Media, C-). Cumulative data comparing baseline activation levels of gastric LPMC CD8 + (C) and CD4 + (D) T RM (white portion of the bar) to PBMC (black portion of the bar). In (C,D) significant differences between T EM and T RM are indicated with asterisks on top of each bar; * p
Figure Legend Snippet: Activation of CD8 + and CD4 + tissue-resident memory T (T RM ) cells in gastric LPMC . Representative plot of the activation of gastric LPMC CD8 + (A) or CD4 + (B) T RM (CD62L − CD45RA − CD69 + CD103 + ) by two stimulants: (1) staphylococcal enterotoxin B (SEB; 10 μg/ml) and (2) anti-CD3/CD28 beads (α-CD3 α-CD28) to produce IL-2, IFN-γ, MIP-1β, TNF-α, IL-17A, and up-regulation of the expression of CD107a. Cells left unstimulated were used as negative control (Media, C-). Cumulative data comparing baseline activation levels of gastric LPMC CD8 + (C) and CD4 + (D) T RM (white portion of the bar) to PBMC (black portion of the bar). In (C,D) significant differences between T EM and T RM are indicated with asterisks on top of each bar; * p

Techniques Used: Activation Assay, Expressing, Negative Control

Multifunctional gastric LPMC CD4 + T RM and PBMC CD4 + T EM responses to SEB stimulation in adults, children, and the elderly . Multifunctionality was determined by simultaneous detection of two or more functions performed by CD4 + T RM (LPMC) or CD4 + T EM (PBMC). Six functions were evaluated: production of five cytokines/chemokines (IFN-γ, TNF-α, IL-2, IL-17A, MIP-1β) and expression of CD107a in response to SEB stimulation. (A) Scatter plot showing the six predominant function patterns in LPMC CD4 + T RM and (B) in PBMC CD4 + T EM cells from adults (red circles, n = 9), children (black squares, n = 7), and the elderly (blue triangles, n = 8). Multifunctionality was analyzed using the FCOM feature of WinList. Significant differences between age groups were denoted by asterisks (* p
Figure Legend Snippet: Multifunctional gastric LPMC CD4 + T RM and PBMC CD4 + T EM responses to SEB stimulation in adults, children, and the elderly . Multifunctionality was determined by simultaneous detection of two or more functions performed by CD4 + T RM (LPMC) or CD4 + T EM (PBMC). Six functions were evaluated: production of five cytokines/chemokines (IFN-γ, TNF-α, IL-2, IL-17A, MIP-1β) and expression of CD107a in response to SEB stimulation. (A) Scatter plot showing the six predominant function patterns in LPMC CD4 + T RM and (B) in PBMC CD4 + T EM cells from adults (red circles, n = 9), children (black squares, n = 7), and the elderly (blue triangles, n = 8). Multifunctionality was analyzed using the FCOM feature of WinList. Significant differences between age groups were denoted by asterisks (* p

Techniques Used: Expressing

Characterization of memory T (T M ) cells in gastric LPMC and PBMC from children, adults, and the elderly . (A) Representative scatter plots of the gating strategy used to characterize T cell subsets in LPMC and PBMC: naïve (CD62L + CD45RA + ), central memory (T CM , CD62L + CD45RA − ), effector memory (T EM , CD62L − CD45RA − ), and effector memory expressing CD45RA (T EMRA , CD62L − CD45RA + ). Data shown are from a 12 year-old child. (B) Cumulative data showing the median % and range of CD3, CD4, CD8, T EM populations in gastric LPMC and PBMC from all three age groups. (C) Identification of tissue-resident memory T (T RM ) cells in gastric LPMC. Representative plots showing expression of T RM cells as defined by the concomitant expression of CD103 and CD69 markers on CD8 + and CD4 + T cells in gastric LPMC and PBMC. (D) Cumulative data ( n = 10) showing the percentage of gastric T RM cells among CD8 + and CD4 + T cells from LPMC and PBMC (*** p
Figure Legend Snippet: Characterization of memory T (T M ) cells in gastric LPMC and PBMC from children, adults, and the elderly . (A) Representative scatter plots of the gating strategy used to characterize T cell subsets in LPMC and PBMC: naïve (CD62L + CD45RA + ), central memory (T CM , CD62L + CD45RA − ), effector memory (T EM , CD62L − CD45RA − ), and effector memory expressing CD45RA (T EMRA , CD62L − CD45RA + ). Data shown are from a 12 year-old child. (B) Cumulative data showing the median % and range of CD3, CD4, CD8, T EM populations in gastric LPMC and PBMC from all three age groups. (C) Identification of tissue-resident memory T (T RM ) cells in gastric LPMC. Representative plots showing expression of T RM cells as defined by the concomitant expression of CD103 and CD69 markers on CD8 + and CD4 + T cells in gastric LPMC and PBMC. (D) Cumulative data ( n = 10) showing the percentage of gastric T RM cells among CD8 + and CD4 + T cells from LPMC and PBMC (*** p

Techniques Used: Expressing

Characterization of integrin α4β7 expression on memory T (T RM and T EM ) cells in gastric LPMC and PBMC . (A) Representative scatter plots showing integrin α4β7 expression on CD8 + and CD4 + gastric LPMC and PBMC (data shown are from a 12-year-old child). (B) Cumulative data ( n = 14) showing the percentage of CD8 + and CD4 + T RM (LPMC) as well as CD8 + and CD4 + T EM (PBMC) cells expressing integrin α4β7. Significant differences are denoted as follows: ** p
Figure Legend Snippet: Characterization of integrin α4β7 expression on memory T (T RM and T EM ) cells in gastric LPMC and PBMC . (A) Representative scatter plots showing integrin α4β7 expression on CD8 + and CD4 + gastric LPMC and PBMC (data shown are from a 12-year-old child). (B) Cumulative data ( n = 14) showing the percentage of CD8 + and CD4 + T RM (LPMC) as well as CD8 + and CD4 + T EM (PBMC) cells expressing integrin α4β7. Significant differences are denoted as follows: ** p

Techniques Used: Expressing

Multifunctional gastric LPMC CD8 + T RM and PBMC CD8 + T EM responses to SEB stimulation in adults, children, and the elderly . Multifunctionality was determined by simultaneous detection of two or more functions performed by CD8 + T RM (LPMC) or CD8 + T EM (PBMC). Six functions were evaluated: production of five cytokines/chemokines (IFN-γ, TNF-α, IL-2, IL-17A, MIP-1β) and expression of CD107a in response to SEB stimulation. (A) Scatter plot showing the six predominant function patterns in LPMC CD8 + T RM and (B) in PBMC CD8 + T EM cells from adults (red circles, n = 9), children (black squares, n = 7), and the elderly (blue triangles, n = 8). Multifunctionality was analyzed using the FCOM feature of WinList. Significant differences between age groups were denoted by asterisks (* p
Figure Legend Snippet: Multifunctional gastric LPMC CD8 + T RM and PBMC CD8 + T EM responses to SEB stimulation in adults, children, and the elderly . Multifunctionality was determined by simultaneous detection of two or more functions performed by CD8 + T RM (LPMC) or CD8 + T EM (PBMC). Six functions were evaluated: production of five cytokines/chemokines (IFN-γ, TNF-α, IL-2, IL-17A, MIP-1β) and expression of CD107a in response to SEB stimulation. (A) Scatter plot showing the six predominant function patterns in LPMC CD8 + T RM and (B) in PBMC CD8 + T EM cells from adults (red circles, n = 9), children (black squares, n = 7), and the elderly (blue triangles, n = 8). Multifunctionality was analyzed using the FCOM feature of WinList. Significant differences between age groups were denoted by asterisks (* p

Techniques Used: Expressing

14) Product Images from "STAT3 Regulates Monocyte TNF-Alpha Production in Systemic Inflammation Caused by Cardiac Surgery with Cardiopulmonary Bypass"

Article Title: STAT3 Regulates Monocyte TNF-Alpha Production in Systemic Inflammation Caused by Cardiac Surgery with Cardiopulmonary Bypass

Journal: PLoS ONE

doi: 10.1371/journal.pone.0035070

STAT3 signaling is required for the suppressive effects of post-perfusion plasma on TNF-α production. A . Pre-treatment of 4 h post-surgery plasma samples with anti-IL-10 partially restored TNF-α production by patient monocytes in response to LPS (n = 10). Control: plasma from healthy donors. B . Activation of STAT3 in monocytes by incubation with suppressive (4 h post-perfusion) but not control (24 h post-perfusion) plasma. Cells were incubated in the absence or presence of LPS to match the experimental setup as in Fig. 2 . C . Pre-treatment of patient PBMC with active STAT3 inhibitor (pY-STAT3i) but not control peptide (STAT3i) before LPS stimulation in the presence of post-surgery plasma restored TNF-α synthesis (left panel), in contrast to IL-6 (right panel). Shown are percentages of TNF-α and IL-6 producing monocytes normalized to control (24 h post-surgery) plasma (n = 8). D . TNF-α and IL-6 levels measured in supernatants of LPS-stimulated mononuclear cells after pre-treatment with STAT3 inhibitor or control peptide, in the presence of 4 h post-surgery plasma (n = 8). Cytokine levels were normalized to LPS stimulation in control plasma from healthy donors due to interassay variability. All results are depicted as mean ± SEM. * P
Figure Legend Snippet: STAT3 signaling is required for the suppressive effects of post-perfusion plasma on TNF-α production. A . Pre-treatment of 4 h post-surgery plasma samples with anti-IL-10 partially restored TNF-α production by patient monocytes in response to LPS (n = 10). Control: plasma from healthy donors. B . Activation of STAT3 in monocytes by incubation with suppressive (4 h post-perfusion) but not control (24 h post-perfusion) plasma. Cells were incubated in the absence or presence of LPS to match the experimental setup as in Fig. 2 . C . Pre-treatment of patient PBMC with active STAT3 inhibitor (pY-STAT3i) but not control peptide (STAT3i) before LPS stimulation in the presence of post-surgery plasma restored TNF-α synthesis (left panel), in contrast to IL-6 (right panel). Shown are percentages of TNF-α and IL-6 producing monocytes normalized to control (24 h post-surgery) plasma (n = 8). D . TNF-α and IL-6 levels measured in supernatants of LPS-stimulated mononuclear cells after pre-treatment with STAT3 inhibitor or control peptide, in the presence of 4 h post-surgery plasma (n = 8). Cytokine levels were normalized to LPS stimulation in control plasma from healthy donors due to interassay variability. All results are depicted as mean ± SEM. * P

Techniques Used: Activation Assay, Incubation

15) Product Images from "Robust gene expression changes in the ganglia following subclinical reactivation in rhesus macaques infected with Simian Varicella Virus"

Article Title: Robust gene expression changes in the ganglia following subclinical reactivation in rhesus macaques infected with Simian Varicella Virus

Journal: Journal of neurovirology

doi: 10.1007/s13365-017-0522-3

SVV reactivation is detected in depleted animals in the absence of robust changes in immunity (a) SVV DNA viral loads were assessed in PBMC (200ng/sample). SVV-specific IgG titers were determined in (b) Controls (c) CD4 depleted and (d) CD8 depleted animals. Frequencies of PBMC SVV-specific (e) CD4 and (f) CD8 T cells producing IFNγ in animals that experienced SVV reactivation (red), depleted animals that did not experience a reactivation (black), and controls (blue). Dashed line represents the reactivation period
Figure Legend Snippet: SVV reactivation is detected in depleted animals in the absence of robust changes in immunity (a) SVV DNA viral loads were assessed in PBMC (200ng/sample). SVV-specific IgG titers were determined in (b) Controls (c) CD4 depleted and (d) CD8 depleted animals. Frequencies of PBMC SVV-specific (e) CD4 and (f) CD8 T cells producing IFNγ in animals that experienced SVV reactivation (red), depleted animals that did not experience a reactivation (black), and controls (blue). Dashed line represents the reactivation period

Techniques Used:

16) Product Images from "Differential Effects of Human Immunodeficiency Virus Isolates on ?-Chemokine and Gamma Interferon Production and on Cell Proliferation"

Article Title: Differential Effects of Human Immunodeficiency Virus Isolates on ?-Chemokine and Gamma Interferon Production and on Cell Proliferation

Journal: Journal of Virology

doi:

PBMC or purified CD4 + cells were stimulated for 3 days with 3 μg of PHA per ml, washed, and subsequently passaged every 2 to 3 days by exchanging the entire amount of culture fluid with fresh culture medium. Purified CD8 + cells were obtained from PBMC stimulated with PHA for 3 days. On day 7 and thereafter, cells were replated at a density of 2 × 10 6 /ml. The concentration of chemokine in the cell culture fluids was measured by ELISA in duplicate (Quantikine; R D Systems). The results show the maximal level of chemokines produced (adjusted per 10 6 viable cells) as measured in cultured fluids on day 13. The data are representative of two to four different experiments.
Figure Legend Snippet: PBMC or purified CD4 + cells were stimulated for 3 days with 3 μg of PHA per ml, washed, and subsequently passaged every 2 to 3 days by exchanging the entire amount of culture fluid with fresh culture medium. Purified CD8 + cells were obtained from PBMC stimulated with PHA for 3 days. On day 7 and thereafter, cells were replated at a density of 2 × 10 6 /ml. The concentration of chemokine in the cell culture fluids was measured by ELISA in duplicate (Quantikine; R D Systems). The results show the maximal level of chemokines produced (adjusted per 10 6 viable cells) as measured in cultured fluids on day 13. The data are representative of two to four different experiments.

Techniques Used: Purification, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Produced

17) Product Images from "Type I IFN signaling blockade by a PASylated antagonist during chronic SIV infection suppresses specific inflammatory pathways but does not alter T cell activation or virus replication"

Article Title: Type I IFN signaling blockade by a PASylated antagonist during chronic SIV infection suppresses specific inflammatory pathways but does not alter T cell activation or virus replication

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007246

Effect of PASylated IFN-1ant on cell-associated virus. ( a ) PBMC-associated SIV gag DNA at week 8 p.i., week 16 p.i. (pre-IFN-1ant) and week 24 p.i. (post-IFN-1ant) in sorted CD4 T cell subsets (CCR5 + , central memory: CM, effector memory: EM and total) from macaques treated with placebo saline, PASylated IFN-1ant injected 2 times weekly (IFN-1ant2x) or 3 times weekly (IFN-1ant3x). Animals receiving ART starting week 8 p.i. are presented in the lower panel. Horizontal bars represent median values. P values were calculated by Mann–Whitney U test. ( b ) LN-associated SIV gag DNA at week 7 p.i., week 15 p.i. (pre-IFN-1ant) and week 23 p.i. (post-IFN-1ant) in sorted CD4 T cell subsets (effector memory: EM, central memory: CM, germinal center T follicular helper: GC Tfh; non-GC Tfh and total) from macaques treated with placebo saline, IFN-1ant injections 2 times weekly (IFN-1ant2x) or 3 times weekly (IFN-1ant3x). Animals receiving ART starting week 8 p.i. are presented in the lower panel. Horizontal bars represent median values. P values were calculated by Mann–Whitney U test. ( c ) PBMC and ( d ) LN subset percentages of total SIV gag DNA before and after 2 or 3 times weekly IFN-1ant administration in macaques with ART-untreated or ART-treated SIV infection.
Figure Legend Snippet: Effect of PASylated IFN-1ant on cell-associated virus. ( a ) PBMC-associated SIV gag DNA at week 8 p.i., week 16 p.i. (pre-IFN-1ant) and week 24 p.i. (post-IFN-1ant) in sorted CD4 T cell subsets (CCR5 + , central memory: CM, effector memory: EM and total) from macaques treated with placebo saline, PASylated IFN-1ant injected 2 times weekly (IFN-1ant2x) or 3 times weekly (IFN-1ant3x). Animals receiving ART starting week 8 p.i. are presented in the lower panel. Horizontal bars represent median values. P values were calculated by Mann–Whitney U test. ( b ) LN-associated SIV gag DNA at week 7 p.i., week 15 p.i. (pre-IFN-1ant) and week 23 p.i. (post-IFN-1ant) in sorted CD4 T cell subsets (effector memory: EM, central memory: CM, germinal center T follicular helper: GC Tfh; non-GC Tfh and total) from macaques treated with placebo saline, IFN-1ant injections 2 times weekly (IFN-1ant2x) or 3 times weekly (IFN-1ant3x). Animals receiving ART starting week 8 p.i. are presented in the lower panel. Horizontal bars represent median values. P values were calculated by Mann–Whitney U test. ( c ) PBMC and ( d ) LN subset percentages of total SIV gag DNA before and after 2 or 3 times weekly IFN-1ant administration in macaques with ART-untreated or ART-treated SIV infection.

