pha  (Thermo Fisher)


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    Structured Review

    Thermo Fisher pha
    WJ-MSC inhibit CD3 + CD4 + and CD3 + CD8 + T cell proliferation. WJ-MSC were seeded, after 24 hours. <t>PBMC</t> was added and stimulated with <t>PHA</t> for 3 days in the presence of WJ-MSC; the ratio used was 1 : 10 (WJ-MSC : PBMC). The WJ-MSC passages used in experiments were either P5 or P6. T cells were collected, stained with anti-CD3, anti-CD4, and anti-CD8 antibodies, and proliferation was measured by flow cytometry using the Click It Kit. (a) % proliferation of CD3 + CD4 + Click It + T cell and (b) % proliferation of CD3 + CD8 + Click It + T cell. The percentage of inhibition (Inhib) was calculated using the percentage of proliferation of PBMC + PHA + WJ-MSC compared to the control PBMC + PHA. Experiments were performed in triplicate. Results are represented by mean ± SD. Statistically significant differences are shown as ∗∗∗ p
    Pha, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Intrinsic Variability Present in Wharton's Jelly Mesenchymal Stem Cells and T Cell Responses May Impact Cell Therapy"

    Article Title: Intrinsic Variability Present in Wharton's Jelly Mesenchymal Stem Cells and T Cell Responses May Impact Cell Therapy

    Journal: Stem Cells International

    doi: 10.1155/2017/8492797

    WJ-MSC inhibit CD3 + CD4 + and CD3 + CD8 + T cell proliferation. WJ-MSC were seeded, after 24 hours. PBMC was added and stimulated with PHA for 3 days in the presence of WJ-MSC; the ratio used was 1 : 10 (WJ-MSC : PBMC). The WJ-MSC passages used in experiments were either P5 or P6. T cells were collected, stained with anti-CD3, anti-CD4, and anti-CD8 antibodies, and proliferation was measured by flow cytometry using the Click It Kit. (a) % proliferation of CD3 + CD4 + Click It + T cell and (b) % proliferation of CD3 + CD8 + Click It + T cell. The percentage of inhibition (Inhib) was calculated using the percentage of proliferation of PBMC + PHA + WJ-MSC compared to the control PBMC + PHA. Experiments were performed in triplicate. Results are represented by mean ± SD. Statistically significant differences are shown as ∗∗∗ p
    Figure Legend Snippet: WJ-MSC inhibit CD3 + CD4 + and CD3 + CD8 + T cell proliferation. WJ-MSC were seeded, after 24 hours. PBMC was added and stimulated with PHA for 3 days in the presence of WJ-MSC; the ratio used was 1 : 10 (WJ-MSC : PBMC). The WJ-MSC passages used in experiments were either P5 or P6. T cells were collected, stained with anti-CD3, anti-CD4, and anti-CD8 antibodies, and proliferation was measured by flow cytometry using the Click It Kit. (a) % proliferation of CD3 + CD4 + Click It + T cell and (b) % proliferation of CD3 + CD8 + Click It + T cell. The percentage of inhibition (Inhib) was calculated using the percentage of proliferation of PBMC + PHA + WJ-MSC compared to the control PBMC + PHA. Experiments were performed in triplicate. Results are represented by mean ± SD. Statistically significant differences are shown as ∗∗∗ p

    Techniques Used: Staining, Flow Cytometry, Cytometry, Inhibition

    WJ-MSC licensed with IFN- γ inhibit CD3 + T cell proliferation. WJ-MSC were seeded, and IFN- γ was added for 24 hours. PBMC were stimulated with PHA for 3 days in the presence of WJ-MSC; the ratio used was 1 : 10 (WJ-MSC : PBMC). The WJ-MSC passages used in the experiments were P5 and P10. T cells were collected, stained with anti-CD3, anti-CD4, and anti-CD8 antibodies, and proliferation was measured by flow cytometry using the Click It Kit. The percentage of inhibition (Inhib) was calculated using the percentage of proliferation of PBMC + PHA + WJ-MSC compared to the control PBMC + PHA. Experiments were performed in triplicate. Results are represented by mean ± SD. Statistically significant differences are shown as ∗ p
    Figure Legend Snippet: WJ-MSC licensed with IFN- γ inhibit CD3 + T cell proliferation. WJ-MSC were seeded, and IFN- γ was added for 24 hours. PBMC were stimulated with PHA for 3 days in the presence of WJ-MSC; the ratio used was 1 : 10 (WJ-MSC : PBMC). The WJ-MSC passages used in the experiments were P5 and P10. T cells were collected, stained with anti-CD3, anti-CD4, and anti-CD8 antibodies, and proliferation was measured by flow cytometry using the Click It Kit. The percentage of inhibition (Inhib) was calculated using the percentage of proliferation of PBMC + PHA + WJ-MSC compared to the control PBMC + PHA. Experiments were performed in triplicate. Results are represented by mean ± SD. Statistically significant differences are shown as ∗ p

    Techniques Used: Staining, Flow Cytometry, Cytometry, Inhibition

    Gene expression of IL-10 and IDO is increased in WJ-MSC after coculture with PBMC. WJ-MSC were seeded, after 24 hours. PBMC was added and stimulated with PHA for 3 days in the presence of MSC; the ratio used was 1 : 10 (MSC : PBMC). The WJ-MSC passages used in the experiments were P5 and P10. After 3 days, WJ-MSC were lysed, total RNA extracted, and real-time PCR performed. (a) IDO and (b) IL-10 gene expression of WJ-MSC1, WJ-MSC2, and WJ-MSC3. Gene expression was normalized by GAPDH and expressed as fold change compared to the control WJ-MSC. Experiments were performed in triplicate. Results are represented as mean ± SD. Statistically significant differences are shown as ∗ p
    Figure Legend Snippet: Gene expression of IL-10 and IDO is increased in WJ-MSC after coculture with PBMC. WJ-MSC were seeded, after 24 hours. PBMC was added and stimulated with PHA for 3 days in the presence of MSC; the ratio used was 1 : 10 (MSC : PBMC). The WJ-MSC passages used in the experiments were P5 and P10. After 3 days, WJ-MSC were lysed, total RNA extracted, and real-time PCR performed. (a) IDO and (b) IL-10 gene expression of WJ-MSC1, WJ-MSC2, and WJ-MSC3. Gene expression was normalized by GAPDH and expressed as fold change compared to the control WJ-MSC. Experiments were performed in triplicate. Results are represented as mean ± SD. Statistically significant differences are shown as ∗ p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    WJ-MSC inhibit CD3 + T cell proliferation. WJ-MSC were seeded, after 24 hours. PBMC were added and stimulated with PHA for 3 days in the presence of MSC; the ratio used was 1 : 10 (WJ-MSC : PBMC). The WJ-MSC passages used in experiments were either P5 or P6. T cells were collected and stained with anti-CD3 antibody and proliferation measured by flow cytometry using the Click It Kit. The percentage of inhibition (Inhib) was calculated using the percentage of proliferation of PBMC + PHA + WJ-MSC compared to the control PBMC + PHA. Experiments were performed in triplicate. Results are represented by mean ± SD. Statistically significant differences are shown as ∗∗∗ p
    Figure Legend Snippet: WJ-MSC inhibit CD3 + T cell proliferation. WJ-MSC were seeded, after 24 hours. PBMC were added and stimulated with PHA for 3 days in the presence of MSC; the ratio used was 1 : 10 (WJ-MSC : PBMC). The WJ-MSC passages used in experiments were either P5 or P6. T cells were collected and stained with anti-CD3 antibody and proliferation measured by flow cytometry using the Click It Kit. The percentage of inhibition (Inhib) was calculated using the percentage of proliferation of PBMC + PHA + WJ-MSC compared to the control PBMC + PHA. Experiments were performed in triplicate. Results are represented by mean ± SD. Statistically significant differences are shown as ∗∗∗ p

    Techniques Used: Staining, Flow Cytometry, Cytometry, Inhibition

    2) Product Images from "Functional and molecular aspects of transient T cell unresponsiveness: role of selective interleukin-2 deficiency"

    Article Title: Functional and molecular aspects of transient T cell unresponsiveness: role of selective interleukin-2 deficiency

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.2003.02150.x

    T cell proliferation of US and anti-CD3 PA PBMC in response to anti-CD3, IL-2 and other mitogens. Proliferative response of US and PA PBMC to anti-CD3, IL-2 + anti-CD3, PHA or Con A. [ 3 H]-thymidine uptake is given in cpm (* P
    Figure Legend Snippet: T cell proliferation of US and anti-CD3 PA PBMC in response to anti-CD3, IL-2 and other mitogens. Proliferative response of US and PA PBMC to anti-CD3, IL-2 + anti-CD3, PHA or Con A. [ 3 H]-thymidine uptake is given in cpm (* P

    Techniques Used:

    3) Product Images from "Spontaneous Immunity Against the Receptor Tyrosine Kinase ROR1 in Patients with Chronic Lymphocytic Leukemia"

    Article Title: Spontaneous Immunity Against the Receptor Tyrosine Kinase ROR1 in Patients with Chronic Lymphocytic Leukemia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0142310

    Frequency of T cells (spots/10 6 T cells) (mean + SEM) secreting (A) IFN-γ and (B) IL-17A (ELISPOT) and (C) stimulation index (SI) (proliferation) (mean + SEM) in response to PHA, PPD and autologous DC loaded with ROR1 derived peptides (p1, p2, p8, p10, p16) or an influenza peptide. (■) CLL patients (n = 9). (□) control donors (n = 6). (D) Frequency of IFN-γ T cells (spots/10 6 ■ ) patients. Background values i.e. number of spots as well as SI after stimulation with an HIV (9 aa) and a Ras (16 aa) peptide were deducted in each experiment. *p
    Figure Legend Snippet: Frequency of T cells (spots/10 6 T cells) (mean + SEM) secreting (A) IFN-γ and (B) IL-17A (ELISPOT) and (C) stimulation index (SI) (proliferation) (mean + SEM) in response to PHA, PPD and autologous DC loaded with ROR1 derived peptides (p1, p2, p8, p10, p16) or an influenza peptide. (■) CLL patients (n = 9). (□) control donors (n = 6). (D) Frequency of IFN-γ T cells (spots/10 6 ■ ) patients. Background values i.e. number of spots as well as SI after stimulation with an HIV (9 aa) and a Ras (16 aa) peptide were deducted in each experiment. *p

    Techniques Used: Enzyme-linked Immunospot, Derivative Assay

    4) Product Images from "Different Expressions of Specific Transcription Factors of Th1 (T-bet) and Th2 cells (GATA-3) by Peripheral Blood Mononuclear Cells From Patients With Multiple Sclerosis"

    Article Title: Different Expressions of Specific Transcription Factors of Th1 (T-bet) and Th2 cells (GATA-3) by Peripheral Blood Mononuclear Cells From Patients With Multiple Sclerosis

    Journal: Basic and Clinical Neuroscience

    doi: 10.32598/bcn.9.6.458

    The comparison of T-bet expression by PBMCs in patients with MS, based on gender In non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from patients with MS, no differences were observed between males and females in terms of T-bet expression.
    Figure Legend Snippet: The comparison of T-bet expression by PBMCs in patients with MS, based on gender In non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from patients with MS, no differences were observed between males and females in terms of T-bet expression.