Techniques Used: Injection, MANN-WHITNEY, Infection

18) Product Images from "Increased bovine Tim-3 and its ligand expressions during bovine leukemia virus infection"

Article Title: Increased bovine Tim-3 and its ligand expressions during bovine leukemia virus infection

Journal: Veterinary Research

doi: 10.1186/1297-9716-43-45

Inhibition of the Tim-3/Gal-9 pathway increases cytokine expression in PBMC from BLV-infected cattle. (A and B) The expression of bovine Tim-3 on Cos7 cells. The detections were performed by (A) Western blotting analysis and (B) Flowcytometric analysis with anti-FLAG antibody as described elsewhere. Empty plasmid-transfected cells were used as mock for each assay. (C, D and E) Real-time PCR quantification of mRNA expression levels for cytokines in the treated-PBMC from BLV-infected cattle ( n = 3). Up-regulations of IL-2 (C) and IFN-γ (D) expressions in PBMC by the inhibition of the Tim-3/Gal-9 pathway using Tim-3 expressing cells (Tim-3-FLAG). Up-regulations of IFN-γ (E) expressions by blockade of the PD-1/PD-L1 pathway using anti-PD-L1 antibody (α-PD-L1) and enhancement of the expression by combination PD-L1 blockade with Tim-3 expressing cells. Each cytokine mRNA level was normalized by the bovine β-actin mRNA as above, and the relative index was determined in comparison to the cytokine mRNA level in the PBMC without Tim-3 expressing cells and antibodies. The results are means of three independent experiments using cells from three individual cattle. Asterisks donate significant differences between the types of the cells (* p
Figure Legend Snippet: Inhibition of the Tim-3/Gal-9 pathway increases cytokine expression in PBMC from BLV-infected cattle. (A and B) The expression of bovine Tim-3 on Cos7 cells. The detections were performed by (A) Western blotting analysis and (B) Flowcytometric analysis with anti-FLAG antibody as described elsewhere. Empty plasmid-transfected cells were used as mock for each assay. (C, D and E) Real-time PCR quantification of mRNA expression levels for cytokines in the treated-PBMC from BLV-infected cattle ( n = 3). Up-regulations of IL-2 (C) and IFN-γ (D) expressions in PBMC by the inhibition of the Tim-3/Gal-9 pathway using Tim-3 expressing cells (Tim-3-FLAG). Up-regulations of IFN-γ (E) expressions by blockade of the PD-1/PD-L1 pathway using anti-PD-L1 antibody (α-PD-L1) and enhancement of the expression by combination PD-L1 blockade with Tim-3 expressing cells. Each cytokine mRNA level was normalized by the bovine β-actin mRNA as above, and the relative index was determined in comparison to the cytokine mRNA level in the PBMC without Tim-3 expressing cells and antibodies. The results are means of three independent experiments using cells from three individual cattle. Asterisks donate significant differences between the types of the cells (* p

Techniques Used: Inhibition, Expressing, Infection, Western Blot, Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction

Quantification of Tim-3 mRNA expression in PBMC and subpopulations of CD4 + , CD8 + , CD5 + , CD14 + and CD21 + cells derived from BLV-uninfected and BLV-infected cattle using real-rime PCR analysis. The concentration of Tim-3 mRNA was divided by that of β-actin mRNA. Each line indicates the mean percentages in each group. Asterisks donate significant differences between the types of the cells (* p
Figure Legend Snippet: Quantification of Tim-3 mRNA expression in PBMC and subpopulations of CD4 + , CD8 + , CD5 + , CD14 + and CD21 + cells derived from BLV-uninfected and BLV-infected cattle using real-rime PCR analysis. The concentration of Tim-3 mRNA was divided by that of β-actin mRNA. Each line indicates the mean percentages in each group. Asterisks donate significant differences between the types of the cells (* p

Techniques Used: Expressing, Derivative Assay, Infection, Polymerase Chain Reaction, Concentration Assay

19) Product Images from "Lymphocyte proliferation modulated by glutamine: involved in the endogenous redox reaction"

Article Title: Lymphocyte proliferation modulated by glutamine: involved in the endogenous redox reaction

Journal: Clinical and Experimental Immunology

doi: 10.1046/j.1365-2249.1999.01009.x

Effect of exogenous glutathione (GSH) supplementation on lymphocyte proliferation. Peripheral blood mononuclear cells (PBMC; 2 × 10 6 cells/ml) were cultured in the presence GSH at various concentrations and stimulated with phytohaemagglutinin (PHA; 5 μg/ml) for 72 h. Results presented are mean ±s.e.m. ( n = 6). *Significant difference between presence and absence of GSH ( P
Figure Legend Snippet: Effect of exogenous glutathione (GSH) supplementation on lymphocyte proliferation. Peripheral blood mononuclear cells (PBMC; 2 × 10 6 cells/ml) were cultured in the presence GSH at various concentrations and stimulated with phytohaemagglutinin (PHA; 5 μg/ml) for 72 h. Results presented are mean ±s.e.m. ( n = 6). *Significant difference between presence and absence of GSH ( P

Techniques Used: Cell Culture

20) Product Images from "Membrane-Associated Proteinase 3 on Granulocytes and Acute Myeloid Leukemia Inhibits T Cell Proliferation"

Article Title: Membrane-Associated Proteinase 3 on Granulocytes and Acute Myeloid Leukemia Inhibits T Cell Proliferation

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1800324

mP3 + PMN mediate the inhibition of T cell proliferation. CFSE-labeled PBMC were activated with anti-CD3/CD28 mAbs in all cell cultures. Sort-purified PMN were fixed with 0.5% PFA. ( A ) Sorted CD177-expressing and CD177-negative PMN were cocultured with PBMC at a ratio of 3:1. T cells alone were used as control. Data from three different donors ( n = 3) are shown as mean ± SEM. *** p
Figure Legend Snippet: mP3 + PMN mediate the inhibition of T cell proliferation. CFSE-labeled PBMC were activated with anti-CD3/CD28 mAbs in all cell cultures. Sort-purified PMN were fixed with 0.5% PFA. ( A ) Sorted CD177-expressing and CD177-negative PMN were cocultured with PBMC at a ratio of 3:1. T cells alone were used as control. Data from three different donors ( n = 3) are shown as mean ± SEM. *** p

Techniques Used: Inhibition, Labeling, Purification, Expressing

PMN coculture does not affect cytokine production in stimulated T cells. ( A and B ) PBMC stimulated with or without anti-CD3/CD28 mAbs were cocultured with increasing numbers of PMN for 16 h, and the percentage of T cells producing cytokines was determined using intracellular staining of flow cytometry. Live CD8 and CD4 T cells were gated. Results are shown as mean ± SEM ( n = 3) from three different donors. p = NS. ( C and D ) Dot plots show representative data of three different donors as shown in the cumulative data graph (top panels). Fixed PMN were used.
Figure Legend Snippet: PMN coculture does not affect cytokine production in stimulated T cells. ( A and B ) PBMC stimulated with or without anti-CD3/CD28 mAbs were cocultured with increasing numbers of PMN for 16 h, and the percentage of T cells producing cytokines was determined using intracellular staining of flow cytometry. Live CD8 and CD4 T cells were gated. Results are shown as mean ± SEM ( n = 3) from three different donors. p = NS. ( C and D ) Dot plots show representative data of three different donors as shown in the cumulative data graph (top panels). Fixed PMN were used.

Techniques Used: Staining, Flow Cytometry, Cytometry

PMN-mediated inhibition of T cell proliferation requires cell-cell contact. Flow cytometric analysis of T cell proliferation in the presence of PMN using a Transwell system. ( A ) PMN were mixed with CFSE-labeled PBMC at a 4:1 ratio in coculture with anti-CD3/CD28 (coculture). To separate PBMC and PMN, PBMC were cultured in the bottom chamber and PMN were placed in the top chamber of a 96-well Transwell culture plate. Representative results from one of three different donors performed in triplicate are expressed as mean ± SEM. **** p
Figure Legend Snippet: PMN-mediated inhibition of T cell proliferation requires cell-cell contact. Flow cytometric analysis of T cell proliferation in the presence of PMN using a Transwell system. ( A ) PMN were mixed with CFSE-labeled PBMC at a 4:1 ratio in coculture with anti-CD3/CD28 (coculture). To separate PBMC and PMN, PBMC were cultured in the bottom chamber and PMN were placed in the top chamber of a 96-well Transwell culture plate. Representative results from one of three different donors performed in triplicate are expressed as mean ± SEM. **** p

Techniques Used: Inhibition, Flow Cytometry, Labeling, Cell Culture

LRP1 blockade prevents PMN-mediated inhibition of T cell proliferation. ( A ) PBMC from healthy donors were stimulated with anti-CD3/CD28 mAbs for up to 5 d, and LRP1 surface expression was determined by flow cytometry. Graph shows the median fluorescence intensity (MFI) of LRP1 on CD8 and CD4 T cells, respectively. Representative results from one of two different donors performed in duplicates are shown. Error bars represent mean ± SEM. * p
Figure Legend Snippet: LRP1 blockade prevents PMN-mediated inhibition of T cell proliferation. ( A ) PBMC from healthy donors were stimulated with anti-CD3/CD28 mAbs for up to 5 d, and LRP1 surface expression was determined by flow cytometry. Graph shows the median fluorescence intensity (MFI) of LRP1 on CD8 and CD4 T cells, respectively. Representative results from one of two different donors performed in duplicates are shown. Error bars represent mean ± SEM. * p

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

PMN-mediated T cell inhibition is prevented by anti-P3 blocking Ab and requires P3 enzyme activity. CFSE-labeled PBMC stimulated with anti-CD3/CD28 mAbs were cocultured with PMN at the indicated PMN/PBMC ratio. ( A ) PMN were treated with anti-P3 mAb (clone WGM2) or mIgG (clone 11711). The PMN/PBMC ratio is 3:1. Results from five different donors ( n = 5) are shown as mean ± SEM. ( B ) PMN were preincubated with or without PMSF, elafin, or AAT before adding to PBMC cultures. The PMN/PBMC ratio is 4:1. Results shown as mean ± SEM are representative of three different donors performed in triplicates. Fixed PMN were used. **** p
Figure Legend Snippet: PMN-mediated T cell inhibition is prevented by anti-P3 blocking Ab and requires P3 enzyme activity. CFSE-labeled PBMC stimulated with anti-CD3/CD28 mAbs were cocultured with PMN at the indicated PMN/PBMC ratio. ( A ) PMN were treated with anti-P3 mAb (clone WGM2) or mIgG (clone 11711). The PMN/PBMC ratio is 3:1. Results from five different donors ( n = 5) are shown as mean ± SEM. ( B ) PMN were preincubated with or without PMSF, elafin, or AAT before adding to PBMC cultures. The PMN/PBMC ratio is 4:1. Results shown as mean ± SEM are representative of three different donors performed in triplicates. Fixed PMN were used. **** p

Techniques Used: Inhibition, Blocking Assay, Activity Assay, Labeling

PMN inhibit T cell proliferation in a dose-dependent manner. CFSE-labeled PBMC stimulated with or without anti-CD3/CD28 mAbs were cocultured with increasing numbers of fixed PMN ( A and B ) or unfixed PMN ( C ) for 5 d. Flow cytometry was used to determine CFSE dilution in live CD8 and CD4 T cells. (A) The result is representative of nine different donors. Numbers on histograms represent the percentage of proliferating T cells. (B) Results from nine different donors ( n = 9) are expressed as mean ± SEM. **** p
Figure Legend Snippet: PMN inhibit T cell proliferation in a dose-dependent manner. CFSE-labeled PBMC stimulated with or without anti-CD3/CD28 mAbs were cocultured with increasing numbers of fixed PMN ( A and B ) or unfixed PMN ( C ) for 5 d. Flow cytometry was used to determine CFSE dilution in live CD8 and CD4 T cells. (A) The result is representative of nine different donors. Numbers on histograms represent the percentage of proliferating T cells. (B) Results from nine different donors ( n = 9) are expressed as mean ± SEM. **** p

Techniques Used: Labeling, Flow Cytometry, Cytometry

mP3 + AML mediates the inhibition of T cell proliferation, and proliferation is restored via P3 blockade. ( A ) Flow cytometric analysis of CD177 and mP3 expression on AML blasts and healthy donor PMN were performed. ( B ) Representative plots of AML blasts from AML patients ( n = 19) and PMN of healthy donor ( n = 20) are shown. ( C ) Following fixation with 0.5% PFA, allogeneic AML was preincubated with or without anti-P3 Ab for 30 min before they were cocultured with PBMC at a 5:1 ratio (experiment was performed in duplicates) for up to 5 d. PBMC stimulated with or without anti-CD3/CD28 mAbs were used as positive and negative controls, respectively. Error bars represent mean ± SEM. ** p
Figure Legend Snippet: mP3 + AML mediates the inhibition of T cell proliferation, and proliferation is restored via P3 blockade. ( A ) Flow cytometric analysis of CD177 and mP3 expression on AML blasts and healthy donor PMN were performed. ( B ) Representative plots of AML blasts from AML patients ( n = 19) and PMN of healthy donor ( n = 20) are shown. ( C ) Following fixation with 0.5% PFA, allogeneic AML was preincubated with or without anti-P3 Ab for 30 min before they were cocultured with PBMC at a 5:1 ratio (experiment was performed in duplicates) for up to 5 d. PBMC stimulated with or without anti-CD3/CD28 mAbs were used as positive and negative controls, respectively. Error bars represent mean ± SEM. ** p

Techniques Used: Inhibition, Flow Cytometry, Expressing

21) Product Images from "Expansion in CD39+ CD4+ Immunoregulatory T Cells and Rarity of Th17 Cells in HTLV-1 Infected Patients Is Associated with Neurological Complications"

Article Title: Expansion in CD39+ CD4+ Immunoregulatory T Cells and Rarity of Th17 Cells in HTLV-1 Infected Patients Is Associated with Neurological Complications

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0002028

IL-17 and IFN-γ production by CD4 + T cells subsets. Subsets were numbered according to CD39 and CD25 expression (A): 1 = CD39 + CD25 + ; 2 = CD39 + CD25 − ; 3 = CD39 − CD25 + . Graph shows median of proportion of (B) IL-17-producing CD4 + T cells and (C) IFN-γ-producing CD4 + T cells subsets after PMA and ionomycin stimulation on PBMCs from 9 uninfected, 8 HTLV-1-infected asymptomatic carriers and 10 HAM/TSP patients. The statistical difference was deemed significant using a Mann-Whitney U test analysis if p
Figure Legend Snippet: IL-17 and IFN-γ production by CD4 + T cells subsets. Subsets were numbered according to CD39 and CD25 expression (A): 1 = CD39 + CD25 + ; 2 = CD39 + CD25 − ; 3 = CD39 − CD25 + . Graph shows median of proportion of (B) IL-17-producing CD4 + T cells and (C) IFN-γ-producing CD4 + T cells subsets after PMA and ionomycin stimulation on PBMCs from 9 uninfected, 8 HTLV-1-infected asymptomatic carriers and 10 HAM/TSP patients. The statistical difference was deemed significant using a Mann-Whitney U test analysis if p

Techniques Used: Expressing, Infection, MANN-WHITNEY, IF-P

22) Product Images from "Nickel, palladium and rhodium induced IFN-gamma and IL-10 production as assessed by in vitro ELISpot-analysis in contact dermatitis patients"

Article Title: Nickel, palladium and rhodium induced IFN-gamma and IL-10 production as assessed by in vitro ELISpot-analysis in contact dermatitis patients

Journal: BMC Immunology

doi: 10.1186/1471-2172-9-19

Scatter plot analysis of individual IFN-γ and IL-10 induction by PHA, Ni or Pd in the different group of patients . PHA induces PBMC release of both IFN-γ and IL-10 in all subjects, while metal salts induces IFN-γ or IL-10 production according to the in vivo positive or negative response. IFN-γ or IL-10 production was alternatively produced and consequently a highly significant negative correlation was found (Spearman analysis P
Figure Legend Snippet: Scatter plot analysis of individual IFN-γ and IL-10 induction by PHA, Ni or Pd in the different group of patients . PHA induces PBMC release of both IFN-γ and IL-10 in all subjects, while metal salts induces IFN-γ or IL-10 production according to the in vivo positive or negative response. IFN-γ or IL-10 production was alternatively produced and consequently a highly significant negative correlation was found (Spearman analysis P

Techniques Used: In Vivo, Produced

Cytokine response elicited by metals or PHA by ELISpot analysis . Elispot results of detection of IFN-γ or IL-10 producing PBMC upon stimulation with Ni (20 μg/ml), Pd (2.5 μg/ml), Rh (5 μg/ml) or PHA (1 μg/ml) were expressed as Index values. Index is expressed by the ratio between the number of spot forming cells (IFN-γ and IL-10 producing PBMC) upon stimulation with metal salts and those present in the absence of stimuli (spontaneous cytokine production). The mean values of Index values ± SD were obtained for the 4 different groups of 10 subjects according to their in vivo reactivity to metal salts. Cytokine producing cells in control triplicate wells with medium alone was the baseline value (Index = 1). Panel A : patch test positive to Ni (group 1); B : patch test positive to Pd (group 2); C : patch test positive to Ni and Pd (group 3); D : negative patch test (group 4). The statistical analysis between IFN-g and IL-10 producing cells upon stimulation with Ni or Pd between patients belonging to group C and A or B was performed using the Mann-Whitney test. The highly significant differences (P
Figure Legend Snippet: Cytokine response elicited by metals or PHA by ELISpot analysis . Elispot results of detection of IFN-γ or IL-10 producing PBMC upon stimulation with Ni (20 μg/ml), Pd (2.5 μg/ml), Rh (5 μg/ml) or PHA (1 μg/ml) were expressed as Index values. Index is expressed by the ratio between the number of spot forming cells (IFN-γ and IL-10 producing PBMC) upon stimulation with metal salts and those present in the absence of stimuli (spontaneous cytokine production). The mean values of Index values ± SD were obtained for the 4 different groups of 10 subjects according to their in vivo reactivity to metal salts. Cytokine producing cells in control triplicate wells with medium alone was the baseline value (Index = 1). Panel A : patch test positive to Ni (group 1); B : patch test positive to Pd (group 2); C : patch test positive to Ni and Pd (group 3); D : negative patch test (group 4). The statistical analysis between IFN-g and IL-10 producing cells upon stimulation with Ni or Pd between patients belonging to group C and A or B was performed using the Mann-Whitney test. The highly significant differences (P

Techniques Used: Enzyme-linked Immunospot, In Vivo, MANN-WHITNEY

23) Product Images from "Increased secretion of IL-18 in vitro by peripheral blood mononuclear cells of patients with bronchial asthma and atopic dermatitis"

Article Title: Increased secretion of IL-18 in vitro by peripheral blood mononuclear cells of patients with bronchial asthma and atopic dermatitis

Journal: Clinical and Experimental Immunology

doi: 10.1046/j.1365-2249.2001.01664.x

Correlation between plasma IgE concentration and in vitro IL-18 secretion by LPS-stimulated PBMC in BA patients (a), and that between IFN-γ secretion and IL-13 secretion in PHA-stimulated PBMC cultures in non-allergic controls (b). The differences in the slopes between groups were assessed by single regression analyses. A significant positive correlation between IL-18 secretion and plasma IgE was found ( r = 0·556, P = 0·017). Secretion of IFN-γ was significantly correlated with secretion of IL-13 ( r = 0·533, P = 0·019).
Figure Legend Snippet: Correlation between plasma IgE concentration and in vitro IL-18 secretion by LPS-stimulated PBMC in BA patients (a), and that between IFN-γ secretion and IL-13 secretion in PHA-stimulated PBMC cultures in non-allergic controls (b). The differences in the slopes between groups were assessed by single regression analyses. A significant positive correlation between IL-18 secretion and plasma IgE was found ( r = 0·556, P = 0·017). Secretion of IFN-γ was significantly correlated with secretion of IL-13 ( r = 0·533, P = 0·019).

Techniques Used: Concentration Assay, In Vitro

24) Product Images from "A high-frequency polymorphism in exon 6 of the CD45 tyrosine phosphatase gene (PTPRC) resulting in altered isoform expression"

Article Title: A high-frequency polymorphism in exon 6 of the CD45 tyrosine phosphatase gene (PTPRC) resulting in altered isoform expression

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

doi: 10.1073/pnas.0931490100

CD45 RNA expression in PBMC from homozygous G138G and homozygous A138A individuals. Total RNA was extracted from unstimulated PBMC. After reverse transcription the resulting cDNA was amplified with primers spanning exons 2–7 of the CD45 gene. PBMC from both homozygotes for G138G and common variant A138A allele individuals contained mRNA for the CD45R0 (197 bp), CD45RB (337 bp), CD45RBC (480 bp), CD45RAB (534 bp), and CD45RABC (677 bp) isoforms. Bands in each lane were quantitated, and densitograms are shown on top of the gel corresponding to the respective isoform. The ratio between the intensity of the CD45R0 and CD45RB bands is shown at the bottom of the gel. Data of three representative analyses of three G138G homozygotes and three control samples for the common variant A138A allele are shown.
Figure Legend Snippet: CD45 RNA expression in PBMC from homozygous G138G and homozygous A138A individuals. Total RNA was extracted from unstimulated PBMC. After reverse transcription the resulting cDNA was amplified with primers spanning exons 2–7 of the CD45 gene. PBMC from both homozygotes for G138G and common variant A138A allele individuals contained mRNA for the CD45R0 (197 bp), CD45RB (337 bp), CD45RBC (480 bp), CD45RAB (534 bp), and CD45RABC (677 bp) isoforms. Bands in each lane were quantitated, and densitograms are shown on top of the gel corresponding to the respective isoform. The ratio between the intensity of the CD45R0 and CD45RB bands is shown at the bottom of the gel. Data of three representative analyses of three G138G homozygotes and three control samples for the common variant A138A allele are shown.

Techniques Used: RNA Expression, Amplification, Variant Assay

25) Product Images from "Major Depletion of Plasmacytoid Dendritic Cells in HIV-2 Infection, an Attenuated Form of HIV Disease"

Article Title: Major Depletion of Plasmacytoid Dendritic Cells in HIV-2 Infection, an Attenuated Form of HIV Disease

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000667

pDC Phenotype. ( A ) Phenotype of circulating pDC in HIV-1 and HIV-2 infections. Levels of expression of CD86 and PD-L1 on pDC from freshly isolated PBMC assessed as proportion within pDC and as geomean FI within total pDC measured by flow cytometry. Each dot represents one individual and bars represent mean. ( B ) pDC phenotype upon CpG stimulation. Freshly isolated PBMC were cultured with medium alone, CpG or the non-CpG ODN control. pDC phenotype was assessed by flow cytometry after 22 hours. Histograms represent the analysis of CD40 and CD80 expression upon CpG stimulation within pDC in a representative HIV-2 infected patient with 508 CD4 T cells/µl and 13627 HIV-2 RNA copies/ml. After a large gate including lymphocytes and monocytes defined by forward and side scatter, cells were sequentially gated on HLA-DR + cells that do not express lineage markers and on CD123 + CD11c − cells. ( C ) The up-regulation of CD40 expression on pDC is showed as stimulation index defined as ratio between the geomean FI in the presence of CpG or ODN and in its absence (medium). Data are presented within total HIV-1 and HIV-2 cohorts (left graph) and within the infected cohorts grouped according to viremia status (right graph). Bars represent mean±SEM. Numbers under the bars represent the total HIV-1 and HIV-2 infected individuals as well as healthy controls analyzed. The subgroups of patients analyzed are representative of their respective patient population described in Table 1 with respect to CD4 counts and viral load. There are no significant differences between HIV-1 and HIV-2 cohorts. * p
Figure Legend Snippet: pDC Phenotype. ( A ) Phenotype of circulating pDC in HIV-1 and HIV-2 infections. Levels of expression of CD86 and PD-L1 on pDC from freshly isolated PBMC assessed as proportion within pDC and as geomean FI within total pDC measured by flow cytometry. Each dot represents one individual and bars represent mean. ( B ) pDC phenotype upon CpG stimulation. Freshly isolated PBMC were cultured with medium alone, CpG or the non-CpG ODN control. pDC phenotype was assessed by flow cytometry after 22 hours. Histograms represent the analysis of CD40 and CD80 expression upon CpG stimulation within pDC in a representative HIV-2 infected patient with 508 CD4 T cells/µl and 13627 HIV-2 RNA copies/ml. After a large gate including lymphocytes and monocytes defined by forward and side scatter, cells were sequentially gated on HLA-DR + cells that do not express lineage markers and on CD123 + CD11c − cells. ( C ) The up-regulation of CD40 expression on pDC is showed as stimulation index defined as ratio between the geomean FI in the presence of CpG or ODN and in its absence (medium). Data are presented within total HIV-1 and HIV-2 cohorts (left graph) and within the infected cohorts grouped according to viremia status (right graph). Bars represent mean±SEM. Numbers under the bars represent the total HIV-1 and HIV-2 infected individuals as well as healthy controls analyzed. The subgroups of patients analyzed are representative of their respective patient population described in Table 1 with respect to CD4 counts and viral load. There are no significant differences between HIV-1 and HIV-2 cohorts. * p

Techniques Used: Expressing, Isolation, Flow Cytometry, Cytometry, Cell Culture, Infection

26) Product Images from "Major Depletion of Plasmacytoid Dendritic Cells in HIV-2 Infection, an Attenuated Form of HIV Disease"

Article Title: Major Depletion of Plasmacytoid Dendritic Cells in HIV-2 Infection, an Attenuated Form of HIV Disease

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000667

IFN-α production upon CpG stimulation. Freshly isolated PBMC were stimulated with CpG or the non-CpG ODN control. After 22 hours IFN-α was quantified in culture supernatants by ELISA. ( A ) Levels of IFN-α upon CpG or the non-CpG ODN control in healthy, HIV-1 and HIV-2 cohorts. The infected cohorts were further stratified according to CD4 T cell counts ( B ) or the presence or absence of detectable viremia ( C ) and the levels of IFN-α upon CpG stimulation are shown. Results expressed as “Net IFN-α” refer to the amount of IFN-α produced upon CpG stimulation subtracted by the IFN-α measured with medium alone. The “Net IFN-α” was divided by the total number of pDC in the culture and results are shown for the healthy, HIV-1 and HIV-2 cohorts ( D ), as well as for the infected cohorts stratified according to CD4 T cell counts ( E ) or the presence or absence of detectable viremia ( F ). Each dot represents one individual. Bars represent mean±SEM. Numbers under the bars represent the total individuals analyzed. The subgroups of patients analyzed are representative of their respective patient population described in Table 1 with respect to CD4 counts and viral load. ** p
Figure Legend Snippet: IFN-α production upon CpG stimulation. Freshly isolated PBMC were stimulated with CpG or the non-CpG ODN control. After 22 hours IFN-α was quantified in culture supernatants by ELISA. ( A ) Levels of IFN-α upon CpG or the non-CpG ODN control in healthy, HIV-1 and HIV-2 cohorts. The infected cohorts were further stratified according to CD4 T cell counts ( B ) or the presence or absence of detectable viremia ( C ) and the levels of IFN-α upon CpG stimulation are shown. Results expressed as “Net IFN-α” refer to the amount of IFN-α produced upon CpG stimulation subtracted by the IFN-α measured with medium alone. The “Net IFN-α” was divided by the total number of pDC in the culture and results are shown for the healthy, HIV-1 and HIV-2 cohorts ( D ), as well as for the infected cohorts stratified according to CD4 T cell counts ( E ) or the presence or absence of detectable viremia ( F ). Each dot represents one individual. Bars represent mean±SEM. Numbers under the bars represent the total individuals analyzed. The subgroups of patients analyzed are representative of their respective patient population described in Table 1 with respect to CD4 counts and viral load. ** p

Techniques Used: Isolation, Enzyme-linked Immunosorbent Assay, Infection, Produced

IL-10, IL-12p40, MIP-1β and TNF-α levels upon CpG stimulation in Healthy, HIV-1 and HIV-2 cohorts. Freshly isolated PBMC were cultured in the absence or in the presence of CpG. After 22 hours, culture supernatants were harvested and analyzed for the secretion of IL-10, IL-12p40, MIP-1β and TNF-α using the Luminex multiplex assay. ( A ) Cytokine levels. Each dot represents one individual. The levels observed in non-stimulated cultures (open symbols) are connected with those documented in the presence of CpG (closed symbols), and were compared using Wilcoxon test. ( B ) Stimulation indexes defined as ratio between the level of cytokine in the supernatant of the culture in the presence of CpG and in its absence (medium) in the three cohorts. ( C ) The HIV-2 and the HIV-1 cohorts were split according to the presence of detectable and undectable viremia and the stimulation indexes for TNF-α are shown. Bars represent mean±SEM. Numbers under the bars represent the total individuals analyzed. The subgroups of patients analyzed are representative of their respective patient population described in Table 1 with respect to CD4 counts and viral load. The Mann-Whitney test was used to compare the data of the HIV-2, HIV-1 and healthy cohorts, and the significant p values (
Figure Legend Snippet: IL-10, IL-12p40, MIP-1β and TNF-α levels upon CpG stimulation in Healthy, HIV-1 and HIV-2 cohorts. Freshly isolated PBMC were cultured in the absence or in the presence of CpG. After 22 hours, culture supernatants were harvested and analyzed for the secretion of IL-10, IL-12p40, MIP-1β and TNF-α using the Luminex multiplex assay. ( A ) Cytokine levels. Each dot represents one individual. The levels observed in non-stimulated cultures (open symbols) are connected with those documented in the presence of CpG (closed symbols), and were compared using Wilcoxon test. ( B ) Stimulation indexes defined as ratio between the level of cytokine in the supernatant of the culture in the presence of CpG and in its absence (medium) in the three cohorts. ( C ) The HIV-2 and the HIV-1 cohorts were split according to the presence of detectable and undectable viremia and the stimulation indexes for TNF-α are shown. Bars represent mean±SEM. Numbers under the bars represent the total individuals analyzed. The subgroups of patients analyzed are representative of their respective patient population described in Table 1 with respect to CD4 counts and viral load. The Mann-Whitney test was used to compare the data of the HIV-2, HIV-1 and healthy cohorts, and the significant p values (