    Techniques Used: Expressing, Mass Spectrometry

    The comparison of T-bet / GATA-3 mRNA Ratio by PBMCs between healthy females and female patients with MS The T-bet / GATA-3 expression ratio in non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from female patients with MS was significantly higher than that of the same cell cultures from the healthy females.
    Figure Legend Snippet: The comparison of T-bet / GATA-3 mRNA Ratio by PBMCs between healthy females and female patients with MS The T-bet / GATA-3 expression ratio in non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from female patients with MS was significantly higher than that of the same cell cultures from the healthy females.

    Techniques Used: Mass Spectrometry, Expressing

    The comparison of GATA-3 expression by PBMC in patients with MS, based on gender. The of GATA-3 expression in non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from male patients with MS were significantly higher than that of the same cell cultures from female patients with MS.
    Figure Legend Snippet: The comparison of GATA-3 expression by PBMC in patients with MS, based on gender. The of GATA-3 expression in non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from male patients with MS were significantly higher than that of the same cell cultures from female patients with MS.

    Techniques Used: Expressing, Mass Spectrometry

    The comparison of T-bet / GATA-3 mRNA ratio by PBMCs in healthy subjects, based on gender In non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from healthy controls, no significant differences were observed between males and females in terms of T-bet / GATA-3 expression ratio.
    Figure Legend Snippet: The comparison of T-bet / GATA-3 mRNA ratio by PBMCs in healthy subjects, based on gender In non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from healthy controls, no significant differences were observed between males and females in terms of T-bet / GATA-3 expression ratio.

    Techniques Used: Expressing

    The comparison of T-bet expression by PBMCs between healthy females and female patients with MS The expression of T-bet in non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from female patients with MS was significantly higher than that of the same cell cultures from the healthy females.
    Figure Legend Snippet: The comparison of T-bet expression by PBMCs between healthy females and female patients with MS The expression of T-bet in non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from female patients with MS was significantly higher than that of the same cell cultures from the healthy females.

    Techniques Used: Expressing, Mass Spectrometry

    The comparison of GATA-3 expression by PBMCs between healthy females and female patients with MS The GATA-3 expression in non-stimulated, MOG-stimulated and PHA-stimulated PBMCs from MS female patients was significantly lower than that of the same cell cultures from the healthy females.
    Figure Legend Snippet: The comparison of GATA-3 expression by PBMCs between healthy females and female patients with MS The GATA-3 expression in non-stimulated, MOG-stimulated and PHA-stimulated PBMCs from MS female patients was significantly lower than that of the same cell cultures from the healthy females.

    Techniques Used: Expressing, Mass Spectrometry

    The comparison of GATA-3 expression by PBMCs in healthy individuals, based on gender In non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from healthy controls, no significant differences were observed between males and females in terms of GATA-3 expression.
    Figure Legend Snippet: The comparison of GATA-3 expression by PBMCs in healthy individuals, based on gender In non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from healthy controls, no significant differences were observed between males and females in terms of GATA-3 expression.

    Techniques Used: Expressing

    The comparison of T-bet / GATA-3 expression ratio by PBMC between the MS and control groups The non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from MS patients expressed higher amounts of T-bet / GATA-3 mRNA ratio in comparison with same cell cultures from healthy individuals.
    Figure Legend Snippet: The comparison of T-bet / GATA-3 expression ratio by PBMC between the MS and control groups The non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from MS patients expressed higher amounts of T-bet / GATA-3 mRNA ratio in comparison with same cell cultures from healthy individuals.

    Techniques Used: Expressing, Mass Spectrometry

    The comparison of GATA-3 expression by PBMC between the MS and control groups The non-stimulated, MOG-simulated, and PHA-stimulated PBMCs from MS patients expressed lower amounts of GATA-3 in comparison with same cell cultures from healthy individuals.
    Figure Legend Snippet: The comparison of GATA-3 expression by PBMC between the MS and control groups The non-stimulated, MOG-simulated, and PHA-stimulated PBMCs from MS patients expressed lower amounts of GATA-3 in comparison with same cell cultures from healthy individuals.

    Techniques Used: Expressing, Mass Spectrometry

    The comparison of GATA-3 expression by PBMCs between healthy males and male patients with MS The GATA-3 expression in MOG-stimulated PBMCs from males with MS was significantly lower than that of the same culture from healthy males. The GATA-3 expression in non-stimulated and PHA-stimulated PBMCs from male patients with MS was lower than that of the same cultures from the healthy males, but the differences were insignificant.
    Figure Legend Snippet: The comparison of GATA-3 expression by PBMCs between healthy males and male patients with MS The GATA-3 expression in MOG-stimulated PBMCs from males with MS was significantly lower than that of the same culture from healthy males. The GATA-3 expression in non-stimulated and PHA-stimulated PBMCs from male patients with MS was lower than that of the same cultures from the healthy males, but the differences were insignificant.

    Techniques Used: Expressing, Mass Spectrometry

    The comparison of T-bet expression by PBMC between the MS and control groups The non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from MS patients expressed higher amounts of T-bet in comparison with the same cell cultures from healthy individuals.
    Figure Legend Snippet: The comparison of T-bet expression by PBMC between the MS and control groups The non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from MS patients expressed higher amounts of T-bet in comparison with the same cell cultures from healthy individuals.

    Techniques Used: Expressing, Mass Spectrometry

    The comparison of T-bet / GATA-3 mRNA ratio by PBMCs in patients with MS, based on gender The of T-bet / GATA-3 expression ratio in MOG-stimulated and PHA-stimulated PBMCs from females with MS were significantly higher than that of the same cultures from male patients with MS.
    Figure Legend Snippet: The comparison of T-bet / GATA-3 mRNA ratio by PBMCs in patients with MS, based on gender The of T-bet / GATA-3 expression ratio in MOG-stimulated and PHA-stimulated PBMCs from females with MS were significantly higher than that of the same cultures from male patients with MS.

    Techniques Used: Mass Spectrometry, Expressing

    The Comparison of T-bet expression by PBMCs in healthy individuals, based on gender In non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs, no significant differences were observed between healthy males and females in terms of T-bet expression.
    Figure Legend Snippet: The Comparison of T-bet expression by PBMCs in healthy individuals, based on gender In non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs, no significant differences were observed between healthy males and females in terms of T-bet expression.

    Techniques Used: Expressing

    5) Product Images from "Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy"

    Article Title: Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy

    Journal: Nature

    doi: 10.1038/nature11286

    The relative HIV-1 RNA copy number in resting CD4+ T cells of 13 ART-treated HIV+ patients with plasma HIV RNA BDL Values are calculated by cycle number, and limit of quantitation of cell-associated RNA is 10 copies. Cells were cultured alone (untreated), with VOR 335 nM (VOR), or activated with 3 µg/ml PHA and 60 U/ml IL2 for 6 hours. Data from 16 patients ( a ), and those who later received in vivo dosing [1 (Δ), 2 (□), 3 (◊), 4 (X), 5 (▽), 6 (❍), 7 (+), and 8 (⨀)] are shown in detail ( b, mean and s.d.).
    Figure Legend Snippet: The relative HIV-1 RNA copy number in resting CD4+ T cells of 13 ART-treated HIV+ patients with plasma HIV RNA BDL Values are calculated by cycle number, and limit of quantitation of cell-associated RNA is 10 copies. Cells were cultured alone (untreated), with VOR 335 nM (VOR), or activated with 3 µg/ml PHA and 60 U/ml IL2 for 6 hours. Data from 16 patients ( a ), and those who later received in vivo dosing [1 (Δ), 2 (□), 3 (◊), 4 (X), 5 (▽), 6 (❍), 7 (+), and 8 (⨀)] are shown in detail ( b, mean and s.d.).

    Techniques Used: Quantitation Assay, Cell Culture, In Vivo

    6) Product Images from "The First Dose of Fingolimod Affects Circulating Extracellular Vesicles in Multiple Sclerosis Patients"

    Article Title: The First Dose of Fingolimod Affects Circulating Extracellular Vesicles in Multiple Sclerosis Patients

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19082448

    Circulating EVs inhibit lymphocyte activation. Cultured peripheral blood mononuclear cells (PBMCs) were treated with EVs isolated from one donor for each condition (HC, UNT, FGM 0 h, and FGM 5 h) and then activated with PHA addition. In all cases, EVs inhibit lymphocyte activation as shown by flow cytometry analysis of CD25+. Besides, FGM 5 h have impaired inhibition of lymphocyte activation. The fold change (FC) was calculated in respect to the PBS treated sample, and is shown under each bar.
    Figure Legend Snippet: Circulating EVs inhibit lymphocyte activation. Cultured peripheral blood mononuclear cells (PBMCs) were treated with EVs isolated from one donor for each condition (HC, UNT, FGM 0 h, and FGM 5 h) and then activated with PHA addition. In all cases, EVs inhibit lymphocyte activation as shown by flow cytometry analysis of CD25+. Besides, FGM 5 h have impaired inhibition of lymphocyte activation. The fold change (FC) was calculated in respect to the PBS treated sample, and is shown under each bar.

    Techniques Used: Activation Assay, Cell Culture, Isolation, Flow Cytometry, Cytometry, Inhibition

    7) Product Images from "Loss of Multi-Epitope Specificity in Memory CD4+ T Cell Responses to B. Pertussis with Age"

    Article Title: Loss of Multi-Epitope Specificity in Memory CD4+ T Cell Responses to B. Pertussis with Age

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0083583

    Immunogenicity of the Prn- and Ptx-peptide panel. Fresh PBMC of (ex-)pertussis patients and household contacts (n = 91) were stimulated with 1 µM peptide or 1 µg/ml protein for 7 days and [ 3 H]thymidine incorporation was assed in the last 18 hours. The responsiveness of participants to PHA was 100% (Data not shown). (A) Epitope specific responsiveness shown as Stimulation Index (S.I. = geomean CPM peptide/geomean CPM medium) of (ex-)pertussis patients and their household contacts. Epitope specific responses with a S.I.≥2 were regarded as positive. Lines indicate geometric means. (B) Responsiveness to the tested Prn- or Ptx- peptides (S.I.≥2) in participants with proliferative responses to Prn (n = 69) or Ptx (n = 78) protein.
    Figure Legend Snippet: Immunogenicity of the Prn- and Ptx-peptide panel. Fresh PBMC of (ex-)pertussis patients and household contacts (n = 91) were stimulated with 1 µM peptide or 1 µg/ml protein for 7 days and [ 3 H]thymidine incorporation was assed in the last 18 hours. The responsiveness of participants to PHA was 100% (Data not shown). (A) Epitope specific responsiveness shown as Stimulation Index (S.I. = geomean CPM peptide/geomean CPM medium) of (ex-)pertussis patients and their household contacts. Epitope specific responses with a S.I.≥2 were regarded as positive. Lines indicate geometric means. (B) Responsiveness to the tested Prn- or Ptx- peptides (S.I.≥2) in participants with proliferative responses to Prn (n = 69) or Ptx (n = 78) protein.