Techniques Used: Isolation, Cell Culture, Luminex, Multiplex Assay, MANN-WHITNEY

27) Product Images from "Innate-Like and Conventional T Cell Populations from Hemodialyzed and Kidney Transplanted Patients Are Equally Compromised"

Article Title: Innate-Like and Conventional T Cell Populations from Hemodialyzed and Kidney Transplanted Patients Are Equally Compromised

Journal: PLoS ONE

doi: 10.1371/journal.pone.0105422

Global Foxp3 + T cell numbers are reduced in HD patients. (A) Representative FACS analysis of CD4 + CD25 + Foxp3 + T cells and their distinct subsets on PBMC from control (top) and HD patients (bottom). (B) CD4 + CD25 + Foxp3 + T cell numbers are reduced in HD patients. (C to E) Among these gated cells, cytokine-secreting CD45RA − Foxp3 lo nonsuppressive (C), resting suppressive CD45RA + Foxp3 lo (D) and activated suppressive CD45RA − Foxp3 hi (E) cells, all these subsets are reduced in HD patients compared to controls. Bars represent the median. *, P
Figure Legend Snippet: Global Foxp3 + T cell numbers are reduced in HD patients. (A) Representative FACS analysis of CD4 + CD25 + Foxp3 + T cells and their distinct subsets on PBMC from control (top) and HD patients (bottom). (B) CD4 + CD25 + Foxp3 + T cell numbers are reduced in HD patients. (C to E) Among these gated cells, cytokine-secreting CD45RA − Foxp3 lo nonsuppressive (C), resting suppressive CD45RA + Foxp3 lo (D) and activated suppressive CD45RA − Foxp3 hi (E) cells, all these subsets are reduced in HD patients compared to controls. Bars represent the median. *, P

Techniques Used: FACS

28) Product Images from "Poke Weed Mitogen Requires Toll-Like Receptor Ligands for Proliferative Activity in Human and Murine B Lymphocytes"

Article Title: Poke Weed Mitogen Requires Toll-Like Receptor Ligands for Proliferative Activity in Human and Murine B Lymphocytes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0029806

TLR-dependency of Poke weed mitogen (PWM)-induced cell stimulation. A: B cell proliferation. Human CD19+ peripheral blood B cells were stained with CFSE and stimulated with 1 or 10 µg/ml PWM in the presence or absence of 5 µg/ml Staphylococcus aureus protein A (SpA) or 5 µg/ml anti-human Ig F(ab′) 2 fragments (aIg) as B cell receptor stimuli, with SpA or aIg alone, or left unstimulated. After 5 days B cells were harvested, stained with anti-CD20 and live gated cells were analyzed for CFSE dilution. The percentages of live gated cells (upper right corner) and proliferating cells (left) are indicated in each diagram. The diagrams depict the results from one representative experiment of n = 3. B: TNF-induction. Human PBMC were stimulated for four hours with or without PWM, TLR2 ligands FSL-1R (FSL) and Pam 3 CSK 4 (P3), phosphorothioate-modified DNA ODN CpG 2006 (CpG) and 2006 GC (GC) and LPS in the presence of Brefeldin A (BfA) or with LPS in the absence of BfA. Subsequently, intracellular staining was performed with an anti-human TNF mAb and TNF expression was analyzed by flow cytometry. The results show a summary of the data obtained in n = 4 experiments. Mean values of anti-TNF mean fluorescence intensities are provided ± SEM. C: Antagonization with Polymyxin B. CFSE-stained human CD19+ B cells were stimulated with PWM in the presence and absence of polymyxin B. Proliferation was assessed by flow cytometric analysis of CFSE dilution on day 5. The results obtained in two representative donors of n = 3 are shown. D: MyD88-dependency of B cell stimulation. B220+ B cells were isolated from the spleens of MyD88 −/− mice and their wild type counterparts. B cells were stimulated with CpG, P3, PWM and PMA/Ionomycin (P/I) and harvested after 72 hours and a 18 hour pulse with 3 H-thymidine. The diagram shows the average values in counts per minute (cpm) of n = 4 experiments ± SEM. E: TLR-dependency. Wild type, TLR2−/− and TLR9−/− B cells were isolated from murine spleen with anti-CD19 microbeads, labelled with CFSE and stimulated with TLR2 ligands Pam 3 CSK 4 (P3) and FSL-1R (FSL), PWM (10 µg/ml), TLR9 ligand CpG ODN 1668 or 1668 GC control ODN. After 4 days B cell proliferation (CFSE dilution) was quantified by flow cytometry. The diagram depicts the mean fluorescence intensity (MFI) for CFSE in live gated cells as mean value ± SEM from n = 4 experiments. Note that low MFI corresponds to strong proliferation while high MFI values reveal absence of proliferation.
Figure Legend Snippet: TLR-dependency of Poke weed mitogen (PWM)-induced cell stimulation. A: B cell proliferation. Human CD19+ peripheral blood B cells were stained with CFSE and stimulated with 1 or 10 µg/ml PWM in the presence or absence of 5 µg/ml Staphylococcus aureus protein A (SpA) or 5 µg/ml anti-human Ig F(ab′) 2 fragments (aIg) as B cell receptor stimuli, with SpA or aIg alone, or left unstimulated. After 5 days B cells were harvested, stained with anti-CD20 and live gated cells were analyzed for CFSE dilution. The percentages of live gated cells (upper right corner) and proliferating cells (left) are indicated in each diagram. The diagrams depict the results from one representative experiment of n = 3. B: TNF-induction. Human PBMC were stimulated for four hours with or without PWM, TLR2 ligands FSL-1R (FSL) and Pam 3 CSK 4 (P3), phosphorothioate-modified DNA ODN CpG 2006 (CpG) and 2006 GC (GC) and LPS in the presence of Brefeldin A (BfA) or with LPS in the absence of BfA. Subsequently, intracellular staining was performed with an anti-human TNF mAb and TNF expression was analyzed by flow cytometry. The results show a summary of the data obtained in n = 4 experiments. Mean values of anti-TNF mean fluorescence intensities are provided ± SEM. C: Antagonization with Polymyxin B. CFSE-stained human CD19+ B cells were stimulated with PWM in the presence and absence of polymyxin B. Proliferation was assessed by flow cytometric analysis of CFSE dilution on day 5. The results obtained in two representative donors of n = 3 are shown. D: MyD88-dependency of B cell stimulation. B220+ B cells were isolated from the spleens of MyD88 −/− mice and their wild type counterparts. B cells were stimulated with CpG, P3, PWM and PMA/Ionomycin (P/I) and harvested after 72 hours and a 18 hour pulse with 3 H-thymidine. The diagram shows the average values in counts per minute (cpm) of n = 4 experiments ± SEM. E: TLR-dependency. Wild type, TLR2−/− and TLR9−/− B cells were isolated from murine spleen with anti-CD19 microbeads, labelled with CFSE and stimulated with TLR2 ligands Pam 3 CSK 4 (P3) and FSL-1R (FSL), PWM (10 µg/ml), TLR9 ligand CpG ODN 1668 or 1668 GC control ODN. After 4 days B cell proliferation (CFSE dilution) was quantified by flow cytometry. The diagram depicts the mean fluorescence intensity (MFI) for CFSE in live gated cells as mean value ± SEM from n = 4 experiments. Note that low MFI corresponds to strong proliferation while high MFI values reveal absence of proliferation.

Techniques Used: Cell Stimulation, Staining, Modification, Expressing, Flow Cytometry, Cytometry, Fluorescence, Isolation, Mouse Assay

29) Product Images from "Mucosal-associated invariant T cells are a profibrogenic immune cell population in the liver"

Article Title: Mucosal-associated invariant T cells are a profibrogenic immune cell population in the liver

Journal: Nature Communications

doi: 10.1038/s41467-018-04450-y

Frequency and functions of circulating MAIT cells are impaired during cirrhosis. a Representative dot plots showing reduction of CD161 hi Vα7.2 + double positive (MAIT) cells in PBMC and summary data from cirrhotic patients ( n = 74), as compared to that in healthy donors ( n = 47), and repartition of cirrhotic patients into disease etiology (alcoholic cirrhosis, n = 63 and NASH cirrhosis, n = 11) and severity (compensated cirrhosis, n = 15 and decompensated cirrhosis, n = 59). Representative dot plots and cumulative data of b increased surface expression of CD25 and CD69 on MAIT cells from healthy donors ( n = 25) and cirrhotic patients ( n = 29) and c cytokine profile of cirrhotic ( n = 20–23) vs. healthy ( n = 13–16) blood MAIT cells. Statistical analysis was performed using Mann–Whitney ( a , b , c ) or Kruskal–Wallis followed by Dunn’s post test analysis ( a ). ** p ≤ 0.01; **** p ≤ 0.0001; ns p > 0.05
Figure Legend Snippet: Frequency and functions of circulating MAIT cells are impaired during cirrhosis. a Representative dot plots showing reduction of CD161 hi Vα7.2 + double positive (MAIT) cells in PBMC and summary data from cirrhotic patients ( n = 74), as compared to that in healthy donors ( n = 47), and repartition of cirrhotic patients into disease etiology (alcoholic cirrhosis, n = 63 and NASH cirrhosis, n = 11) and severity (compensated cirrhosis, n = 15 and decompensated cirrhosis, n = 59). Representative dot plots and cumulative data of b increased surface expression of CD25 and CD69 on MAIT cells from healthy donors ( n = 25) and cirrhotic patients ( n = 29) and c cytokine profile of cirrhotic ( n = 20–23) vs. healthy ( n = 13–16) blood MAIT cells. Statistical analysis was performed using Mann–Whitney ( a , b , c ) or Kruskal–Wallis followed by Dunn’s post test analysis ( a ). ** p ≤ 0.01; **** p ≤ 0.0001; ns p > 0.05

Techniques Used: Expressing, MANN-WHITNEY

30) Product Images from "Identifying a Novel Role for Fractalkine (CX3CL1) in Memory CD8+ T Cell Accumulation in the Omentum of Obesity-Associated Cancer Patients"

Article Title: Identifying a Novel Role for Fractalkine (CX3CL1) in Memory CD8+ T Cell Accumulation in the Omentum of Obesity-Associated Cancer Patients

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01867

Frequencies of CX3CR1 + CD8 + T cells are significantly lower in omentum of esophagogastric adenocarcinoma (EAC) patients compared to frequencies of both peripheral blood CX3CR1 + CD8 + T cells and CX3CR1 + CD4 + T cells. Peripheral blood mononuclear cells and stromal vascular fraction of omentum were isolated from EAC patients. (A) Scatterplots show the frequencies of CX3CR1 + CD8 + and CD4 + T cells in the blood and omentum of a total of 24 EAC patients. (B) Representative dot plots of CX3CR1 + CD8 + and CX3CR1 + CD4 + T cells (gated on and shown as a percentage of total CD3 + population) in blood and omentum. (C) Bar chart showing the frequencies of CD8 + cells expressing no CX3CR1 (CX3CR1 NEG ), intermediate (CX3CR1 INT ), and high levels of CX3CR1 (CX3CR1 HI ) in the blood (white) and omentum (black) of 24 EAC patients. (D) Representative dot plot of circulating CD8 + T cells expressing no CX3CR1 (CX3CR1 NEG ), intermediate levels of CX3CR1 (CX3CR1 INT ), and high levels of CX3CR1 (CX3CR1 HI ). (E) Bar charts showing the CX3CR1 + CD8 + and CD4 + T cells in the blood and omentum of 8 non-cancer control subjects and 24 EAC patients (left) and 14 non-obese and 10 obese EAC patients (right). * p
Figure Legend Snippet: Frequencies of CX3CR1 + CD8 + T cells are significantly lower in omentum of esophagogastric adenocarcinoma (EAC) patients compared to frequencies of both peripheral blood CX3CR1 + CD8 + T cells and CX3CR1 + CD4 + T cells. Peripheral blood mononuclear cells and stromal vascular fraction of omentum were isolated from EAC patients. (A) Scatterplots show the frequencies of CX3CR1 + CD8 + and CD4 + T cells in the blood and omentum of a total of 24 EAC patients. (B) Representative dot plots of CX3CR1 + CD8 + and CX3CR1 + CD4 + T cells (gated on and shown as a percentage of total CD3 + population) in blood and omentum. (C) Bar chart showing the frequencies of CD8 + cells expressing no CX3CR1 (CX3CR1 NEG ), intermediate (CX3CR1 INT ), and high levels of CX3CR1 (CX3CR1 HI ) in the blood (white) and omentum (black) of 24 EAC patients. (D) Representative dot plot of circulating CD8 + T cells expressing no CX3CR1 (CX3CR1 NEG ), intermediate levels of CX3CR1 (CX3CR1 INT ), and high levels of CX3CR1 (CX3CR1 HI ). (E) Bar charts showing the CX3CR1 + CD8 + and CD4 + T cells in the blood and omentum of 8 non-cancer control subjects and 24 EAC patients (left) and 14 non-obese and 10 obese EAC patients (right). * p

Techniques Used: Isolation, Expressing

CX3CR1 is endocytosed following fractalkine treatment and is not subsequently recycled to the surface of CD8 + T cells or secreted but, intracellular accumulations of the protein are detectable. (A) Frequencies of CX3CR1 + cells, as a percentage of CD8 + T cells following treatment with M199 media alone (NT), 30 ng/ml recombinant fractalkine alone (treatment alone), 30 ng/ml recombinant fractalkine at 4°C and 30 ng/ml recombinant fractalkine plus 80 µM Dynasore ( n = 10). (B) Bar chart showing frequencies of CX3CR1 + cells, as a percentage of CD8 + T cells following treatment with M199 media alone (NT) or 30 ng/ml of recombinant fractalkine for 24 h (fractalkine, light gray dot pattern) followed by removal from a fractalkine-free environment for 24 h (fractalkine, dark gray no pattern) or 48 h (fractalkine, light gray no pattern) ( n = 6). (C) Bar chart showing frequencies of CX3CR1 + cells, as a percentage of CD8 + T cells following treatment with M199 media alone (NT) or 30 ng/ml of recombinant fractalkine for 24 h (fractalkine, light gray dot pattern) followed by removal from a fractalkine-free environment for 24 h and treatment with 50 ng/ml of TNF-α, 100 ng/ml of IFN-γ, IL-17, IL-6, IL-7, IL-15, IL-18, IL-36, and 50 U/ml of IL-2 ( n = 3). (D) Western Blot (left) showing CX3CR1 and beta-actin protein in peripheral blood-derived CD8 + T cells from three donors following 24 h of no treatment (N1, N2, and N3) or fractalkine treatment (F1, F2, and F3) and densitometry data (right) from this western blot displayed as fold change bar chart. (E) Soluble CX3CR1 levels in the supernatant of peripheral blood mononuclear cells treated with M199 alone or fractalkine for 24 or 48 h ( n = 3). * p
Figure Legend Snippet: CX3CR1 is endocytosed following fractalkine treatment and is not subsequently recycled to the surface of CD8 + T cells or secreted but, intracellular accumulations of the protein are detectable. (A) Frequencies of CX3CR1 + cells, as a percentage of CD8 + T cells following treatment with M199 media alone (NT), 30 ng/ml recombinant fractalkine alone (treatment alone), 30 ng/ml recombinant fractalkine at 4°C and 30 ng/ml recombinant fractalkine plus 80 µM Dynasore ( n = 10). (B) Bar chart showing frequencies of CX3CR1 + cells, as a percentage of CD8 + T cells following treatment with M199 media alone (NT) or 30 ng/ml of recombinant fractalkine for 24 h (fractalkine, light gray dot pattern) followed by removal from a fractalkine-free environment for 24 h (fractalkine, dark gray no pattern) or 48 h (fractalkine, light gray no pattern) ( n = 6). (C) Bar chart showing frequencies of CX3CR1 + cells, as a percentage of CD8 + T cells following treatment with M199 media alone (NT) or 30 ng/ml of recombinant fractalkine for 24 h (fractalkine, light gray dot pattern) followed by removal from a fractalkine-free environment for 24 h and treatment with 50 ng/ml of TNF-α, 100 ng/ml of IFN-γ, IL-17, IL-6, IL-7, IL-15, IL-18, IL-36, and 50 U/ml of IL-2 ( n = 3). (D) Western Blot (left) showing CX3CR1 and beta-actin protein in peripheral blood-derived CD8 + T cells from three donors following 24 h of no treatment (N1, N2, and N3) or fractalkine treatment (F1, F2, and F3) and densitometry data (right) from this western blot displayed as fold change bar chart. (E) Soluble CX3CR1 levels in the supernatant of peripheral blood mononuclear cells treated with M199 alone or fractalkine for 24 or 48 h ( n = 3). * p