    Techniques Used:

    8) Product Images from "2,5-Deoxyfructosazine, a D-glucosamine derivative, inhibits T-cell interleukin-2 production better than D-glucosamine"

    Article Title: 2,5-Deoxyfructosazine, a D-glucosamine derivative, inhibits T-cell interleukin-2 production better than D-glucosamine

    Journal: Carbohydrate research

    doi: 10.1016/j.carres.2007.08.025

    Fluorescence microscopy of PHA binding to the Jurkat cell surface. (A) and (C) are differential interference contrast images of cells, whereas (B) and (D) are fluorescence micrographs. Alexa-488-conjugated PHA was added to cells with buffer alone (A) and (B) or fructosazines (C) and (D). No differences in binding were found.
    Figure Legend Snippet: Fluorescence microscopy of PHA binding to the Jurkat cell surface. (A) and (C) are differential interference contrast images of cells, whereas (B) and (D) are fluorescence micrographs. Alexa-488-conjugated PHA was added to cells with buffer alone (A) and (B) or fructosazines (C) and (D). No differences in binding were found.

    Techniques Used: Fluorescence, Microscopy, Binding Assay

    Effect of fructosazines on cytokine release by Jurkat cells. Fructosazines (A) and D-GlcN (B) reduce IL-2 production by Jurkat cells in a dose-dependent fashion. Cells were activated with PHA in the presence of various doses of fructosazines or D-GlcN. Fructosazines caused a dramatic reduction in IL-2 production by cells. As these data illustrate, fructosazines are substantially more effective in blocking IL-2 release than its parent compound, D-GlcN. Cell viability was also measured in the presence of fructosazines (C) and D-GlcN (D). In all cases, there was no significant effect of these compounds or of the stimulatory PHA molecules on the viability of Jurkat cells.
    Figure Legend Snippet: Effect of fructosazines on cytokine release by Jurkat cells. Fructosazines (A) and D-GlcN (B) reduce IL-2 production by Jurkat cells in a dose-dependent fashion. Cells were activated with PHA in the presence of various doses of fructosazines or D-GlcN. Fructosazines caused a dramatic reduction in IL-2 production by cells. As these data illustrate, fructosazines are substantially more effective in blocking IL-2 release than its parent compound, D-GlcN. Cell viability was also measured in the presence of fructosazines (C) and D-GlcN (D). In all cases, there was no significant effect of these compounds or of the stimulatory PHA molecules on the viability of Jurkat cells.

    Techniques Used: Blocking Assay

    9) Product Images from "Significance of PD1 Alternative Splicing in Celiac Disease as a Novel Source for Diagnostic and Therapeutic Target"

    Article Title: Significance of PD1 Alternative Splicing in Celiac Disease as a Novel Source for Diagnostic and Therapeutic Target

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.678400

    Gel electrophoresis of PD-1 cDNA amplification products from PHA stimulated PBMCs of celiac patients as control positive of T cell stimulation.
    Figure Legend Snippet: Gel electrophoresis of PD-1 cDNA amplification products from PHA stimulated PBMCs of celiac patients as control positive of T cell stimulation.

    Techniques Used: Nucleic Acid Electrophoresis, Amplification, Cell Stimulation

    IFN - gamma release by PBMCs of CD patients stimulated with different peptides and PHA as positive control and of healthy controls stimulated and unstimulated with PHA. In axis X the patients and in axis Y the levels of IFN-gamma expressed in pg/ml.
    Figure Legend Snippet: IFN - gamma release by PBMCs of CD patients stimulated with different peptides and PHA as positive control and of healthy controls stimulated and unstimulated with PHA. In axis X the patients and in axis Y the levels of IFN-gamma expressed in pg/ml.

    Techniques Used: Positive Control

    Detection of PD1 in Human PBMCs from healthy controls by flow cytometry either unstimulated (A) and stimulated with PHA (B) .
    Figure Legend Snippet: Detection of PD1 in Human PBMCs from healthy controls by flow cytometry either unstimulated (A) and stimulated with PHA (B) .

    Techniques Used: Flow Cytometry

    10) Product Images from "NCKAP1L defects lead to a novel syndrome combining immunodeficiency, lymphoproliferation, and hyperinflammation"

    Article Title: NCKAP1L defects lead to a novel syndrome combining immunodeficiency, lymphoproliferation, and hyperinflammation

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20192275

    Immunophenotyping, T and NK cell stimulation, proliferation, degranulation, and cytotoxicity. (A) Immunophenotyping of CD8 + T cells: gating of naive (CD45RA + , CCR7 + ), CM (CD45RA − , CCR7 + ), EM (CD45RA − , CCR7 − ) and EM RA (CD45RA + , CCR7 − ) populations and expression of CD57, CD69, CD95, HLA-DR, and PD-1 in CD8 + T cells. In each dot plot, the numbers indicate the percentage of the gated populations. The experiments were repeated on three separate samples from each patient (unless otherwise specified). The shown control sample is one representative example of eight age-matched controls analyzed for each patient. (B) T cell activation markers: PBMCs were stimulated with PHA or anti-CD3/anti-CD28–coated beads for 20 h, and up-regulation of CD69 and CD25 was determined by flow cytometry. Summarized results of determinations done on cells from three different shipments of patient 1 (P1, filled, up facing triangles) or two shipments of patient 2 (P2, filled, down-facing triangles) are compared with reference values obtained from 50 in-house controls (gray area indicates 5th–95th percentile) and travel controls (C, open circles). (C) T cell proliferation. Histograms show CFSE dilution of live CD4 + T cells after 5 d of stimulation of PBMC from patient 1 (P1) or her father (C1; upper panels) or patient 2 (P2) or his father (C2; lower panels) with PHA, plate-bound anti-CD3, or plate-bound anti-CD3/anti-CD28. Gray line, medium; black line, stimulated. (D) Ex vivo T cell cytokine expression. Dot plots showing IL-17 and IFN-γ expression of patient 1 (P1) and patient 2 (P2) CD4 + CD45RO + memory T cells together with a representative age-matched control (C1 and C2, respectively; left panels). Summary graph shows the percentage of IFN-γ + memory T cells from patient 1 (filled, up-facing triangle) and patient 2 (filled down-facing triangles) compared with reference values obtained from in-house healthy controls ( n = 21, 0–2 yr old; n = 90, > 10 yr old; gray area indicates 5th–95th percentile). (E) Serum cytokine levels of IFN-γ–regulated cytokines/chemokines. Summarized results show levels of the indicated cytokine or chemokine of patient 1 (P1, filled up-facing triangles) in comparison with healthy controls (C, open circles) and patients with primary HLH (open up-facing triangles). (F) NK cell cytokine production. PBMC from patient 1 (P1), an age-matched shipping control (C1), and an internal in-house control (C2) were cultured with IL-15 and IL-18 for 24 h or in the presence of K562 for 6 h before intracellular IFN-γ and MIP-1β staining. Dot plots show cytokine expression of CD56 + CD3 − NK cells. (G) NK cytotoxicity. Lytic activity of overnight rested PBMCs on 51 Cr-labeled K562 cells. The percentage of NK cells among PBMCs as determined by flow cytometry was used to calculate the NK:target cell ratio. Representative graph of two experiments performed with two different blood shipments shows patient 1 (P1) compared with her father (C1), mother (C2), and an unrelated control (C3). (H) CTL degranulation. Histograms show CD107a expression of T cell blasts from patient 1 (P1) or her father (C1) kept for 24 h in medium without IL-2 followed by stimulation with anti-CD3/CD28 beads or the indicated concentrations of plate-bound anti-CD3. Gray line: medium, black line: stimulated. (I) CTL cytotoxicity. Lytic activity of long-term T cell blasts overnight rested from IL-2 on 51 Cr-labeled K562 cells. The percentage of CD8 + cells determined by flow cytometry was used to calculate the T cell:target ratio. Representative graph of two experiments shows patient 1 (P1) in comparison with her father (C1) and an unrelated control (C) or patient 2 (P2) in comparison with his father (C2) and an unrelated control (C).
    Figure Legend Snippet: Immunophenotyping, T and NK cell stimulation, proliferation, degranulation, and cytotoxicity. (A) Immunophenotyping of CD8 + T cells: gating of naive (CD45RA + , CCR7 + ), CM (CD45RA − , CCR7 + ), EM (CD45RA − , CCR7 − ) and EM RA (CD45RA + , CCR7 − ) populations and expression of CD57, CD69, CD95, HLA-DR, and PD-1 in CD8 + T cells. In each dot plot, the numbers indicate the percentage of the gated populations. The experiments were repeated on three separate samples from each patient (unless otherwise specified). The shown control sample is one representative example of eight age-matched controls analyzed for each patient. (B) T cell activation markers: PBMCs were stimulated with PHA or anti-CD3/anti-CD28–coated beads for 20 h, and up-regulation of CD69 and CD25 was determined by flow cytometry. Summarized results of determinations done on cells from three different shipments of patient 1 (P1, filled, up facing triangles) or two shipments of patient 2 (P2, filled, down-facing triangles) are compared with reference values obtained from 50 in-house controls (gray area indicates 5th–95th percentile) and travel controls (C, open circles). (C) T cell proliferation. Histograms show CFSE dilution of live CD4 + T cells after 5 d of stimulation of PBMC from patient 1 (P1) or her father (C1; upper panels) or patient 2 (P2) or his father (C2; lower panels) with PHA, plate-bound anti-CD3, or plate-bound anti-CD3/anti-CD28. Gray line, medium; black line, stimulated. (D) Ex vivo T cell cytokine expression. Dot plots showing IL-17 and IFN-γ expression of patient 1 (P1) and patient 2 (P2) CD4 + CD45RO + memory T cells together with a representative age-matched control (C1 and C2, respectively; left panels). Summary graph shows the percentage of IFN-γ + memory T cells from patient 1 (filled, up-facing triangle) and patient 2 (filled down-facing triangles) compared with reference values obtained from in-house healthy controls ( n = 21, 0–2 yr old; n = 90, > 10 yr old; gray area indicates 5th–95th percentile). (E) Serum cytokine levels of IFN-γ–regulated cytokines/chemokines. Summarized results show levels of the indicated cytokine or chemokine of patient 1 (P1, filled up-facing triangles) in comparison with healthy controls (C, open circles) and patients with primary HLH (open up-facing triangles). (F) NK cell cytokine production. PBMC from patient 1 (P1), an age-matched shipping control (C1), and an internal in-house control (C2) were cultured with IL-15 and IL-18 for 24 h or in the presence of K562 for 6 h before intracellular IFN-γ and MIP-1β staining. Dot plots show cytokine expression of CD56 + CD3 − NK cells. (G) NK cytotoxicity. Lytic activity of overnight rested PBMCs on 51 Cr-labeled K562 cells. The percentage of NK cells among PBMCs as determined by flow cytometry was used to calculate the NK:target cell ratio. Representative graph of two experiments performed with two different blood shipments shows patient 1 (P1) compared with her father (C1), mother (C2), and an unrelated control (C3). (H) CTL degranulation. Histograms show CD107a expression of T cell blasts from patient 1 (P1) or her father (C1) kept for 24 h in medium without IL-2 followed by stimulation with anti-CD3/CD28 beads or the indicated concentrations of plate-bound anti-CD3. Gray line: medium, black line: stimulated. (I) CTL cytotoxicity. Lytic activity of long-term T cell blasts overnight rested from IL-2 on 51 Cr-labeled K562 cells. The percentage of CD8 + cells determined by flow cytometry was used to calculate the T cell:target ratio. Representative graph of two experiments shows patient 1 (P1) in comparison with her father (C1) and an unrelated control (C) or patient 2 (P2) in comparison with his father (C2) and an unrelated control (C).