Techniques Used: Recombinant, Western Blot, Derivative Assay

31) Product Images from "Human CD4+ CD25+ Regulatory T Cells Control T-Cell Responses to Human Immunodeficiency Virus and Cytomegalovirus Antigens"

Article Title: Human CD4+ CD25+ Regulatory T Cells Control T-Cell Responses to Human Immunodeficiency Virus and Cytomegalovirus Antigens

Journal: Journal of Virology

doi: 10.1128/JVI.78.5.2454-2459.2004

Suppressive CD25 + T cells can be induced from PBMC depleted of CD25 + T cells after activation with SEB. PBMC were depleted of CD25 + cells (a), labeled with CFSE, and cultured in the presence of SEB for 7 days (b). At day 7 (c), the CD25 + and CD25 − cell fractions were added into fresh PBMC cultures from the same donor. The cocultures were stimulated for 18 h with SEB. Brefeldin A was added for the last 5 h to promote cytokine accumulation. The CFSE-stained cells added into the fresh PBMC at day 7 were gated out of the analysis. Representative data are shown.
Figure Legend Snippet: Suppressive CD25 + T cells can be induced from PBMC depleted of CD25 + T cells after activation with SEB. PBMC were depleted of CD25 + cells (a), labeled with CFSE, and cultured in the presence of SEB for 7 days (b). At day 7 (c), the CD25 + and CD25 − cell fractions were added into fresh PBMC cultures from the same donor. The cocultures were stimulated for 18 h with SEB. Brefeldin A was added for the last 5 h to promote cytokine accumulation. The CFSE-stained cells added into the fresh PBMC at day 7 were gated out of the analysis. Representative data are shown.

Techniques Used: Activation Assay, Labeling, Cell Culture, Staining

T R cells suppress superantigen-induced cytokine production. IFN-γ expression in T cells in PBMC was compared with that of T cells in PBMC depleted of CD25 + cells (a). The cultures were stimulated for 18 h with SEB. Brefeldin A was added for the last 5 h to promote cytokine accumulation. Means ± standard errors of the mean are shown ( n = 3). (b) CD25 + T cells were added back into PBMC depleted of CD25 + cells at increasing ratios. The CD25 + T cells were stained with CFSE before being added back and were gated out of the analysis. (c) CD25 + T cells and sorted CD25 + CD4 + T cells ( > 99% pure) were added back into PBMC depleted of CD25 + cells. Means ± standard errors of the mean are shown ( n = 2).
Figure Legend Snippet: T R cells suppress superantigen-induced cytokine production. IFN-γ expression in T cells in PBMC was compared with that of T cells in PBMC depleted of CD25 + cells (a). The cultures were stimulated for 18 h with SEB. Brefeldin A was added for the last 5 h to promote cytokine accumulation. Means ± standard errors of the mean are shown ( n = 3). (b) CD25 + T cells were added back into PBMC depleted of CD25 + cells at increasing ratios. The CD25 + T cells were stained with CFSE before being added back and were gated out of the analysis. (c) CD25 + T cells and sorted CD25 + CD4 + T cells ( > 99% pure) were added back into PBMC depleted of CD25 + cells. Means ± standard errors of the mean are shown ( n = 2).

Techniques Used: Expressing, Staining

T R cells suppress antiviral immune responses. PBMC from a healthy CMV-infected individual were stained with a CMV pp65 tetramer and with cell surface markers (a). The frequency of tetramer-positive CD8 + T cells was compared with the frequency of IFN-γ-expressing T cells in PBMC, PBMC cultures depleted of CD25 + cells, and PBMC cultures depleted of CD25 + cells to which the depleted cells were added back in a 1:3 ratio. PBMC from an HIV-infected subject were stimulated with HIV antigens (b). The frequencies of IFN-γ- and TNF-α-expressing T cells in PMBC cultures and PBMC cultures depleted of CD25 + cells were compared. Representative data are shown.
Figure Legend Snippet: T R cells suppress antiviral immune responses. PBMC from a healthy CMV-infected individual were stained with a CMV pp65 tetramer and with cell surface markers (a). The frequency of tetramer-positive CD8 + T cells was compared with the frequency of IFN-γ-expressing T cells in PBMC, PBMC cultures depleted of CD25 + cells, and PBMC cultures depleted of CD25 + cells to which the depleted cells were added back in a 1:3 ratio. PBMC from an HIV-infected subject were stimulated with HIV antigens (b). The frequencies of IFN-γ- and TNF-α-expressing T cells in PMBC cultures and PBMC cultures depleted of CD25 + cells were compared. Representative data are shown.

Techniques Used: Infection, Staining, Expressing

The frequency of T R cells in HIV patients is unaltered. PBMC from HIV patients ( n = 10) and healthy subjects ( n = 10) were stained with fluorochrome-labeled monoclonal antibodies and analyzed for the frequencies of CD4 + CD25 + T cells and CD8 + CD38 + T cells. Means ± standard errors of the mean are shown.
Figure Legend Snippet: The frequency of T R cells in HIV patients is unaltered. PBMC from HIV patients ( n = 10) and healthy subjects ( n = 10) were stained with fluorochrome-labeled monoclonal antibodies and analyzed for the frequencies of CD4 + CD25 + T cells and CD8 + CD38 + T cells. Means ± standard errors of the mean are shown.

Techniques Used: Staining, Labeling

Depletion of T R cells enhances the antiviral immune response to HIV. PBMC from HIV-infected subjects ( n = 6) were stimulated with HIV and CMV antigens. The frequencies of IFN-γ- and TNF-α-expressing T cells in PMBC cultures and PBMC cultures depleted of CD25 + cells were compared. The cells were gated on the CD3 + CD8 − and CD3 + CD8 + T cells. The shaded area in each graph represents the level of detection. Note: the scale on the y axis differs in the panels.
Figure Legend Snippet: Depletion of T R cells enhances the antiviral immune response to HIV. PBMC from HIV-infected subjects ( n = 6) were stimulated with HIV and CMV antigens. The frequencies of IFN-γ- and TNF-α-expressing T cells in PMBC cultures and PBMC cultures depleted of CD25 + cells were compared. The cells were gated on the CD3 + CD8 − and CD3 + CD8 + T cells. The shaded area in each graph represents the level of detection. Note: the scale on the y axis differs in the panels.

Techniques Used: Infection, Expressing

32) Product Images from "Inhibition of mesenchymal stromal cells by pre-activated lymphocytes and their culture media"

Article Title: Inhibition of mesenchymal stromal cells by pre-activated lymphocytes and their culture media

Journal: Stem Cell Research & Therapy

doi: 10.1186/scrt392

Impedance profiles of MSCs in co-culture with NK-depleted PBMCs. Unstimulated (NS) NK-depleted PBMCs (A) and PHA-stimulated NK-depleted PBMCs (B) were added to MSCs (400/well), after about 72 hours of culture, at four different ratios. The experiment was conducted in triplicate. MSCs, mesenchymal stem cells; NK, natural killer; PBMCs, peripheral blood mononuclear cells; PHA, phytohemagglutinin.
Figure Legend Snippet: Impedance profiles of MSCs in co-culture with NK-depleted PBMCs. Unstimulated (NS) NK-depleted PBMCs (A) and PHA-stimulated NK-depleted PBMCs (B) were added to MSCs (400/well), after about 72 hours of culture, at four different ratios. The experiment was conducted in triplicate. MSCs, mesenchymal stem cells; NK, natural killer; PBMCs, peripheral blood mononuclear cells; PHA, phytohemagglutinin.

Techniques Used: Co-Culture Assay

NK degranulation assay. Surface expression of CD107a in resting (A) or pre-activated (B) PBMCs. Three culture conditions were analyzed: PBMCs alone (negative control), PBMCs with the addition of K562 (positive control) and PBMCs with MSCs. For this flow cytometry assay, CD3 - CD56 + NK cells were gated. MSCs, mesenchymal stem cells; NK, natural killer, PBMCs, peripheral blood mononuclear cells.
Figure Legend Snippet: NK degranulation assay. Surface expression of CD107a in resting (A) or pre-activated (B) PBMCs. Three culture conditions were analyzed: PBMCs alone (negative control), PBMCs with the addition of K562 (positive control) and PBMCs with MSCs. For this flow cytometry assay, CD3 - CD56 + NK cells were gated. MSCs, mesenchymal stem cells; NK, natural killer, PBMCs, peripheral blood mononuclear cells.

Techniques Used: Degranulation Assay, Expressing, Negative Control, Positive Control, Flow Cytometry, Cytometry

Impedance profiles and mean CI values of MSCs treated with lymphocyte conditioned medium (CM). MSCs were stimulated with CM from unstimulated lymphocytes (A) and PHA-stimulated lymphocytes (B) . The CM was added to MSCs (400/well) at four different dilutions after about 72 hours of culture. The experiment was conducted in triplicate. (C) The mean CI values, obtained in the four independent experiments, treating MSCs with CM from resting or pre-activated PBMCs. Results were analyzed and expressed as percentage of the untreated MSCs and presented as mean values ± standard deviations. Statistical analysis was performed using one-way ANOVA (*P
Figure Legend Snippet: Impedance profiles and mean CI values of MSCs treated with lymphocyte conditioned medium (CM). MSCs were stimulated with CM from unstimulated lymphocytes (A) and PHA-stimulated lymphocytes (B) . The CM was added to MSCs (400/well) at four different dilutions after about 72 hours of culture. The experiment was conducted in triplicate. (C) The mean CI values, obtained in the four independent experiments, treating MSCs with CM from resting or pre-activated PBMCs. Results were analyzed and expressed as percentage of the untreated MSCs and presented as mean values ± standard deviations. Statistical analysis was performed using one-way ANOVA (*P

Techniques Used:

MTT cell viability assay. Cell viability was assessed after MSC treatment with the indicated dose of lymphocyte CM. (A) CM from unstimulated PBMCs; (B) CM from PHA-stimulated PBMCs. Results were analyzed and expressed as percentage of the control (untreated MSCs) and presented as mean values ± standard deviations of the three independent experiments performed in duplicate. Statistical analysis was performed using Student’s t test (* P
Figure Legend Snippet: MTT cell viability assay. Cell viability was assessed after MSC treatment with the indicated dose of lymphocyte CM. (A) CM from unstimulated PBMCs; (B) CM from PHA-stimulated PBMCs. Results were analyzed and expressed as percentage of the control (untreated MSCs) and presented as mean values ± standard deviations of the three independent experiments performed in duplicate. Statistical analysis was performed using Student’s t test (* P

Techniques Used: MTT Assay, Viability Assay

Impedance profiles and mean CI values of MSCs in co-culture with PBMCs. Unstimulated (NS) PBMCs (A) and PHA-stimulated PBMCs (B) were added to MSCs (400/well) after about 72 hours of culture at four different ratios. The experiment was conducted in triplicate. (C) The mean CI values, obtained in the four independent experiments, treating MSCs with resting or pre-activated PBMCs. Results were analyzed and expressed as percentage of the untreated MSCs and presented as mean values ± standard deviations. Statistical analysis was performed using one-way ANOVA (* P
Figure Legend Snippet: Impedance profiles and mean CI values of MSCs in co-culture with PBMCs. Unstimulated (NS) PBMCs (A) and PHA-stimulated PBMCs (B) were added to MSCs (400/well) after about 72 hours of culture at four different ratios. The experiment was conducted in triplicate. (C) The mean CI values, obtained in the four independent experiments, treating MSCs with resting or pre-activated PBMCs. Results were analyzed and expressed as percentage of the untreated MSCs and presented as mean values ± standard deviations. Statistical analysis was performed using one-way ANOVA (* P

Techniques Used: Co-Culture Assay

33) Product Images from "Oxidative Stress-Induced DNA Damage and Repair in Human Peripheral Blood Mononuclear Cells: Protective Role of Hemoglobin"

Article Title: Oxidative Stress-Induced DNA Damage and Repair in Human Peripheral Blood Mononuclear Cells: Protective Role of Hemoglobin

Journal: PLoS ONE

doi: 10.1371/journal.pone.0068341

Induction of activation and H2AX phosphorylation in PBMC cells exposed to 200 µM H 2 O2 for 40 min. (a) PBMC were untreated; or (b) treated with 200 µM H 2 O 2 for 40 min;(C) or treated with 200 µmol/L H 2 O 2 for 40 min with hemolysate, each detected immunocytochemically, measured by laser scanning cytometry (scatterplots). The dashed skewed lines show the upper threshold level of PBMC and phosphorylated H2AX expression. % of PBMC expressing H2AX (n = 3) is shown in the graph.
Figure Legend Snippet: Induction of activation and H2AX phosphorylation in PBMC cells exposed to 200 µM H 2 O2 for 40 min. (a) PBMC were untreated; or (b) treated with 200 µM H 2 O 2 for 40 min;(C) or treated with 200 µmol/L H 2 O 2 for 40 min with hemolysate, each detected immunocytochemically, measured by laser scanning cytometry (scatterplots). The dashed skewed lines show the upper threshold level of PBMC and phosphorylated H2AX expression. % of PBMC expressing H2AX (n = 3) is shown in the graph.

Techniques Used: Activation Assay, Cytometry, Expressing

34) Product Images from "HLA-F is a surface marker on activated lymphocytes1"

Article Title: HLA-F is a surface marker on activated lymphocytes1

Journal: European journal of immunology

doi: 10.1002/eji.201040348

], and a normal control was activated with PMA plus ionomycin. FACS profiles were generated using each of the anti-F antibodies 4A11 (solid line), the anti-HLA-E reagent 3D12 (dotted line), and control mouse IgG1 (shaded histogram). Cells were analyzed on days 0, 1, 2 and 5 after stimulation as indicated above the profiles (Tapasin − cells were only analyzed on days 0, 1, and 2). Cell subsets analyzed are indicated to the left of each pair of profiles and the source of the PBMC is indicated to the upper left of each set of FACS profiles as TAP − , TPN − or PBMC indicating normal control cells.
Figure Legend Snippet: ], and a normal control was activated with PMA plus ionomycin. FACS profiles were generated using each of the anti-F antibodies 4A11 (solid line), the anti-HLA-E reagent 3D12 (dotted line), and control mouse IgG1 (shaded histogram). Cells were analyzed on days 0, 1, 2 and 5 after stimulation as indicated above the profiles (Tapasin − cells were only analyzed on days 0, 1, and 2). Cell subsets analyzed are indicated to the left of each pair of profiles and the source of the PBMC is indicated to the upper left of each set of FACS profiles as TAP − , TPN − or PBMC indicating normal control cells.