    Techniques Used: Cell Stimulation, Expressing, Activation Assay, Flow Cytometry, Ex Vivo, Cell Culture, Staining, Activity Assay, Labeling

    11) Product Images from "NCKAP1L defects lead to a novel syndrome combining immunodeficiency, lymphoproliferation, and hyperinflammation"

    Article Title: NCKAP1L defects lead to a novel syndrome combining immunodeficiency, lymphoproliferation, and hyperinflammation

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20192275

    Immunophenotyping, T and NK cell stimulation, proliferation, degranulation, and cytotoxicity. (A) Immunophenotyping of CD8 + T cells: gating of naive (CD45RA + , CCR7 + ), CM (CD45RA − , CCR7 + ), EM (CD45RA − , CCR7 − ) and EM RA (CD45RA + , CCR7 − ) populations and expression of CD57, CD69, CD95, HLA-DR, and PD-1 in CD8 + T cells. In each dot plot, the numbers indicate the percentage of the gated populations. The experiments were repeated on three separate samples from each patient (unless otherwise specified). The shown control sample is one representative example of eight age-matched controls analyzed for each patient. (B) T cell activation markers: PBMCs were stimulated with PHA or anti-CD3/anti-CD28–coated beads for 20 h, and up-regulation of CD69 and CD25 was determined by flow cytometry. Summarized results of determinations done on cells from three different shipments of patient 1 (P1, filled, up facing triangles) or two shipments of patient 2 (P2, filled, down-facing triangles) are compared with reference values obtained from 50 in-house controls (gray area indicates 5th–95th percentile) and travel controls (C, open circles). (C) T cell proliferation. Histograms show CFSE dilution of live CD4 + T cells after 5 d of stimulation of PBMC from patient 1 (P1) or her father (C1; upper panels) or patient 2 (P2) or his father (C2; lower panels) with PHA, plate-bound anti-CD3, or plate-bound anti-CD3/anti-CD28. Gray line, medium; black line, stimulated. (D) Ex vivo T cell cytokine expression. Dot plots showing IL-17 and IFN-γ expression of patient 1 (P1) and patient 2 (P2) CD4 + CD45RO + memory T cells together with a representative age-matched control (C1 and C2, respectively; left panels). Summary graph shows the percentage of IFN-γ + memory T cells from patient 1 (filled, up-facing triangle) and patient 2 (filled down-facing triangles) compared with reference values obtained from in-house healthy controls ( n = 21, 0–2 yr old; n = 90, > 10 yr old; gray area indicates 5th–95th percentile). (E) Serum cytokine levels of IFN-γ–regulated cytokines/chemokines. Summarized results show levels of the indicated cytokine or chemokine of patient 1 (P1, filled up-facing triangles) in comparison with healthy controls (C, open circles) and patients with primary HLH (open up-facing triangles). (F) NK cell cytokine production. PBMC from patient 1 (P1), an age-matched shipping control (C1), and an internal in-house control (C2) were cultured with IL-15 and IL-18 for 24 h or in the presence of K562 for 6 h before intracellular IFN-γ and MIP-1β staining. Dot plots show cytokine expression of CD56 + CD3 − NK cells. (G) NK cytotoxicity. Lytic activity of overnight rested PBMCs on 51 Cr-labeled K562 cells. The percentage of NK cells among PBMCs as determined by flow cytometry was used to calculate the NK:target cell ratio. Representative graph of two experiments performed with two different blood shipments shows patient 1 (P1) compared with her father (C1), mother (C2), and an unrelated control (C3). (H) CTL degranulation. Histograms show CD107a expression of T cell blasts from patient 1 (P1) or her father (C1) kept for 24 h in medium without IL-2 followed by stimulation with anti-CD3/CD28 beads or the indicated concentrations of plate-bound anti-CD3. Gray line: medium, black line: stimulated. (I) CTL cytotoxicity. Lytic activity of long-term T cell blasts overnight rested from IL-2 on 51 Cr-labeled K562 cells. The percentage of CD8 + cells determined by flow cytometry was used to calculate the T cell:target ratio. Representative graph of two experiments shows patient 1 (P1) in comparison with her father (C1) and an unrelated control (C) or patient 2 (P2) in comparison with his father (C2) and an unrelated control (C).
    Figure Legend Snippet: Immunophenotyping, T and NK cell stimulation, proliferation, degranulation, and cytotoxicity. (A) Immunophenotyping of CD8 + T cells: gating of naive (CD45RA + , CCR7 + ), CM (CD45RA − , CCR7 + ), EM (CD45RA − , CCR7 − ) and EM RA (CD45RA + , CCR7 − ) populations and expression of CD57, CD69, CD95, HLA-DR, and PD-1 in CD8 + T cells. In each dot plot, the numbers indicate the percentage of the gated populations. The experiments were repeated on three separate samples from each patient (unless otherwise specified). The shown control sample is one representative example of eight age-matched controls analyzed for each patient. (B) T cell activation markers: PBMCs were stimulated with PHA or anti-CD3/anti-CD28–coated beads for 20 h, and up-regulation of CD69 and CD25 was determined by flow cytometry. Summarized results of determinations done on cells from three different shipments of patient 1 (P1, filled, up facing triangles) or two shipments of patient 2 (P2, filled, down-facing triangles) are compared with reference values obtained from 50 in-house controls (gray area indicates 5th–95th percentile) and travel controls (C, open circles). (C) T cell proliferation. Histograms show CFSE dilution of live CD4 + T cells after 5 d of stimulation of PBMC from patient 1 (P1) or her father (C1; upper panels) or patient 2 (P2) or his father (C2; lower panels) with PHA, plate-bound anti-CD3, or plate-bound anti-CD3/anti-CD28. Gray line, medium; black line, stimulated. (D) Ex vivo T cell cytokine expression. Dot plots showing IL-17 and IFN-γ expression of patient 1 (P1) and patient 2 (P2) CD4 + CD45RO + memory T cells together with a representative age-matched control (C1 and C2, respectively; left panels). Summary graph shows the percentage of IFN-γ + memory T cells from patient 1 (filled, up-facing triangle) and patient 2 (filled down-facing triangles) compared with reference values obtained from in-house healthy controls ( n = 21, 0–2 yr old; n = 90, > 10 yr old; gray area indicates 5th–95th percentile). (E) Serum cytokine levels of IFN-γ–regulated cytokines/chemokines. Summarized results show levels of the indicated cytokine or chemokine of patient 1 (P1, filled up-facing triangles) in comparison with healthy controls (C, open circles) and patients with primary HLH (open up-facing triangles). (F) NK cell cytokine production. PBMC from patient 1 (P1), an age-matched shipping control (C1), and an internal in-house control (C2) were cultured with IL-15 and IL-18 for 24 h or in the presence of K562 for 6 h before intracellular IFN-γ and MIP-1β staining. Dot plots show cytokine expression of CD56 + CD3 − NK cells. (G) NK cytotoxicity. Lytic activity of overnight rested PBMCs on 51 Cr-labeled K562 cells. The percentage of NK cells among PBMCs as determined by flow cytometry was used to calculate the NK:target cell ratio. Representative graph of two experiments performed with two different blood shipments shows patient 1 (P1) compared with her father (C1), mother (C2), and an unrelated control (C3). (H) CTL degranulation. Histograms show CD107a expression of T cell blasts from patient 1 (P1) or her father (C1) kept for 24 h in medium without IL-2 followed by stimulation with anti-CD3/CD28 beads or the indicated concentrations of plate-bound anti-CD3. Gray line: medium, black line: stimulated. (I) CTL cytotoxicity. Lytic activity of long-term T cell blasts overnight rested from IL-2 on 51 Cr-labeled K562 cells. The percentage of CD8 + cells determined by flow cytometry was used to calculate the T cell:target ratio. Representative graph of two experiments shows patient 1 (P1) in comparison with her father (C1) and an unrelated control (C) or patient 2 (P2) in comparison with his father (C2) and an unrelated control (C).

    Techniques Used: Cell Stimulation, Expressing, Activation Assay, Flow Cytometry, Ex Vivo, Cell Culture, Staining, Activity Assay, Labeling

    12) Product Images from "Germline deletion of CIN85 in humans with X chromosome–linked antibody deficiency"

    Article Title: Germline deletion of CIN85 in humans with X chromosome–linked antibody deficiency

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20170534

    Robust TCR signaling and T cell function in the absence of CIN85. (A) Ratiometric Ca 2+ flux profiles and intracellular staining of IκBα degradation (left and middle panels, respectively) of CD45RA and CD45R0 CD4 T cells (upper and lower rows, respectively) upon anti-CD3/-CD28 stimulation (tinted) or treatment with secondary antibodies alone (unfilled). For quantification, graph on the right depicts MFI ratios of IκBα expression in stimulated and unstimulated CD45RA T cells. Dotted line (ratio = 1) indicates no degradation (two independent experiments). (B) Expression of CD69, CD25, and ICOS induced by CD3 or CD3/CD28 stimulation of CD45RA or CD45R0 CD4 T cells from a healthy control or patient no. 1. Representative histograms of inducible CD69 expression are shown on the left where solid and dashed lines indicate anti-CD3 and anti-CD3/-CD28 stimulation, respectively. For quantification, MFI expression ratios of all three T activation markers on naive and memory T cell populations were plotted (middle and right panels; three experiments). (C) Intracellular expression of IL-4, IL-17 or IFN-γ in CD45R0 CD4 T cells of patient no. 1 and a control upon TCR/CD28 ligation (upper left four FACS plots) or PMA/Ionomycin treatment (upper right four FACS plots). Quantification (lower three panels) depicts the percentage of positivity for IL-4 (left), IFN-γ (middle), and IL-17 (right) in resting cells, upon PMA/Ionomycin treatment or CD3/CD28 coligation (left to right in each graph; three experiments). (D) T cell proliferation monitored by CSFE labeling of CD4 or CD8 T cells (upper and lower panels, respectively) that were obtained from patient no. 1 (red) or a healthy control person (blue) and stimulated via CD3/CD28 or treated with PHA for 5 d (one experiment).
    Figure Legend Snippet: Robust TCR signaling and T cell function in the absence of CIN85. (A) Ratiometric Ca 2+ flux profiles and intracellular staining of IκBα degradation (left and middle panels, respectively) of CD45RA and CD45R0 CD4 T cells (upper and lower rows, respectively) upon anti-CD3/-CD28 stimulation (tinted) or treatment with secondary antibodies alone (unfilled). For quantification, graph on the right depicts MFI ratios of IκBα expression in stimulated and unstimulated CD45RA T cells. Dotted line (ratio = 1) indicates no degradation (two independent experiments). (B) Expression of CD69, CD25, and ICOS induced by CD3 or CD3/CD28 stimulation of CD45RA or CD45R0 CD4 T cells from a healthy control or patient no. 1. Representative histograms of inducible CD69 expression are shown on the left where solid and dashed lines indicate anti-CD3 and anti-CD3/-CD28 stimulation, respectively. For quantification, MFI expression ratios of all three T activation markers on naive and memory T cell populations were plotted (middle and right panels; three experiments). (C) Intracellular expression of IL-4, IL-17 or IFN-γ in CD45R0 CD4 T cells of patient no. 1 and a control upon TCR/CD28 ligation (upper left four FACS plots) or PMA/Ionomycin treatment (upper right four FACS plots). Quantification (lower three panels) depicts the percentage of positivity for IL-4 (left), IFN-γ (middle), and IL-17 (right) in resting cells, upon PMA/Ionomycin treatment or CD3/CD28 coligation (left to right in each graph; three experiments). (D) T cell proliferation monitored by CSFE labeling of CD4 or CD8 T cells (upper and lower panels, respectively) that were obtained from patient no. 1 (red) or a healthy control person (blue) and stimulated via CD3/CD28 or treated with PHA for 5 d (one experiment).

    Techniques Used: Cell Function Assay, Staining, Expressing, Activation Assay, Ligation, FACS, Labeling

    13) Product Images from "Mesenchymal Stromal Cell-Derived Exosomes Affect mRNA Expression and Function of B-Lymphocytes"

    Article Title: Mesenchymal Stromal Cell-Derived Exosomes Affect mRNA Expression and Function of B-Lymphocytes

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.03053

    Exosome suppression of activated peripheral blood mononuclear cell (PBMCS) and lymphocytes and internalization assays. (A) Thymidine incorporation assays. Mesenchymal stem cell (MSC)-derived exosomes co-cultured with: (1) PHA-activated PBMCs. The results summarize 3 experiments with 6 batches of exosomes derived from 0.5 × 10 6 MSCs and 9 batches of exosomes derived from 1 × 10 6 MSCs (batches no. 1–9). Control—Activated condition counts set to represent 100%. P
    Figure Legend Snippet: Exosome suppression of activated peripheral blood mononuclear cell (PBMCS) and lymphocytes and internalization assays. (A) Thymidine incorporation assays. Mesenchymal stem cell (MSC)-derived exosomes co-cultured with: (1) PHA-activated PBMCs. The results summarize 3 experiments with 6 batches of exosomes derived from 0.5 × 10 6 MSCs and 9 batches of exosomes derived from 1 × 10 6 MSCs (batches no. 1–9). Control—Activated condition counts set to represent 100%. P

    Techniques Used: Derivative Assay, Cell Culture

    Inhibition of peripheral blood mononuclear cell (PBMC) proliferation by mesenchymal stem cells (MSCs). Carboxyfluorescein succinimidyl ester (CFSE) labeled 1 × 10 5 PBMCs were activated with 2 μg/ml PHA and incubated for 4 days with and without MSCs at a ratio of 1:20, 1:10, and 1:5. (A) A representative FACS analysis of CFSE-labeled PBMCs, activated and non-activated, with and without MSCs at different ratios. (B) A graphic presentation of the percent suppression of activated PBMCs with different MSCs at a ratio of 1:20, 1:10 ( p
    Figure Legend Snippet: Inhibition of peripheral blood mononuclear cell (PBMC) proliferation by mesenchymal stem cells (MSCs). Carboxyfluorescein succinimidyl ester (CFSE) labeled 1 × 10 5 PBMCs were activated with 2 μg/ml PHA and incubated for 4 days with and without MSCs at a ratio of 1:20, 1:10, and 1:5. (A) A representative FACS analysis of CFSE-labeled PBMCs, activated and non-activated, with and without MSCs at different ratios. (B) A graphic presentation of the percent suppression of activated PBMCs with different MSCs at a ratio of 1:20, 1:10 ( p

    Techniques Used: Inhibition, Labeling, Incubation, FACS

    14) Product Images from "Administration of fusion cytokines induces tumor regression and systemic antitumor immunity. Administration of fusion cytokines induces tumor regression and systemic antitumor immunity"

    Article Title: Administration of fusion cytokines induces tumor regression and systemic antitumor immunity. Administration of fusion cytokines induces tumor regression and systemic antitumor immunity

    Journal: MedComm

    doi: 10.1002/mco2.68

    The immune activation capability and antitumor effects of fusion cytokine scIL12IL2GMCSF. (A) Schematic representation of scIL12IL2GMCSF expression cassette and molecule conformation. (B) SDS page electrophoresis of the purified scIL12IL2GMCSF proteins. (C) Splenocytes from the C57BL/6 mice were plated onto 96‐well plates and stimulated with the indicated cytokines plus PHA for 24 h. The levels of IFNγ in the supernatants were measured by ELISA. n = 3. (D) B16F10 cells were subcutaneously inoculated into the flank of C57BL/6 mice. Either scIL12IL2GMCSF or PBS was intratumorally injected into the lesions when the tumor diameters reached 5–9 mm. Tumor growth ( n = 5) and overall survival ( n = 10) were recorded. *** p
    Figure Legend Snippet: The immune activation capability and antitumor effects of fusion cytokine scIL12IL2GMCSF. (A) Schematic representation of scIL12IL2GMCSF expression cassette and molecule conformation. (B) SDS page electrophoresis of the purified scIL12IL2GMCSF proteins. (C) Splenocytes from the C57BL/6 mice were plated onto 96‐well plates and stimulated with the indicated cytokines plus PHA for 24 h. The levels of IFNγ in the supernatants were measured by ELISA. n = 3. (D) B16F10 cells were subcutaneously inoculated into the flank of C57BL/6 mice. Either scIL12IL2GMCSF or PBS was intratumorally injected into the lesions when the tumor diameters reached 5–9 mm. Tumor growth ( n = 5) and overall survival ( n = 10) were recorded. *** p

    Techniques Used: Activation Assay, Expressing, SDS Page, Electrophoresis, Purification, Mouse Assay, Enzyme-linked Immunosorbent Assay, Injection

    The immunocytokine scIL12IL2DiaNFGMCSF stimulated immune activation in vitro and in vivo. (A) Schematic representation of scIL12IL2DiaNFGMCSF expression cassette and molecule conformation. The dashed lines indicate thrombin cleavage sites. (B) Splenocytes from the C57BL/6 mice were plated onto 96‐well plates and stimulated with scIL12IL2GMCSF or scIL12IL2DiaNFGMCSF plus PHA for 12 h. Levels of IFNγ in the supernatants were measured using ELISA. n = 3. (C) Mouse LLC tumor cells were subcutaneously inoculated into the flanks of C57BL/6 mice. When the tumor diameters reached 5–8 mm, 200 μg scIL12IL2DiaNFGMCSF was intravenously injected into mice. Tumor tissues were collected separately at day 0, 1, or 2 after injection and subjected to western blotting analysis, using anti‐IL12p40 antibody. The arrows indicate distinctive bands approximately 75kD in size. (D and E) Mouse LLC tumor cells were subcutaneously inoculated into the flanks of C57BL/6 mice. When the tumor diameters reached 5–8 mm, 200 μg scIL12IL2DiaNFGMCSF was intravenously injected into the mice. At day 0, 1, or 2 after injection, samples of serum were isolated and subjected to ELISA measurement of levels of IFNγ (D), and the splenocytes and tumor‐infiltrating lymphocytes were subjected to flow cytometry analysis (E). The gated CD45+ cells were analyzed with the groups of NK1.1, CD3 or CD11c, MHCII. * p
    Figure Legend Snippet: The immunocytokine scIL12IL2DiaNFGMCSF stimulated immune activation in vitro and in vivo. (A) Schematic representation of scIL12IL2DiaNFGMCSF expression cassette and molecule conformation. The dashed lines indicate thrombin cleavage sites. (B) Splenocytes from the C57BL/6 mice were plated onto 96‐well plates and stimulated with scIL12IL2GMCSF or scIL12IL2DiaNFGMCSF plus PHA for 12 h. Levels of IFNγ in the supernatants were measured using ELISA. n = 3. (C) Mouse LLC tumor cells were subcutaneously inoculated into the flanks of C57BL/6 mice. When the tumor diameters reached 5–8 mm, 200 μg scIL12IL2DiaNFGMCSF was intravenously injected into mice. Tumor tissues were collected separately at day 0, 1, or 2 after injection and subjected to western blotting analysis, using anti‐IL12p40 antibody. The arrows indicate distinctive bands approximately 75kD in size. (D and E) Mouse LLC tumor cells were subcutaneously inoculated into the flanks of C57BL/6 mice. When the tumor diameters reached 5–8 mm, 200 μg scIL12IL2DiaNFGMCSF was intravenously injected into the mice. At day 0, 1, or 2 after injection, samples of serum were isolated and subjected to ELISA measurement of levels of IFNγ (D), and the splenocytes and tumor‐infiltrating lymphocytes were subjected to flow cytometry analysis (E). The gated CD45+ cells were analyzed with the groups of NK1.1, CD3 or CD11c, MHCII. * p

    Techniques Used: Activation Assay, In Vitro, In Vivo, Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay, Injection, Western Blot, Isolation, Flow Cytometry

    15) Product Images from "HIV-Specific, Ex Vivo Expanded T Cell Therapy: Feasibility, Safety, and Efficacy in ART-Suppressed HIV-Infected Individuals"

    Article Title: HIV-Specific, Ex Vivo Expanded T Cell Therapy: Feasibility, Safety, and Efficacy in ART-Suppressed HIV-Infected Individuals

    Journal: Molecular Therapy

    doi: 10.1016/j.ymthe.2018.08.015

    Characterization of HXTC Products (A) HXTC products expanded to clinically relevant levels after two stimulations (n = 6). (B) All HXTC products produced IFNγ in response to HIV Gag, Neg, Pol (GNP) pepmix stimulation, as measured on ELISpot. (C) Product phenotying by flow to show T cells (CD45 + , CD3 + ), CD4 T cells (CD3 + CD4 + ), CD8 + T cells (CD3 + CD8 + ), NK cells (CD3 − CD56 + CD16 + ), natural killer T (NKT) cells (CD3 + CD56 + CD16 + ), monocytes (CD45 + CD14 + ), dendritic cells (CD3 − CD83 + , HLADR + ), and K562 cells (CD45 + CD562 − , CD16 − , CD56 − , CD32 + , CD83 + ). (D) HXTC products displayed minimal exhaustion on CD3 + T cells. (E) 51 Chromium cytotoxicity assay shows specific lysis of HXTC products against autologous PHA blast target cells pulsed with no peptides or HIV peptides (gag, nef, pol peptide pools) at an E:T ratio of 20:1. Each data point represents the mean of three technical replicates. *p
    Figure Legend Snippet: Characterization of HXTC Products (A) HXTC products expanded to clinically relevant levels after two stimulations (n = 6). (B) All HXTC products produced IFNγ in response to HIV Gag, Neg, Pol (GNP) pepmix stimulation, as measured on ELISpot. (C) Product phenotying by flow to show T cells (CD45 + , CD3 + ), CD4 T cells (CD3 + CD4 + ), CD8 + T cells (CD3 + CD8 + ), NK cells (CD3 − CD56 + CD16 + ), natural killer T (NKT) cells (CD3 + CD56 + CD16 + ), monocytes (CD45 + CD14 + ), dendritic cells (CD3 − CD83 + , HLADR + ), and K562 cells (CD45 + CD562 − , CD16 − , CD56 − , CD32 + , CD83 + ). (D) HXTC products displayed minimal exhaustion on CD3 + T cells. (E) 51 Chromium cytotoxicity assay shows specific lysis of HXTC products against autologous PHA blast target cells pulsed with no peptides or HIV peptides (gag, nef, pol peptide pools) at an E:T ratio of 20:1. Each data point represents the mean of three technical replicates. *p