Techniques Used: FACS, Generated

35) Product Images from "Myeloid-Derived Suppressor Cells Specifically Suppress IFN-γ Production and Antitumor Cytotoxic Activity of Vδ2 T Cells"

Article Title: Myeloid-Derived Suppressor Cells Specifically Suppress IFN-γ Production and Antitumor Cytotoxic Activity of Vδ2 T Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01271

PMN-myeloid-derived suppressor cells (MDSC) does not inhibit Vδ2 T cell proliferation. γδ T cells or peripheral blood mononuclear cell (PBMC) labeled with CFDA-SE were stimulated with IPH in the presence of PMN-MDSC (1:1 ratio). After 5 days, Vδ2+ and CD3+ T cells proliferation was evaluated by flow cytometry. Representative gating strategy and histogram plots of one out of three independent experiments showing Vδ2 T cells (A) and CD3+ T cells (B) proliferation in the indicated conditions.
Figure Legend Snippet: PMN-myeloid-derived suppressor cells (MDSC) does not inhibit Vδ2 T cell proliferation. γδ T cells or peripheral blood mononuclear cell (PBMC) labeled with CFDA-SE were stimulated with IPH in the presence of PMN-MDSC (1:1 ratio). After 5 days, Vδ2+ and CD3+ T cells proliferation was evaluated by flow cytometry. Representative gating strategy and histogram plots of one out of three independent experiments showing Vδ2 T cells (A) and CD3+ T cells (B) proliferation in the indicated conditions.

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

36) Product Images from "Lack of evidence for the Th2 predominance in patients with chronic hepatitis C"

Article Title: Lack of evidence for the Th2 predominance in patients with chronic hepatitis C

Journal: Clinical and Experimental Immunology

doi: 10.1046/j.1365-2249.2001.01467.x

Cytokine measurement of mitogen-induced cytokine production by ELISA testing of supernatants from unfractionated peripheral blood mononuclear cells. a, IL-4; b, IL-10; c, IL-13; d, IFN-γ; e, IL-2.Bars represent S.D.
Figure Legend Snippet: Cytokine measurement of mitogen-induced cytokine production by ELISA testing of supernatants from unfractionated peripheral blood mononuclear cells. a, IL-4; b, IL-10; c, IL-13; d, IFN-γ; e, IL-2.Bars represent S.D.

Techniques Used: Enzyme-linked Immunosorbent Assay

37) Product Images from "Interferon-lambda1 induces peripheral blood mononuclear cell-derived chemokines secretion in patients with systemic lupus erythematosus: its correlation with disease activity"

Article Title: Interferon-lambda1 induces peripheral blood mononuclear cell-derived chemokines secretion in patients with systemic lupus erythematosus: its correlation with disease activity

Journal: Arthritis Research & Therapy

doi: 10.1186/ar3363

IFN-λ1-induced chemokines production by human PBMC and dose-relationship of IFN-λ1- induced chemokines by PBMC . Human PBMC were cultured for 72 h in the presence of recombinant IFN-λ1 and supernatants were examined for levels of IP-10, MIG and IL-8 using ELISA. The chemokines' response to LPS was also examined as a positive control. In SLE patients, IFN-λ1-stimulated PBMC displayed higher levels of chemokines IP-10 ( a ) and MIG ( b ) in comparison with positive control LPS. IFN-λ1 could induce more generation levels of chemokines IP-10 (a) and MIG (b) in SLE patients than normal controls, but the secretion levels of IL-8 were lower in SLE patients than normal controls ( c ). Meanwhile, in patients with SLE, IFN-λ1 induced less secretion of chemokine IL-8 than LPS, but it had the ability to stimulate more IL-8 production than medium (c). Then human PBMC were cultured over different IFN-λ1 concentrations for 72 h and supernatants were examined for IP-10 ( d ), MIG ( e ) and IL-8 ( f ) levels using ELISA. Both 10 ng/ml and 50 ng/ml IFN-λ1 had effects on the secretion of IP-10 ( P = 0.033, 0.005) (d). IFN-λ1 displayed its effects at the three different concentration on MIG secretion with dose-dependent relation ( P = 0.043, 0.016, 0.001) (e). Compared with other two concentration, 10 ng/ml IFN-λ1 had the most obvious effect in the secretion of IL-8 ( P = 0.008) (f). *, P
Figure Legend Snippet: IFN-λ1-induced chemokines production by human PBMC and dose-relationship of IFN-λ1- induced chemokines by PBMC . Human PBMC were cultured for 72 h in the presence of recombinant IFN-λ1 and supernatants were examined for levels of IP-10, MIG and IL-8 using ELISA. The chemokines' response to LPS was also examined as a positive control. In SLE patients, IFN-λ1-stimulated PBMC displayed higher levels of chemokines IP-10 ( a ) and MIG ( b ) in comparison with positive control LPS. IFN-λ1 could induce more generation levels of chemokines IP-10 (a) and MIG (b) in SLE patients than normal controls, but the secretion levels of IL-8 were lower in SLE patients than normal controls ( c ). Meanwhile, in patients with SLE, IFN-λ1 induced less secretion of chemokine IL-8 than LPS, but it had the ability to stimulate more IL-8 production than medium (c). Then human PBMC were cultured over different IFN-λ1 concentrations for 72 h and supernatants were examined for IP-10 ( d ), MIG ( e ) and IL-8 ( f ) levels using ELISA. Both 10 ng/ml and 50 ng/ml IFN-λ1 had effects on the secretion of IP-10 ( P = 0.033, 0.005) (d). IFN-λ1 displayed its effects at the three different concentration on MIG secretion with dose-dependent relation ( P = 0.043, 0.016, 0.001) (e). Compared with other two concentration, 10 ng/ml IFN-λ1 had the most obvious effect in the secretion of IL-8 ( P = 0.008) (f). *, P

Techniques Used: Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay, Positive Control, Concentration Assay

IFN-λ1 increased the chemokines response to LPS . Whole PBMC were incubated in the presence of IFN-λ1 for 30 minutes before the addition of LPS at the doses indicated. Cells were then incubated for 72 h before being assessed for levels of IP-10 ( a ), MIG ( b ) and IL-8 ( c ) in culture supernatants using ELISA. only 50 ng/ml IFN-λ1 showed an assistant effect on the chemokine IP-10 secretion ( P = 0.016) (a). Different concentrations of IFN-λ1 all played a synergistic role in the effect of LPS ( P = 0.047, 0.013, 0.038) (b). Both 10 ng/ml and 100 ng/ml IFN-λ1 displayed this synergistic effect on the secretion of chemokine IL-8 ( P
Figure Legend Snippet: IFN-λ1 increased the chemokines response to LPS . Whole PBMC were incubated in the presence of IFN-λ1 for 30 minutes before the addition of LPS at the doses indicated. Cells were then incubated for 72 h before being assessed for levels of IP-10 ( a ), MIG ( b ) and IL-8 ( c ) in culture supernatants using ELISA. only 50 ng/ml IFN-λ1 showed an assistant effect on the chemokine IP-10 secretion ( P = 0.016) (a). Different concentrations of IFN-λ1 all played a synergistic role in the effect of LPS ( P = 0.047, 0.013, 0.038) (b). Both 10 ng/ml and 100 ng/ml IFN-λ1 displayed this synergistic effect on the secretion of chemokine IL-8 ( P

Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

38) Product Images from "Proinflammatory profile of in vitro monocytes in the ageing is affected by lymphocytes presence"

Article Title: Proinflammatory profile of in vitro monocytes in the ageing is affected by lymphocytes presence

Journal: Immunity & Ageing : I & A

doi: 10.1186/1742-4933-10-22

Influence of lymphocytes presence on cytokines production. Note that with the presence of lymphocytes the spontaneous production of IL-10 was higher and of TGF-β was lower than that of monocytes, regardless of age. After stimulation with LPS, the presence of lymphocytes resulted in increased IL-6, IL-1β, MCP-1 and IL-10 and decreased CXCL-8 and TGF-β in comparison to pure culture of monocytes from of young patients. With age, the same differences were observed, except for CXCL-8 and TGF-β which production was the same between monocytes and PBMC stimulated with LPS.
Figure Legend Snippet: Influence of lymphocytes presence on cytokines production. Note that with the presence of lymphocytes the spontaneous production of IL-10 was higher and of TGF-β was lower than that of monocytes, regardless of age. After stimulation with LPS, the presence of lymphocytes resulted in increased IL-6, IL-1β, MCP-1 and IL-10 and decreased CXCL-8 and TGF-β in comparison to pure culture of monocytes from of young patients. With age, the same differences were observed, except for CXCL-8 and TGF-β which production was the same between monocytes and PBMC stimulated with LPS.

Techniques Used:

Measurements of the anti - inflammatory cytokines production by blood monocytes (MON) or PBMC from healthy elderly and young subjects. Peripheral blood was obtained from volunteers, and the cells were purified as described in the Material and Methods section. Monocytes and PBMC were challenged with LPS (100 ng/mL), or not (Basal), for 18 and 24 hours, respectively, and cytokine production was determined by ELISA. Columns represent the median and error bar the interquartile range. The results were evaluated by Mann–Whitney Rank Sum Test. In the same subject group, statistical differences are represented by continuous lines (cells stimulated or not) and letters for different cell types. Asterisks indicate statistical difference between groups of subjects ( p values - * 0,0016; a and b 0,0039; c 0,0159; d 0,0010, e, f and g
Figure Legend Snippet: Measurements of the anti - inflammatory cytokines production by blood monocytes (MON) or PBMC from healthy elderly and young subjects. Peripheral blood was obtained from volunteers, and the cells were purified as described in the Material and Methods section. Monocytes and PBMC were challenged with LPS (100 ng/mL), or not (Basal), for 18 and 24 hours, respectively, and cytokine production was determined by ELISA. Columns represent the median and error bar the interquartile range. The results were evaluated by Mann–Whitney Rank Sum Test. In the same subject group, statistical differences are represented by continuous lines (cells stimulated or not) and letters for different cell types. Asterisks indicate statistical difference between groups of subjects ( p values - * 0,0016; a and b 0,0039; c 0,0159; d 0,0010, e, f and g

Techniques Used: Purification, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Measurements of the proinflammatory cytokines production by blood monocytes (MON) or PBMC from healthy elderly and young subjects. Peripheral blood was obtained from volunteers, and the cells were purified as described in the Material and Methods section. Monocytes and PBMC were challenged with LPS (100 ng/mL), or not (Basal), for 18 and 24 hours, respectively, and cytokine production was determined by ELISA. Columns represent the median and error bar the interquartile range. The results were evaluated by Mann–Whitney Rank Sum Test. In the same subject group, statistical differences are represented by continuous lines (cells stimulated or not) and letters for different cell types. Asterisks indicate statistical difference between groups of subjects ( p values - *0,0487; a 0,0029; b 0,0068; c 0,0061; d 0,0195; e 0,0180; ** 0,0399; f 0,0089; g 0,0098).
Figure Legend Snippet: Measurements of the proinflammatory cytokines production by blood monocytes (MON) or PBMC from healthy elderly and young subjects. Peripheral blood was obtained from volunteers, and the cells were purified as described in the Material and Methods section. Monocytes and PBMC were challenged with LPS (100 ng/mL), or not (Basal), for 18 and 24 hours, respectively, and cytokine production was determined by ELISA. Columns represent the median and error bar the interquartile range. The results were evaluated by Mann–Whitney Rank Sum Test. In the same subject group, statistical differences are represented by continuous lines (cells stimulated or not) and letters for different cell types. Asterisks indicate statistical difference between groups of subjects ( p values - *0,0487; a 0,0029; b 0,0068; c 0,0061; d 0,0195; e 0,0180; ** 0,0399; f 0,0089; g 0,0098).

Techniques Used: Purification, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

39) Product Images from "The CD8-Derived Chemokine XCL1/Lymphotactin Is a Conformation-Dependent, Broad-Spectrum Inhibitor of HIV-1"

Article Title: The CD8-Derived Chemokine XCL1/Lymphotactin Is a Conformation-Dependent, Broad-Spectrum Inhibitor of HIV-1

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003852

XCL1 antiviral activity is equally effective before and after digestion of glycosaminoglycans on PBMC. PBMC were incubated in the presence (‘Heparitinase-treated’) or absence (‘Untreated’) of heparitinase to digest cell surface glycosaminoglycan expression, followed by infection with HIV-1 IIIB (A) or BaL (B) in the presence of XCL1 WT (green bars), W55D (purple bars) or CC3 (light blue bars) at 1 µM. Virus replication was assessed by measuring the amount of p24 Gag antigen in PBMC supernatants via AlphaLISA immunoassay. Data were normalized to the amount of viral replication observed in control cultures (not treated with XCL1). Results represent the mean values (±SD) of replicate wells.
Figure Legend Snippet: XCL1 antiviral activity is equally effective before and after digestion of glycosaminoglycans on PBMC. PBMC were incubated in the presence (‘Heparitinase-treated’) or absence (‘Untreated’) of heparitinase to digest cell surface glycosaminoglycan expression, followed by infection with HIV-1 IIIB (A) or BaL (B) in the presence of XCL1 WT (green bars), W55D (purple bars) or CC3 (light blue bars) at 1 µM. Virus replication was assessed by measuring the amount of p24 Gag antigen in PBMC supernatants via AlphaLISA immunoassay. Data were normalized to the amount of viral replication observed in control cultures (not treated with XCL1). Results represent the mean values (±SD) of replicate wells.

Techniques Used: Activity Assay, Incubation, Expressing, Infection

40) Product Images from "Ex vivo induction of viral antigen-specific CD8+ T cell responses using mRNA-electroporated CD40-activated B cells"

Article Title: Ex vivo induction of viral antigen-specific CD8+ T cell responses using mRNA-electroporated CD40-activated B cells

Journal:

doi: 10.1111/j.1365-2249.2005.02733.x

CD40-activated B cells transfected with mRNA coding for viral antigens can induce viral-specific T cell responses. (Top row) IFN-γ release of PBMC from donors A–C primed with CMV pp65 mRNA-electroporated autologous CD40-B cells after restimulation
Figure Legend Snippet: CD40-activated B cells transfected with mRNA coding for viral antigens can induce viral-specific T cell responses. (Top row) IFN-γ release of PBMC from donors A–C primed with CMV pp65 mRNA-electroporated autologous CD40-B cells after restimulation

Techniques Used: Transfection

Peptide-pulsed CD40-activated B cells can act as alternative antigen-presenting cells. (a) IFN-γ production of PBMC primed with CMV pp65 mRNA-electroporated mature DC after restimulation with unloaded or with CMV pp65 peptide-pulsed T2 or autologous
Figure Legend Snippet: Peptide-pulsed CD40-activated B cells can act as alternative antigen-presenting cells. (a) IFN-γ production of PBMC primed with CMV pp65 mRNA-electroporated mature DC after restimulation with unloaded or with CMV pp65 peptide-pulsed T2 or autologous

Techniques Used: Activated Clotting Time Assay

Related Articles

Flow Cytometry:

Article Title: T-Cell Immunophenotyping Distinguishes Active From Latent Tuberculosis
Article Snippet: .. Intracellular Cytokine Staining and Polychromatic Flow Cytometry Thawed PBMCs (3–5 × 106 per well) were cultured for 16 hours (37°C, 5% CO2 ) in 10% human serum (Sigma–Aldrich) in RPMI-1640 (Sigma-Aldrich) at a concentration of 1 × 107 cells/mL. .. Cells were stimulated with PMA-Ionomycin (positive control) (Sigma–Aldrich; final concentration of 5ng/ml for PMA and 500ng/ml for Ionomycin), PPD (16.7 μg/mL final concentration), or a cocktail of peptides spanning the length of 3 highly immunodominant M. tuberculosis-specific RD1-associated antigens, ESAT-6, CFP-10, and EspC (10 µg/mL final concentration per peptide) [ ].