    Techniques Used: Produced, Enzyme-linked Immunospot, Flow Cytometry, Cytotoxicity Assay, Lysis

    16) Product Images from "Detailed characterization of human Mycobacteriumtuberculosis specific HLA-E restricted CD8+ T cells"

    Article Title: Detailed characterization of human Mycobacteriumtuberculosis specific HLA-E restricted CD8+ T cells

    Journal: European journal of immunology

    doi: 10.1002/eji.201747184

    HLA-E/Mtb TM + CD8 + T-cell frequency after expansion. PBMCs were expanded using mitogenic stimulation, followed by magnetic bead separation of CD8 + T cells and specific peptide stimulation. Cells were stained with TMs first before staining with cell surface markers and intracellular cytokine staining. (A) Representative plots of TM staining of CD8 + T cells after control or combined p62 and p68 stimulation. Dot plots are representative of one HD and one TB patient that were analyzed in the same experiment. (B) Frequency of HLA-E/Mtb TM + CD8 + T cells after PHA expansion and p62 and p68 peptide re-stimulation. 26 out 26 HDs (13 Dutch and 13 Italian HDs), 12 out 14 LTBI, 17 out 24 active TB and 4 out 5 HIV-TB had sufficient cells for in vitro expansion. Black circles: Dutch HDs, open circles: Italian HDs, black triangles: LTBI; black squares: TB patients, black stars: HIV-TB. Each symbol represents one patient. P- values were calculated using Kruskal–Wallis test including multiple-test correction. *** p
    Figure Legend Snippet: HLA-E/Mtb TM + CD8 + T-cell frequency after expansion. PBMCs were expanded using mitogenic stimulation, followed by magnetic bead separation of CD8 + T cells and specific peptide stimulation. Cells were stained with TMs first before staining with cell surface markers and intracellular cytokine staining. (A) Representative plots of TM staining of CD8 + T cells after control or combined p62 and p68 stimulation. Dot plots are representative of one HD and one TB patient that were analyzed in the same experiment. (B) Frequency of HLA-E/Mtb TM + CD8 + T cells after PHA expansion and p62 and p68 peptide re-stimulation. 26 out 26 HDs (13 Dutch and 13 Italian HDs), 12 out 14 LTBI, 17 out 24 active TB and 4 out 5 HIV-TB had sufficient cells for in vitro expansion. Black circles: Dutch HDs, open circles: Italian HDs, black triangles: LTBI; black squares: TB patients, black stars: HIV-TB. Each symbol represents one patient. P- values were calculated using Kruskal–Wallis test including multiple-test correction. *** p

    Techniques Used: Staining, In Vitro

    17) Product Images from "Soluble T Cell Immunoglobulin Mucin Domain 3 Is Shed from CD8+ T Cells by the Sheddase ADAM10, Is Increased in Plasma during Untreated HIV Infection, and Correlates with HIV Disease Progression"

    Article Title: Soluble T Cell Immunoglobulin Mucin Domain 3 Is Shed from CD8+ T Cells by the Sheddase ADAM10, Is Increased in Plasma during Untreated HIV Infection, and Correlates with HIV Disease Progression

    Journal: Journal of Virology

    doi: 10.1128/JVI.00006-15

    Detection of sTim-3 in culture. (A) Measurements of sTim-3 levels in culture. PBMCs from HIV-uninfected volunteers were activated in vitro using media, anti-CD3 and anti-CD28, PHA, or LPS (1, 10, 100, or 1,000 ng/ml) for 4 days. Following activation,
    Figure Legend Snippet: Detection of sTim-3 in culture. (A) Measurements of sTim-3 levels in culture. PBMCs from HIV-uninfected volunteers were activated in vitro using media, anti-CD3 and anti-CD28, PHA, or LPS (1, 10, 100, or 1,000 ng/ml) for 4 days. Following activation,

    Techniques Used: In Vitro, Activation Assay

    18) Product Images from "The Hexosamine Biosynthesis Pathway Negatively Regulates IL-2 Production by Jurkat T Cells"

    Article Title: The Hexosamine Biosynthesis Pathway Negatively Regulates IL-2 Production by Jurkat T Cells

    Journal:

    doi: 10.1016/j.cellimm.2007.03.006

    O-GlcNAcylation of Jurkat cell proteins at 24hrs. (A) Western blot of O-GlcNAc-labeled proteins was prepared at 3 × 10 6 cells/lane for all lanes. Lane 1: control. Lane 2: PHA stimulated. Lane 3: PHA + 40 mM GlcN. (B) Actin loading controls are
    Figure Legend Snippet: O-GlcNAcylation of Jurkat cell proteins at 24hrs. (A) Western blot of O-GlcNAc-labeled proteins was prepared at 3 × 10 6 cells/lane for all lanes. Lane 1: control. Lane 2: PHA stimulated. Lane 3: PHA + 40 mM GlcN. (B) Actin loading controls are

    Techniques Used: Western Blot, Labeling

    GlcN (A), but not Glc (B), reduces IL-2 production by Jurkat cells. Cells were activated with PHA in the presence of various doses of GlcN or Glc as indicated on the charts. Overnight incubations were performed. GlcN caused a dramatic reduction in IL-2
    Figure Legend Snippet: GlcN (A), but not Glc (B), reduces IL-2 production by Jurkat cells. Cells were activated with PHA in the presence of various doses of GlcN or Glc as indicated on the charts. Overnight incubations were performed. GlcN caused a dramatic reduction in IL-2

    Techniques Used: Gas Chromatography

    19) Product Images from "The First Dose of Fingolimod Affects Circulating Extracellular Vesicles in Multiple Sclerosis Patients"

    Article Title: The First Dose of Fingolimod Affects Circulating Extracellular Vesicles in Multiple Sclerosis Patients

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19082448

    Circulating EVs inhibit lymphocyte activation. Cultured peripheral blood mononuclear cells (PBMCs) were treated with EVs isolated from one donor for each condition (HC, UNT, FGM 0 h, and FGM 5 h) and then activated with PHA addition. In all cases, EVs inhibit lymphocyte activation as shown by flow cytometry analysis of CD25+. Besides, FGM 5 h have impaired inhibition of lymphocyte activation. The fold change (FC) was calculated in respect to the PBS treated sample, and is shown under each bar.
    Figure Legend Snippet: Circulating EVs inhibit lymphocyte activation. Cultured peripheral blood mononuclear cells (PBMCs) were treated with EVs isolated from one donor for each condition (HC, UNT, FGM 0 h, and FGM 5 h) and then activated with PHA addition. In all cases, EVs inhibit lymphocyte activation as shown by flow cytometry analysis of CD25+. Besides, FGM 5 h have impaired inhibition of lymphocyte activation. The fold change (FC) was calculated in respect to the PBS treated sample, and is shown under each bar.

    Techniques Used: Activation Assay, Cell Culture, Isolation, Flow Cytometry, Cytometry, Inhibition

    20) Product Images from "Sex hormone levels correlate with the activity of cytokine-secreting cells in vivo"

    Article Title: Sex hormone levels correlate with the activity of cytokine-secreting cells in vivo

    Journal: Immunology

    doi: 10.1046/j.1365-2567.2000.00047.x

    Frequency of cytokine-secreting PBMC in women before and after menopause. The number cells secreting cytokines per 10 5 PBMC, spontaneously or in response to stimulation with PHA, in 38 pre-menopause and 22 post-menopause normal volunteers was determined by ELIspot. Results represent the mean ±SEM of 34 individual experiments. All samples were tested blind and decoded retrospectively. Mann–Whitney U -test.
    Figure Legend Snippet: Frequency of cytokine-secreting PBMC in women before and after menopause. The number cells secreting cytokines per 10 5 PBMC, spontaneously or in response to stimulation with PHA, in 38 pre-menopause and 22 post-menopause normal volunteers was determined by ELIspot. Results represent the mean ±SEM of 34 individual experiments. All samples were tested blind and decoded retrospectively. Mann–Whitney U -test.

    Techniques Used: Enzyme-linked Immunospot, MANN-WHITNEY

    21) Product Images from "Different Expressions of Specific Transcription Factors of Th1 (T-bet) and Th2 cells (GATA-3) by Peripheral Blood Mononuclear Cells From Patients With Multiple Sclerosis"

    Article Title: Different Expressions of Specific Transcription Factors of Th1 (T-bet) and Th2 cells (GATA-3) by Peripheral Blood Mononuclear Cells From Patients With Multiple Sclerosis

    Journal: Basic and Clinical Neuroscience

    doi: 10.32598/bcn.9.6.458

    The comparison of T-bet expression by PBMCs in patients with MS, based on gender In non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from patients with MS, no differences were observed between males and females in terms of T-bet expression.
    Figure Legend Snippet: The comparison of T-bet expression by PBMCs in patients with MS, based on gender In non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from patients with MS, no differences were observed between males and females in terms of T-bet expression.

    Techniques Used: Expressing, Mass Spectrometry

    The comparison of GATA-3 expression by PBMC in patients with MS, based on gender. The of GATA-3 expression in non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from male patients with MS were significantly higher than that of the same cell cultures from female patients with MS.
    Figure Legend Snippet: The comparison of GATA-3 expression by PBMC in patients with MS, based on gender. The of GATA-3 expression in non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from male patients with MS were significantly higher than that of the same cell cultures from female patients with MS.

    Techniques Used: Expressing, Mass Spectrometry

    The comparison of GATA-3 expression by PBMC between the MS and control groups The non-stimulated, MOG-simulated, and PHA-stimulated PBMCs from MS patients expressed lower amounts of GATA-3 in comparison with same cell cultures from healthy individuals.
    Figure Legend Snippet: The comparison of GATA-3 expression by PBMC between the MS and control groups The non-stimulated, MOG-simulated, and PHA-stimulated PBMCs from MS patients expressed lower amounts of GATA-3 in comparison with same cell cultures from healthy individuals.

    Techniques Used: Expressing, Mass Spectrometry

    The comparison of T-bet / GATA-3 mRNA Ratio by PBMCs between healthy females and female patients with MS The T-bet / GATA-3 expression ratio in non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from female patients with MS was significantly higher than that of the same cell cultures from the healthy females.
    Figure Legend Snippet: The comparison of T-bet / GATA-3 mRNA Ratio by PBMCs between healthy females and female patients with MS The T-bet / GATA-3 expression ratio in non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from female patients with MS was significantly higher than that of the same cell cultures from the healthy females.