Article Title: Expression and Activation by Epstein Barr Virus of Human Endogenous Retroviruses-W in Blood Cells and Astrocytes: Inference for Multiple Sclerosis
Article Snippet: .. Treatment with cytokines and PMA The PBMC were exposed overnight to either human recombinant TNFα, IFNγ, or PMA, at the doses of 1 ng/ml, 1000 IU/ml, and 50 nM, respectively (Sigma-Aldrich, St. Louis, MO), then were harvested and processed for RT-PCR and flow cytometry. .. RNA extraction and Real-time PCR RNAs were extracted from 50.000 cells, by mRNA Dynabeads® kit (Dynal Biotech, Oslo, NO) and retrotranscribed; selective amplification of MSRVenv and syncytin-1 sequences was obtained by utilizing the corresponding primer pairs, as published in .

Binding Assay:

Article Title: Peptide-Derivatized SB105-A10 Dendrimer Inhibits the Infectivity of R5 and X4 HIV-1 Strains in Primary PBMCs and Cervicovaginal Histocultures
Article Snippet: .. Alternatively, the activated PBMCs were either incubated with heparinase III (40 mU/ml; Sigma, St Louis, MO, USA) in 20 mM TrisHCl pH 7.5, 0.1 mg/ml BSA and 4 mM CaCl2 for 2 h at 37°C or left untreated prior to being assayed for binding between the activated PBMCs and 25 µg/ml (5.3 µM) of FITC-SB105-A10 dendrimer. .. The samples were analyzed by FACScan flow cytometry.

Cytometry:

Article Title: T-Cell Immunophenotyping Distinguishes Active From Latent Tuberculosis
Article Snippet: .. Intracellular Cytokine Staining and Polychromatic Flow Cytometry Thawed PBMCs (3–5 × 106 per well) were cultured for 16 hours (37°C, 5% CO2 ) in 10% human serum (Sigma–Aldrich) in RPMI-1640 (Sigma-Aldrich) at a concentration of 1 × 107 cells/mL. .. Cells were stimulated with PMA-Ionomycin (positive control) (Sigma–Aldrich; final concentration of 5ng/ml for PMA and 500ng/ml for Ionomycin), PPD (16.7 μg/mL final concentration), or a cocktail of peptides spanning the length of 3 highly immunodominant M. tuberculosis-specific RD1-associated antigens, ESAT-6, CFP-10, and EspC (10 µg/mL final concentration per peptide) [ ].

Article Title: Expression and Activation by Epstein Barr Virus of Human Endogenous Retroviruses-W in Blood Cells and Astrocytes: Inference for Multiple Sclerosis
Article Snippet: .. Treatment with cytokines and PMA The PBMC were exposed overnight to either human recombinant TNFα, IFNγ, or PMA, at the doses of 1 ng/ml, 1000 IU/ml, and 50 nM, respectively (Sigma-Aldrich, St. Louis, MO), then were harvested and processed for RT-PCR and flow cytometry. .. RNA extraction and Real-time PCR RNAs were extracted from 50.000 cells, by mRNA Dynabeads® kit (Dynal Biotech, Oslo, NO) and retrotranscribed; selective amplification of MSRVenv and syncytin-1 sequences was obtained by utilizing the corresponding primer pairs, as published in .

Infection:

Article Title: Infection of human lymphomononuclear cells by SARS-CoV-2
Article Snippet: .. In parallel, PBMCs were treated with 0.5ug/ml Camostat (Sigma Aldrich, cat. SML005) or with 10 uM of anti-ACE2 antibody (Rhea Biotech, cat. IM-0060) starting 1 hour before infection. ..

Concentration Assay:

Article Title: T-Cell Immunophenotyping Distinguishes Active From Latent Tuberculosis
Article Snippet: .. Intracellular Cytokine Staining and Polychromatic Flow Cytometry Thawed PBMCs (3–5 × 106 per well) were cultured for 16 hours (37°C, 5% CO2 ) in 10% human serum (Sigma–Aldrich) in RPMI-1640 (Sigma-Aldrich) at a concentration of 1 × 107 cells/mL. .. Cells were stimulated with PMA-Ionomycin (positive control) (Sigma–Aldrich; final concentration of 5ng/ml for PMA and 500ng/ml for Ionomycin), PPD (16.7 μg/mL final concentration), or a cocktail of peptides spanning the length of 3 highly immunodominant M. tuberculosis-specific RD1-associated antigens, ESAT-6, CFP-10, and EspC (10 µg/mL final concentration per peptide) [ ].

Incubation:

Article Title: Peptide-Derivatized SB105-A10 Dendrimer Inhibits the Infectivity of R5 and X4 HIV-1 Strains in Primary PBMCs and Cervicovaginal Histocultures
Article Snippet: .. Alternatively, the activated PBMCs were either incubated with heparinase III (40 mU/ml; Sigma, St Louis, MO, USA) in 20 mM TrisHCl pH 7.5, 0.1 mg/ml BSA and 4 mM CaCl2 for 2 h at 37°C or left untreated prior to being assayed for binding between the activated PBMCs and 25 µg/ml (5.3 µM) of FITC-SB105-A10 dendrimer. .. The samples were analyzed by FACScan flow cytometry.

Cell Culture:

Article Title: Estrogen and progesterone play pivotal roles in endothelial progenitor cell proliferation
Article Snippet: .. EPC culture with E2 β or P4 To assess the effects of E2 β or P4 on EPC proliferation of EPCs, 8 × 105 PBMCs, derived from the peripheral blood of women in the menstrual or luteal phases, were seeded into each well of 96-well culture plates coated with human fibronectin (Sigma-Aldrich), and cultured in endothelial basal medium (phenol red free EBM-2, Clonetics; San Diego, CA) supplemented with EGM-2MV (Clonetics) consisting of 5% charcoal stripped serum (Invitrogen), VEGF, FGF2, EGF, IGF1, and ascorbic acid with or without 10-9 -10-7 M of E2 β or P4 . .. ICI 182,780 (10-5 M, Wako Pure Chemical Industries, Ltd., Osaka, Japan) or RU486 (10-5 M, Sigma-Aldrich) was used as an inhibitor of ER or PR, respectively [ ].

Article Title: T-Cell Immunophenotyping Distinguishes Active From Latent Tuberculosis
Article Snippet: .. Intracellular Cytokine Staining and Polychromatic Flow Cytometry Thawed PBMCs (3–5 × 106 per well) were cultured for 16 hours (37°C, 5% CO2 ) in 10% human serum (Sigma–Aldrich) in RPMI-1640 (Sigma-Aldrich) at a concentration of 1 × 107 cells/mL. .. Cells were stimulated with PMA-Ionomycin (positive control) (Sigma–Aldrich; final concentration of 5ng/ml for PMA and 500ng/ml for Ionomycin), PPD (16.7 μg/mL final concentration), or a cocktail of peptides spanning the length of 3 highly immunodominant M. tuberculosis-specific RD1-associated antigens, ESAT-6, CFP-10, and EspC (10 µg/mL final concentration per peptide) [ ].

Expressing:

Article Title: Circulating CD4+CD28null T Cells May Increase the Risk of an Atherosclerotic Vascular Event Shortly after Kidney Transplantation
Article Snippet: .. In order to analyze percentages of interferon-gamma (IFN-γ ) or TNF-alpha (TNF-α ) producing CD4+ T cells and that of CD4+ T cells expressing or lacking CD28, PBMC were stimulated with standard culture medium alone or combined with a mixture of PMA (50 ng/mL; Sigma Aldrich, St. Louis, MO, USA) and ionomycin (1 μ g/mL, Sigma Aldrich) for 6 hours in presence of the cytokine secretion inhibitor golgiplug (BD). ..

Reverse Transcription Polymerase Chain Reaction:

Article Title: Expression and Activation by Epstein Barr Virus of Human Endogenous Retroviruses-W in Blood Cells and Astrocytes: Inference for Multiple Sclerosis
Article Snippet: .. Treatment with cytokines and PMA The PBMC were exposed overnight to either human recombinant TNFα, IFNγ, or PMA, at the doses of 1 ng/ml, 1000 IU/ml, and 50 nM, respectively (Sigma-Aldrich, St. Louis, MO), then were harvested and processed for RT-PCR and flow cytometry. .. RNA extraction and Real-time PCR RNAs were extracted from 50.000 cells, by mRNA Dynabeads® kit (Dynal Biotech, Oslo, NO) and retrotranscribed; selective amplification of MSRVenv and syncytin-1 sequences was obtained by utilizing the corresponding primer pairs, as published in .

Staining:

Article Title: T-Cell Immunophenotyping Distinguishes Active From Latent Tuberculosis
Article Snippet: .. Intracellular Cytokine Staining and Polychromatic Flow Cytometry Thawed PBMCs (3–5 × 106 per well) were cultured for 16 hours (37°C, 5% CO2 ) in 10% human serum (Sigma–Aldrich) in RPMI-1640 (Sigma-Aldrich) at a concentration of 1 × 107 cells/mL. .. Cells were stimulated with PMA-Ionomycin (positive control) (Sigma–Aldrich; final concentration of 5ng/ml for PMA and 500ng/ml for Ionomycin), PPD (16.7 μg/mL final concentration), or a cocktail of peptides spanning the length of 3 highly immunodominant M. tuberculosis-specific RD1-associated antigens, ESAT-6, CFP-10, and EspC (10 µg/mL final concentration per peptide) [ ].

Recombinant:

Article Title: Expression and Activation by Epstein Barr Virus of Human Endogenous Retroviruses-W in Blood Cells and Astrocytes: Inference for Multiple Sclerosis
Article Snippet: .. Treatment with cytokines and PMA The PBMC were exposed overnight to either human recombinant TNFα, IFNγ, or PMA, at the doses of 1 ng/ml, 1000 IU/ml, and 50 nM, respectively (Sigma-Aldrich, St. Louis, MO), then were harvested and processed for RT-PCR and flow cytometry. .. RNA extraction and Real-time PCR RNAs were extracted from 50.000 cells, by mRNA Dynabeads® kit (Dynal Biotech, Oslo, NO) and retrotranscribed; selective amplification of MSRVenv and syncytin-1 sequences was obtained by utilizing the corresponding primer pairs, as published in .

Derivative Assay:

Article Title: Estrogen and progesterone play pivotal roles in endothelial progenitor cell proliferation
Article Snippet: .. EPC culture with E2 β or P4 To assess the effects of E2 β or P4 on EPC proliferation of EPCs, 8 × 105 PBMCs, derived from the peripheral blood of women in the menstrual or luteal phases, were seeded into each well of 96-well culture plates coated with human fibronectin (Sigma-Aldrich), and cultured in endothelial basal medium (phenol red free EBM-2, Clonetics; San Diego, CA) supplemented with EGM-2MV (Clonetics) consisting of 5% charcoal stripped serum (Invitrogen), VEGF, FGF2, EGF, IGF1, and ascorbic acid with or without 10-9 -10-7 M of E2 β or P4 . .. ICI 182,780 (10-5 M, Wako Pure Chemical Industries, Ltd., Osaka, Japan) or RU486 (10-5 M, Sigma-Aldrich) was used as an inhibitor of ER or PR, respectively [ ].

Generated:

Article Title: Human IL2RA null mutation mediates immunodeficiency with lymphoproliferation and autoimmunity
Article Snippet: .. 2.3 T cell line generation and stimulation Healthy donor cell lines were generated by stimulating 1 × 106 PBMCs with PHA 1 μg/ml (Sigma) in X-Vivo media (Biowhitaker) containing 5% human serum (Biowhitaker), 1% penicillin and streptomycin (Lonza), IL-2 (40 U/ml, Proleukin (Novartis)). ..

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  • 89
    Millipore culture pbmcs
    Features of ssRNA40-induced LILRA3 expression. a Kinetics of LILRA3 expression was compared to other ssRNA40 inducible cytokines. <t>PBMCs</t> from two donors were stimulated with ssRNA40 or ssRNA41 and the RNA was harvested at various time points. qPCR for LILRA3 , TNF , IFNb , IFNg , and IL1b expression, normalized to RPLP0 and relatively quantified to calibrator cDNA (pooled cDNA from 10. b Effect of cytokine inhibition on LILRA3 expression. PBMCs from two donors were stimulated overnight with ssRNA40 together with neutralizing agents anakinra, certolizumab, tocilizumab and IFN-γ neutralizing antibodies and analysed for LILRA3 and IL6 expression by qPCR. Repeated measures ANOVA was used to calculate difference of ssRNA40+ neutralization to ssRNA40 alone and results displayed as mean ± SD
    Culture Pbmcs, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 2549 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore pbmc specimens against sd1 lps
    Fecal s-IgA antibody titers to <t>SD1-LPS</t> in group1 (cross symbol), group 2 (open square symbol), and group 3 (closed round symbol) monkeys, expressed as MN with MN ± SE, after challenge on day 00 and re-challenge on day 31 with SD11617 strain.
    Pbmc Specimens Against Sd1 Lps, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore human pbmcs
    Imatinib inhibits TNF-α production in human myeloid cells. Human <t>PBMCs,</t> monocytes, or macrophages were exposed to either saline or imatinib (1 h, 1 μM) followed by 3-h stimulation with 100 ng/ml <t>LPS.</t> ( A ) LPS-induced TNF-α levels in supernatants of all three cell types as determined by ELISA ( n = 5; * , P ≤ 0.01). ( B and C ) Imatinib dose-dependently inhibits LPS-induced TNF-α protein ( B ) ( n = 5; * , P ≤ 0.01) and TNF-α mRNA expression ( C ) ( n = 3; * , P ≤ 0.01) in human PBMCs.
    Human Pbmcs, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Features of ssRNA40-induced LILRA3 expression. a Kinetics of LILRA3 expression was compared to other ssRNA40 inducible cytokines. PBMCs from two donors were stimulated with ssRNA40 or ssRNA41 and the RNA was harvested at various time points. qPCR for LILRA3 , TNF , IFNb , IFNg , and IL1b expression, normalized to RPLP0 and relatively quantified to calibrator cDNA (pooled cDNA from 10. b Effect of cytokine inhibition on LILRA3 expression. PBMCs from two donors were stimulated overnight with ssRNA40 together with neutralizing agents anakinra, certolizumab, tocilizumab and IFN-γ neutralizing antibodies and analysed for LILRA3 and IL6 expression by qPCR. Repeated measures ANOVA was used to calculate difference of ssRNA40+ neutralization to ssRNA40 alone and results displayed as mean ± SD