    Techniques Used: Mass Spectrometry, Expressing

    The comparison of T-bet / GATA-3 expression ratio by PBMC between the MS and control groups The non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from MS patients expressed higher amounts of T-bet / GATA-3 mRNA ratio in comparison with same cell cultures from healthy individuals.
    Figure Legend Snippet: The comparison of T-bet / GATA-3 expression ratio by PBMC between the MS and control groups The non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from MS patients expressed higher amounts of T-bet / GATA-3 mRNA ratio in comparison with same cell cultures from healthy individuals.

    Techniques Used: Expressing, Mass Spectrometry

    The comparison of T-bet / GATA-3 mRNA ratio by PBMCs in healthy subjects, based on gender In non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from healthy controls, no significant differences were observed between males and females in terms of T-bet / GATA-3 expression ratio.
    Figure Legend Snippet: The comparison of T-bet / GATA-3 mRNA ratio by PBMCs in healthy subjects, based on gender In non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from healthy controls, no significant differences were observed between males and females in terms of T-bet / GATA-3 expression ratio.

    Techniques Used: Expressing

    The comparison of T-bet expression by PBMCs between healthy females and female patients with MS The expression of T-bet in non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from female patients with MS was significantly higher than that of the same cell cultures from the healthy females.
    Figure Legend Snippet: The comparison of T-bet expression by PBMCs between healthy females and female patients with MS The expression of T-bet in non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from female patients with MS was significantly higher than that of the same cell cultures from the healthy females.

    Techniques Used: Expressing, Mass Spectrometry

    The comparison of GATA-3 expression by PBMCs in healthy individuals, based on gender In non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from healthy controls, no significant differences were observed between males and females in terms of GATA-3 expression.
    Figure Legend Snippet: The comparison of GATA-3 expression by PBMCs in healthy individuals, based on gender In non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from healthy controls, no significant differences were observed between males and females in terms of GATA-3 expression.

    Techniques Used: Expressing

    The comparison of GATA-3 expression by PBMCs between healthy females and female patients with MS The GATA-3 expression in non-stimulated, MOG-stimulated and PHA-stimulated PBMCs from MS female patients was significantly lower than that of the same cell cultures from the healthy females.
    Figure Legend Snippet: The comparison of GATA-3 expression by PBMCs between healthy females and female patients with MS The GATA-3 expression in non-stimulated, MOG-stimulated and PHA-stimulated PBMCs from MS female patients was significantly lower than that of the same cell cultures from the healthy females.

    Techniques Used: Expressing, Mass Spectrometry

    The comparison of GATA-3 expression by PBMCs between healthy males and male patients with MS The GATA-3 expression in MOG-stimulated PBMCs from males with MS was significantly lower than that of the same culture from healthy males. The GATA-3 expression in non-stimulated and PHA-stimulated PBMCs from male patients with MS was lower than that of the same cultures from the healthy males, but the differences were insignificant.
    Figure Legend Snippet: The comparison of GATA-3 expression by PBMCs between healthy males and male patients with MS The GATA-3 expression in MOG-stimulated PBMCs from males with MS was significantly lower than that of the same culture from healthy males. The GATA-3 expression in non-stimulated and PHA-stimulated PBMCs from male patients with MS was lower than that of the same cultures from the healthy males, but the differences were insignificant.

    Techniques Used: Expressing, Mass Spectrometry

    The comparison of T-bet expression by PBMC between the MS and control groups The non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from MS patients expressed higher amounts of T-bet in comparison with the same cell cultures from healthy individuals.
    Figure Legend Snippet: The comparison of T-bet expression by PBMC between the MS and control groups The non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs from MS patients expressed higher amounts of T-bet in comparison with the same cell cultures from healthy individuals.

    Techniques Used: Expressing, Mass Spectrometry

    The comparison of T-bet / GATA-3 mRNA ratio by PBMCs in patients with MS, based on gender The of T-bet / GATA-3 expression ratio in MOG-stimulated and PHA-stimulated PBMCs from females with MS were significantly higher than that of the same cultures from male patients with MS.
    Figure Legend Snippet: The comparison of T-bet / GATA-3 mRNA ratio by PBMCs in patients with MS, based on gender The of T-bet / GATA-3 expression ratio in MOG-stimulated and PHA-stimulated PBMCs from females with MS were significantly higher than that of the same cultures from male patients with MS.

    Techniques Used: Mass Spectrometry, Expressing

    The Comparison of T-bet expression by PBMCs in healthy individuals, based on gender In non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs, no significant differences were observed between healthy males and females in terms of T-bet expression.
    Figure Legend Snippet: The Comparison of T-bet expression by PBMCs in healthy individuals, based on gender In non-stimulated, MOG-stimulated, and PHA-stimulated PBMCs, no significant differences were observed between healthy males and females in terms of T-bet expression.

    Techniques Used: Expressing

    22) Product Images from "Circulating HLA-DR+CD4+ effector memory T cells resistant to CCR5 and PD-L1 mediated suppression compromise regulatory T cell function in tuberculosis"

    Article Title: Circulating HLA-DR+CD4+ effector memory T cells resistant to CCR5 and PD-L1 mediated suppression compromise regulatory T cell function in tuberculosis

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007289

    HLA-DR + and HLA-DR - Teff cells in PTB differ in their capacity to secrete pro-inflammatory cytokines. PBMC from PTB and IGRA-ve subjects were activated with either PHA or Mtb whole cell lysate. Brefeldin and monensin were added to cultures to prevent cytokine secretion. After 16 hrs of activation, cells were fixed, permeabilised and stained with an antibody cocktail comprising Avid, anti-CD3, anti-CD4, anti-CD45RA, anti-CD127, anti-CD25, anti-HLA-DR, anti-IFNγ anti-IL-2, anti-IL-17A and anti-IL-22. Expression of cytokines was measured in the Avid - CD3 + CD4 + CD45RA - CD127 hi CD25 lo HLA-DR + and Avid - CD3 + CD4 + CD45RA - CD127 hi CD25 lo HLA-DR - Teff compartments. A representative FACS plot of PHA activated PBMC from a PTB donor with cytokine positive cells in the HLA-DR + and HLA-DR - Teff fractions is shown (A). IFNγ + , IL-2 + , IL-17A + and IL-22 + cells in response to stimulation were measured as a frequency of HLA-DR + and HLA-DR- Teff cells (B). A total of 9–11 PTB and 6 IGRA-ve donors were used. Paired Wilcoxon matched-pairs signed rank test with Bonferroni correction was used to determine P value. *p ≤ 0.013.
    Figure Legend Snippet: HLA-DR + and HLA-DR - Teff cells in PTB differ in their capacity to secrete pro-inflammatory cytokines. PBMC from PTB and IGRA-ve subjects were activated with either PHA or Mtb whole cell lysate. Brefeldin and monensin were added to cultures to prevent cytokine secretion. After 16 hrs of activation, cells were fixed, permeabilised and stained with an antibody cocktail comprising Avid, anti-CD3, anti-CD4, anti-CD45RA, anti-CD127, anti-CD25, anti-HLA-DR, anti-IFNγ anti-IL-2, anti-IL-17A and anti-IL-22. Expression of cytokines was measured in the Avid - CD3 + CD4 + CD45RA - CD127 hi CD25 lo HLA-DR + and Avid - CD3 + CD4 + CD45RA - CD127 hi CD25 lo HLA-DR - Teff compartments. A representative FACS plot of PHA activated PBMC from a PTB donor with cytokine positive cells in the HLA-DR + and HLA-DR - Teff fractions is shown (A). IFNγ + , IL-2 + , IL-17A + and IL-22 + cells in response to stimulation were measured as a frequency of HLA-DR + and HLA-DR- Teff cells (B). A total of 9–11 PTB and 6 IGRA-ve donors were used. Paired Wilcoxon matched-pairs signed rank test with Bonferroni correction was used to determine P value. *p ≤ 0.013.

    Techniques Used: Activation Assay, Staining, Expressing, FACS

    23) Product Images from "Germline deletion of CIN85 in humans with X chromosome–linked antibody deficiency"

    Article Title: Germline deletion of CIN85 in humans with X chromosome–linked antibody deficiency

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20170534

    Robust TCR signaling and T cell function in the absence of CIN85. (A) Ratiometric Ca 2+ flux profiles and intracellular staining of IκBα degradation (left and middle panels, respectively) of CD45RA and CD45R0 CD4 T cells (upper and lower rows, respectively) upon anti-CD3/-CD28 stimulation (tinted) or treatment with secondary antibodies alone (unfilled). For quantification, graph on the right depicts MFI ratios of IκBα expression in stimulated and unstimulated CD45RA T cells. Dotted line (ratio = 1) indicates no degradation (two independent experiments). (B) Expression of CD69, CD25, and ICOS induced by CD3 or CD3/CD28 stimulation of CD45RA or CD45R0 CD4 T cells from a healthy control or patient no. 1. Representative histograms of inducible CD69 expression are shown on the left where solid and dashed lines indicate anti-CD3 and anti-CD3/-CD28 stimulation, respectively. For quantification, MFI expression ratios of all three T activation markers on naive and memory T cell populations were plotted (middle and right panels; three experiments). (C) Intracellular expression of IL-4, IL-17 or IFN-γ in CD45R0 CD4 T cells of patient no. 1 and a control upon TCR/CD28 ligation (upper left four FACS plots) or PMA/Ionomycin treatment (upper right four FACS plots). Quantification (lower three panels) depicts the percentage of positivity for IL-4 (left), IFN-γ (middle), and IL-17 (right) in resting cells, upon PMA/Ionomycin treatment or CD3/CD28 coligation (left to right in each graph; three experiments). (D) T cell proliferation monitored by CSFE labeling of CD4 or CD8 T cells (upper and lower panels, respectively) that were obtained from patient no. 1 (red) or a healthy control person (blue) and stimulated via CD3/CD28 or treated with PHA for 5 d (one experiment).
    Figure Legend Snippet: Robust TCR signaling and T cell function in the absence of CIN85. (A) Ratiometric Ca 2+ flux profiles and intracellular staining of IκBα degradation (left and middle panels, respectively) of CD45RA and CD45R0 CD4 T cells (upper and lower rows, respectively) upon anti-CD3/-CD28 stimulation (tinted) or treatment with secondary antibodies alone (unfilled). For quantification, graph on the right depicts MFI ratios of IκBα expression in stimulated and unstimulated CD45RA T cells. Dotted line (ratio = 1) indicates no degradation (two independent experiments). (B) Expression of CD69, CD25, and ICOS induced by CD3 or CD3/CD28 stimulation of CD45RA or CD45R0 CD4 T cells from a healthy control or patient no. 1. Representative histograms of inducible CD69 expression are shown on the left where solid and dashed lines indicate anti-CD3 and anti-CD3/-CD28 stimulation, respectively. For quantification, MFI expression ratios of all three T activation markers on naive and memory T cell populations were plotted (middle and right panels; three experiments). (C) Intracellular expression of IL-4, IL-17 or IFN-γ in CD45R0 CD4 T cells of patient no. 1 and a control upon TCR/CD28 ligation (upper left four FACS plots) or PMA/Ionomycin treatment (upper right four FACS plots). Quantification (lower three panels) depicts the percentage of positivity for IL-4 (left), IFN-γ (middle), and IL-17 (right) in resting cells, upon PMA/Ionomycin treatment or CD3/CD28 coligation (left to right in each graph; three experiments). (D) T cell proliferation monitored by CSFE labeling of CD4 or CD8 T cells (upper and lower panels, respectively) that were obtained from patient no. 1 (red) or a healthy control person (blue) and stimulated via CD3/CD28 or treated with PHA for 5 d (one experiment).