    Journal: Retrovirology

    Article Title: TLR8 regulation of LILRA3 in monocytes is abrogated in human immunodeficiency virus infection and correlates to CD4 counts and virus loads

    doi: 10.1186/s12977-016-0248-y

    Figure Lengend Snippet: Features of ssRNA40-induced LILRA3 expression. a Kinetics of LILRA3 expression was compared to other ssRNA40 inducible cytokines. PBMCs from two donors were stimulated with ssRNA40 or ssRNA41 and the RNA was harvested at various time points. qPCR for LILRA3 , TNF , IFNb , IFNg , and IL1b expression, normalized to RPLP0 and relatively quantified to calibrator cDNA (pooled cDNA from 10. b Effect of cytokine inhibition on LILRA3 expression. PBMCs from two donors were stimulated overnight with ssRNA40 together with neutralizing agents anakinra, certolizumab, tocilizumab and IFN-γ neutralizing antibodies and analysed for LILRA3 and IL6 expression by qPCR. Repeated measures ANOVA was used to calculate difference of ssRNA40+ neutralization to ssRNA40 alone and results displayed as mean ± SD

    Article Snippet: PBMC and monocyte isolation and culture PBMCs were isolated using density gradient centrifugation by layering blood onto Biocoll (Millipore) and centrifugation at 1200×g for 20 min without brake.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Inhibition, Neutralization

    LILRA3 induction by TLRs. a Induction of LILRA3 and IL6 expression by a panel of TLR agonists. LILRA3 and IL6 expression was measured, using qPCR, as fold change to the unstimulated control from PBMCs stimulated for 24 h with Pam3CSK4 (P3C), heat killed Listeria monocytogenes (HKLM), polyinosinic-polycytidylic acid (Poly I:C) in high molecular weight (HMW) and low molecular weight (LMW) forms, lipopolysaccharide (LPS), flagellin, synthetic diacylated lipoprotein FSL-1, Imiquimod (R837), ssRNA40/LyoVec and CpG oligonucleotide ODN 2006. b Induction of LILRA3 by ssRNA40 on the transcriptional level. LILRA3 expression was measured as fold change to the unstimulated control from PBMCs stimulated for 24 h with uridine-rich HIV-derived ssRNA40, using ssRNA41 as control (n = 8). Wilcoxon matched-pairs signed rank test was used to compare the median between ssRNA41 and ssRNA40 stimulated LILRA3 expression. Scatter dot plot overlayed on bar graph displayed as median with interquartile range. c Induction of LILRA3 in PBMCS by ssRNA40 on the protein level. 30 × 10 6 /mL PBMCs from LILRA3 + / + and LILRA3 − / − donors were stimulated with 5 μg/mL ssRNA40. 40 µL from each supernatant was loaded and Ponceau S staining was used as loading control. The membrane was blotted for LILRA3 and IL6, using recombinant LILRA3 as protein control. d Expression of ssRNA40-induced LILRA3 by monocytes. PBMCs stimulated overnight with ssRNA40 and LPS were purified for CD14 + monocytes using magnetic-activated cell sorting. The resulting CD14 + positive and depleted fractions were analysed via qPCR for LILRA3 expression, normalized to RPLP0 and relatively quantified to calibrator cDNA (*p

    Journal: Retrovirology

    Article Title: TLR8 regulation of LILRA3 in monocytes is abrogated in human immunodeficiency virus infection and correlates to CD4 counts and virus loads

    doi: 10.1186/s12977-016-0248-y

    Figure Lengend Snippet: LILRA3 induction by TLRs. a Induction of LILRA3 and IL6 expression by a panel of TLR agonists. LILRA3 and IL6 expression was measured, using qPCR, as fold change to the unstimulated control from PBMCs stimulated for 24 h with Pam3CSK4 (P3C), heat killed Listeria monocytogenes (HKLM), polyinosinic-polycytidylic acid (Poly I:C) in high molecular weight (HMW) and low molecular weight (LMW) forms, lipopolysaccharide (LPS), flagellin, synthetic diacylated lipoprotein FSL-1, Imiquimod (R837), ssRNA40/LyoVec and CpG oligonucleotide ODN 2006. b Induction of LILRA3 by ssRNA40 on the transcriptional level. LILRA3 expression was measured as fold change to the unstimulated control from PBMCs stimulated for 24 h with uridine-rich HIV-derived ssRNA40, using ssRNA41 as control (n = 8). Wilcoxon matched-pairs signed rank test was used to compare the median between ssRNA41 and ssRNA40 stimulated LILRA3 expression. Scatter dot plot overlayed on bar graph displayed as median with interquartile range. c Induction of LILRA3 in PBMCS by ssRNA40 on the protein level. 30 × 10 6 /mL PBMCs from LILRA3 + / + and LILRA3 − / − donors were stimulated with 5 μg/mL ssRNA40. 40 µL from each supernatant was loaded and Ponceau S staining was used as loading control. The membrane was blotted for LILRA3 and IL6, using recombinant LILRA3 as protein control. d Expression of ssRNA40-induced LILRA3 by monocytes. PBMCs stimulated overnight with ssRNA40 and LPS were purified for CD14 + monocytes using magnetic-activated cell sorting. The resulting CD14 + positive and depleted fractions were analysed via qPCR for LILRA3 expression, normalized to RPLP0 and relatively quantified to calibrator cDNA (*p

    Article Snippet: PBMC and monocyte isolation and culture PBMCs were isolated using density gradient centrifugation by layering blood onto Biocoll (Millipore) and centrifugation at 1200×g for 20 min without brake.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Molecular Weight, Derivative Assay, Staining, Recombinant, Purification, FACS

    LILRA3 stimulation of cytokines and antigen presenting cells. a qPCR of LILRA3 induced gene expression of LILRA3 , IL - 6 , IL - 1A , IL - 1B and IL - 10 . Due to the different optimal concentrations of LILRA3 for various donors, the gene expression of all four donors were shown, with the corresponding mean ± SD. b Effect of LILRA3 on the antigen presentation mechanism of monocytes and B-cells. PBMCs were stimulated with varying concentrations of LILRA3 for 48 h and analysed by flow cytometry for expression of CD80, CD86, HLA-DR and HLA-ABC on monocytes (CD14 + CD33 + ) and B-cells (CD3 + CD19 + ). On monocytes, upregulation of HLA-ABC and CD80 was observed, whereas CD86 was downregulated. On B-cells, there was a slight but significant upregulation of HLA-DR and CD86. 1-way ANOVA with repeated measures Dunett post test was used to compare to 0 ng/mL LILRA3 control. Results expressed as mean ± SD from six donors. (*p

    Journal: Retrovirology

    Article Title: TLR8 regulation of LILRA3 in monocytes is abrogated in human immunodeficiency virus infection and correlates to CD4 counts and virus loads

    doi: 10.1186/s12977-016-0248-y

    Figure Lengend Snippet: LILRA3 stimulation of cytokines and antigen presenting cells. a qPCR of LILRA3 induced gene expression of LILRA3 , IL - 6 , IL - 1A , IL - 1B and IL - 10 . Due to the different optimal concentrations of LILRA3 for various donors, the gene expression of all four donors were shown, with the corresponding mean ± SD. b Effect of LILRA3 on the antigen presentation mechanism of monocytes and B-cells. PBMCs were stimulated with varying concentrations of LILRA3 for 48 h and analysed by flow cytometry for expression of CD80, CD86, HLA-DR and HLA-ABC on monocytes (CD14 + CD33 + ) and B-cells (CD3 + CD19 + ). On monocytes, upregulation of HLA-ABC and CD80 was observed, whereas CD86 was downregulated. On B-cells, there was a slight but significant upregulation of HLA-DR and CD86. 1-way ANOVA with repeated measures Dunett post test was used to compare to 0 ng/mL LILRA3 control. Results expressed as mean ± SD from six donors. (*p

    Article Snippet: PBMC and monocyte isolation and culture PBMCs were isolated using density gradient centrifugation by layering blood onto Biocoll (Millipore) and centrifugation at 1200×g for 20 min without brake.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Cytometry

    Validation of chemokine induction in TLR2 stimulated PBMCs. Human PBMCs were stimulated with TLR2 agonists for 16 h, and examined for CCL20 and CXCL6 expression by ELISA. PBMCs responded to both agonists that signal through TLR1/2 heterodimers (PAM 3 CSK 4 ) and TLR2/6 heterodimers (PAM 2 CSK 4 and DBS-2-217C). Means and standard deviations of triplicate samples are shown.

    Journal: Human Vaccines & Immunotherapeutics

    Article Title: Transcriptomal signatures of vaccine adjuvants and accessory immunostimulation of sentinel cells by toll-like receptor 2/6 agonists

    doi: 10.1080/21645515.2018.1480284

    Figure Lengend Snippet: Validation of chemokine induction in TLR2 stimulated PBMCs. Human PBMCs were stimulated with TLR2 agonists for 16 h, and examined for CCL20 and CXCL6 expression by ELISA. PBMCs responded to both agonists that signal through TLR1/2 heterodimers (PAM 3 CSK 4 ) and TLR2/6 heterodimers (PAM 2 CSK 4 and DBS-2-217C). Means and standard deviations of triplicate samples are shown.

    Article Snippet: Multiplexed cytokine analysis in PBMCs, SkMC, HMEC-1 and HFFs Cytokine and chemokine responses of PBMCs, SkMCs, HMEC-1, and HFFs were measured using methods previously reported by us , , with the following Milliplex kits: HCYTMAG-60K-PX41, HCYPMAG-63K, and HCYP2MAG-62K (EMD Millipore, Billerica MA).

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay

    Chemotaxis of PBMCs toward TLR2/6- stimulated human foreskin fibroblasts. HFFs stimulated with TLR2/6, but not TLR1/2 agonists elicited functional chemotactic responses from lymphocytic populations in human PBMCs. Resting, adherent fibroblasts were stimulated with graded concentrations of agonists for 24 h in a chemotaxis plate. Chemotaxis of freshly isolated PBMCs was quantified by flow cytometry using lineage-specific antibodies. Means and SD on triplicate samples are shown.

    Journal: Human Vaccines & Immunotherapeutics

    Article Title: Transcriptomal signatures of vaccine adjuvants and accessory immunostimulation of sentinel cells by toll-like receptor 2/6 agonists

    doi: 10.1080/21645515.2018.1480284

    Figure Lengend Snippet: Chemotaxis of PBMCs toward TLR2/6- stimulated human foreskin fibroblasts. HFFs stimulated with TLR2/6, but not TLR1/2 agonists elicited functional chemotactic responses from lymphocytic populations in human PBMCs. Resting, adherent fibroblasts were stimulated with graded concentrations of agonists for 24 h in a chemotaxis plate. Chemotaxis of freshly isolated PBMCs was quantified by flow cytometry using lineage-specific antibodies. Means and SD on triplicate samples are shown.

    Article Snippet: Multiplexed cytokine analysis in PBMCs, SkMC, HMEC-1 and HFFs Cytokine and chemokine responses of PBMCs, SkMCs, HMEC-1, and HFFs were measured using methods previously reported by us , , with the following Milliplex kits: HCYTMAG-60K-PX41, HCYPMAG-63K, and HCYP2MAG-62K (EMD Millipore, Billerica MA).

    Techniques: Chemotaxis Assay, Functional Assay, Isolation, Flow Cytometry, Cytometry

    Fecal s-IgA antibody titers to SD1-LPS in group1 (cross symbol), group 2 (open square symbol), and group 3 (closed round symbol) monkeys, expressed as MN with MN ± SE, after challenge on day 00 and re-challenge on day 31 with SD11617 strain.

    Journal: Apmis

    Article Title: Evaluation of an intragastric challenge model for Shigella dysenteriae 1 in rhesus monkeys (Macaca mulatta) for the pre-clinical assessment of Shigella vaccine formulations

    doi: 10.1111/apm.12168

    Figure Lengend Snippet: Fecal s-IgA antibody titers to SD1-LPS in group1 (cross symbol), group 2 (open square symbol), and group 3 (closed round symbol) monkeys, expressed as MN with MN ± SE, after challenge on day 00 and re-challenge on day 31 with SD11617 strain.

    Article Snippet: Antibody-secreting cell assay Enzyme-linked immunospot (ELISPOT) assay was performed to enumerate IgA, IgG, and IgM ASCs on fresh PBMC specimens against SD1 LPS and SD1 Invaplex antigens using Multiscreen Immobilon-P filtration, 96-well plates, EMD Millipore Corporation, Billerica, MA, USA .

    Techniques:

    IgA, IgG, and IgM antibody titers against SD1-LPS in group 1 monkeys (cross symbol), group 2 (open square symbol), and group 3 (closed round symbol), expressed as geometric mean titer (GMT) with GMT±SE after challenge on day 00 and re-challenge on day 31 with SD11617 strain.

    Journal: Apmis

    Article Title: Evaluation of an intragastric challenge model for Shigella dysenteriae 1 in rhesus monkeys (Macaca mulatta) for the pre-clinical assessment of Shigella vaccine formulations

    doi: 10.1111/apm.12168

    Figure Lengend Snippet: IgA, IgG, and IgM antibody titers against SD1-LPS in group 1 monkeys (cross symbol), group 2 (open square symbol), and group 3 (closed round symbol), expressed as geometric mean titer (GMT) with GMT±SE after challenge on day 00 and re-challenge on day 31 with SD11617 strain.

    Article Snippet: Antibody-secreting cell assay Enzyme-linked immunospot (ELISPOT) assay was performed to enumerate IgA, IgG, and IgM ASCs on fresh PBMC specimens against SD1 LPS and SD1 Invaplex antigens using Multiscreen Immobilon-P filtration, 96-well plates, EMD Millipore Corporation, Billerica, MA, USA .

    Techniques:

    Imatinib inhibits TNF-α production in human myeloid cells. Human PBMCs, monocytes, or macrophages were exposed to either saline or imatinib (1 h, 1 μM) followed by 3-h stimulation with 100 ng/ml LPS. ( A ) LPS-induced TNF-α levels in supernatants of all three cell types as determined by ELISA ( n = 5; * , P ≤ 0.01). ( B and C ) Imatinib dose-dependently inhibits LPS-induced TNF-α protein ( B ) ( n = 5; * , P ≤ 0.01) and TNF-α mRNA expression ( C ) ( n = 3; * , P ≤ 0.01) in human PBMCs.

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

    Article Title: The kinase inhibitor imatinib mesylate inhibits TNF-? production in vitro and prevents TNF-dependent acute hepatic inflammation

    doi: 10.1073/pnas.0501758102

    Figure Lengend Snippet: Imatinib inhibits TNF-α production in human myeloid cells. Human PBMCs, monocytes, or macrophages were exposed to either saline or imatinib (1 h, 1 μM) followed by 3-h stimulation with 100 ng/ml LPS. ( A ) LPS-induced TNF-α levels in supernatants of all three cell types as determined by ELISA ( n = 5; * , P ≤ 0.01). ( B and C ) Imatinib dose-dependently inhibits LPS-induced TNF-α protein ( B ) ( n = 5; * , P ≤ 0.01) and TNF-α mRNA expression ( C ) ( n = 3; * , P ≤ 0.01) in human PBMCs.

    Article Snippet: Human PBMCs or CD14-selected monocytes were stimulated with LPS at a concentration of 100 ng/ml for 12 h in the presence of either solvent or the indicated kinase inhibitor ( , SB203580, PD98059, and JNK-Inhibitor II, all used in a final concentration of 35 μM and purchased from Calbiochem).

    Techniques: Enzyme-linked Immunosorbent Assay, Expressing