    Techniques Used: Cell Function Assay, Staining, Expressing, Activation Assay, Ligation, FACS, Labeling

    24) Product Images from "Dual Role of miR-21 in CD4+ T-Cells: Activation-Induced miR-21 Supports Survival of Memory T-Cells and Regulates CCR7 Expression in Naive T-Cells"

    Article Title: Dual Role of miR-21 in CD4+ T-Cells: Activation-Induced miR-21 Supports Survival of Memory T-Cells and Regulates CCR7 Expression in Naive T-Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076217

    MiR-21 mediates survival of activated memory CD4+ T-cells. A Resting naive (CD3+ CD8-CD45RO-CD25-) T-cells were stimulated with 5µg/ml PHA and 100 U/mL IL-2, followed by transduction with lentiviral vectors harboring miR-21 or control inhibitor (scrambled hairpin sequence) and GFP. The percentage of GFP+ cells in culture over time was monitored by FACS. Data were normalized to the first measurement at day six. Each line represents a separate donor ( n =5, two-way RM ANOVA with Bonferroni posttests, ns-not significant). B Data from A, depicted as median values with interquartile range. C Resting memory (CD3+ CD8-CD45RO-CD25-) T-cells were stimulated with 5µg/ml PHA and 100 U/mL IL-2, followed by transduction with lentiviral vectors harboring miR-21 or control inhibitor (scrambled hairpin sequence) and GFP. The percentage of GFP+ cells in culture over time was monitored by FACS. Data were normalized to the first measurement at day six. Each line represents a separate donor ( n =5, two-way RM ANOVA with Bonferroni posttests). D Data from C, depicted as median with interquartile range. E Activated naive, and F activated memory GFP+ T-cells harboring miR-21 or control inhibitor were isolated by FACS from mixed cultures (A, and C respectively) at day six post lentiviral transduction. Isolated GFP+ cells, and activated not transduced cells (NT) were cultured in complete media supplemented with 100 U/mL of IL-2 for 48h (until day 8), and percentage of apoptotic cells was assessed by FACS-based measurement of mitochondrial trans-membrane potential loss, using DilC1(5). Each line represents a separate donor (naive: n =4, memory n=5 , RM ANOVA with Bonferroni posttests). *p
    Figure Legend Snippet: MiR-21 mediates survival of activated memory CD4+ T-cells. A Resting naive (CD3+ CD8-CD45RO-CD25-) T-cells were stimulated with 5µg/ml PHA and 100 U/mL IL-2, followed by transduction with lentiviral vectors harboring miR-21 or control inhibitor (scrambled hairpin sequence) and GFP. The percentage of GFP+ cells in culture over time was monitored by FACS. Data were normalized to the first measurement at day six. Each line represents a separate donor ( n =5, two-way RM ANOVA with Bonferroni posttests, ns-not significant). B Data from A, depicted as median values with interquartile range. C Resting memory (CD3+ CD8-CD45RO-CD25-) T-cells were stimulated with 5µg/ml PHA and 100 U/mL IL-2, followed by transduction with lentiviral vectors harboring miR-21 or control inhibitor (scrambled hairpin sequence) and GFP. The percentage of GFP+ cells in culture over time was monitored by FACS. Data were normalized to the first measurement at day six. Each line represents a separate donor ( n =5, two-way RM ANOVA with Bonferroni posttests). D Data from C, depicted as median with interquartile range. E Activated naive, and F activated memory GFP+ T-cells harboring miR-21 or control inhibitor were isolated by FACS from mixed cultures (A, and C respectively) at day six post lentiviral transduction. Isolated GFP+ cells, and activated not transduced cells (NT) were cultured in complete media supplemented with 100 U/mL of IL-2 for 48h (until day 8), and percentage of apoptotic cells was assessed by FACS-based measurement of mitochondrial trans-membrane potential loss, using DilC1(5). Each line represents a separate donor (naive: n =4, memory n=5 , RM ANOVA with Bonferroni posttests). *p

    Techniques Used: Transduction, Sequencing, FACS, Isolation, Cell Culture

    MiR-21 inhibition leads to increased CCR7 expression in activated T-cells. A Representative FACS staining plot depicting CCR7 expression on resting naive (CD3+ CD8-CD45RO-CD25-, Day 0), and on PHA/IL-2 activated, GFP+ cells harboring miR-21 or control inhibitor (scrambled hairpin sequence) at day six post lentiviral transduction. The isotype staining control is depicted. B Quantification of CCR7 staining of GFP+ cells from A, depicted as MFI (geometric mean fluorescent intensity). Each line represents a separate donor ( n=8 , Wilcoxon signed ranked test). C Percentage of CCR7 positive, GFP+ cells from A. Each line represents a separate donor ( n=8 , Wilcoxon signed ranked test). D GFP+, PHA/IL-2 activated naive T-cells harboring miR-21 or control inhibitor were FACS isolated at day six post-lentiviral transduction and CCR7 transcript expression was determined by qRT-PCR. Each line represents a separate donor ( n=3 , paired t-test, ns). Relative expression normalized to the U6 reference gene is shown. E Representative FACS staining plot depicting CCR7 expression on resting memory (CD3+ CD8-CD45RO+CD25-, Day 0), and on PHA/IL-2 activated, GFP+ cells harboring miR-21 or control inhibitor (scrambled hairpin sequence) at day six post lentiviral transduction. The isotype staining control is also depicted. F Quantification of CCR7 staining of GFP+ cells from E, depicted as MFI (geometric mean fluorescent intensity). Each line represents a separate donor ( n=7 , Wilcoxon signed ranked test). G Percentage of CCR7 positive, GFP+ cells from E. Each line represents a separate donor ( n=7 , Wilcoxon signed ranked test). D GFP+, PHA/IL-2 activated memory T-cells harboring miR-21 or control inhibitor were FACS isolated at day six post-lentiviral transduction and CCR7 transcript expression was determined by qRT-PCR. Each line represents a separate donor ( n=3 , paired- t-test, ns). Relative expression normalized to the U6 reference gene is shown. *p
    Figure Legend Snippet: MiR-21 inhibition leads to increased CCR7 expression in activated T-cells. A Representative FACS staining plot depicting CCR7 expression on resting naive (CD3+ CD8-CD45RO-CD25-, Day 0), and on PHA/IL-2 activated, GFP+ cells harboring miR-21 or control inhibitor (scrambled hairpin sequence) at day six post lentiviral transduction. The isotype staining control is depicted. B Quantification of CCR7 staining of GFP+ cells from A, depicted as MFI (geometric mean fluorescent intensity). Each line represents a separate donor ( n=8 , Wilcoxon signed ranked test). C Percentage of CCR7 positive, GFP+ cells from A. Each line represents a separate donor ( n=8 , Wilcoxon signed ranked test). D GFP+, PHA/IL-2 activated naive T-cells harboring miR-21 or control inhibitor were FACS isolated at day six post-lentiviral transduction and CCR7 transcript expression was determined by qRT-PCR. Each line represents a separate donor ( n=3 , paired t-test, ns). Relative expression normalized to the U6 reference gene is shown. E Representative FACS staining plot depicting CCR7 expression on resting memory (CD3+ CD8-CD45RO+CD25-, Day 0), and on PHA/IL-2 activated, GFP+ cells harboring miR-21 or control inhibitor (scrambled hairpin sequence) at day six post lentiviral transduction. The isotype staining control is also depicted. F Quantification of CCR7 staining of GFP+ cells from E, depicted as MFI (geometric mean fluorescent intensity). Each line represents a separate donor ( n=7 , Wilcoxon signed ranked test). G Percentage of CCR7 positive, GFP+ cells from E. Each line represents a separate donor ( n=7 , Wilcoxon signed ranked test). D GFP+, PHA/IL-2 activated memory T-cells harboring miR-21 or control inhibitor were FACS isolated at day six post-lentiviral transduction and CCR7 transcript expression was determined by qRT-PCR. Each line represents a separate donor ( n=3 , paired- t-test, ns). Relative expression normalized to the U6 reference gene is shown. *p

    Techniques Used: Inhibition, Expressing, FACS, Staining, Sequencing, Transduction, Isolation, Quantitative RT-PCR

    25) Product Images from "Comparison of methods to quantify inducible HIV-1 outgrowth"

    Article Title: Comparison of methods to quantify inducible HIV-1 outgrowth

    Journal: Journal of Virus Eradication

    doi: 10.1016/j.jve.2021.100043

    Triplicate qVOA Set-up and Workflow. Total CD4 + T-cells were prepared in a limiting dilution in the presence of PHA and gamma-irradiated feeders on Day 1 before addition of targets cells (PHA-activated allogeneic blasts, SupT1-R5 cells, or MOLT4-R5 cells) on Day 2. Allogeneic blasts were replenished on Days 7 and 14, and p24 measured on Days 7, 14, and 21. Day 21 p24-negative wells were screened for quantitative HIV-1 RNA with qRT-PCR, from which Day 7 and 14 samples were quantified to measure RNA kinetics.
    Figure Legend Snippet: Triplicate qVOA Set-up and Workflow. Total CD4 + T-cells were prepared in a limiting dilution in the presence of PHA and gamma-irradiated feeders on Day 1 before addition of targets cells (PHA-activated allogeneic blasts, SupT1-R5 cells, or MOLT4-R5 cells) on Day 2. Allogeneic blasts were replenished on Days 7 and 14, and p24 measured on Days 7, 14, and 21. Day 21 p24-negative wells were screened for quantitative HIV-1 RNA with qRT-PCR, from which Day 7 and 14 samples were quantified to measure RNA kinetics.

    Techniques Used: Irradiation, Quantitative RT-PCR

    26) Product Images from "Circulating HLA-DR+CD4+ effector memory T cells resistant to CCR5 and PD-L1 mediated suppression compromise regulatory T cell function in tuberculosis"

    Article Title: Circulating HLA-DR+CD4+ effector memory T cells resistant to CCR5 and PD-L1 mediated suppression compromise regulatory T cell function in tuberculosis

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007289

    HLA-DR + and HLA-DR - Teff cells in PTB differ in their capacity to secrete pro-inflammatory cytokines. PBMC from PTB and IGRA-ve subjects were activated with either PHA or Mtb whole cell lysate. Brefeldin and monensin were added to cultures to prevent cytokine secretion. After 16 hrs of activation, cells were fixed, permeabilised and stained with an antibody cocktail comprising Avid, anti-CD3, anti-CD4, anti-CD45RA, anti-CD127, anti-CD25, anti-HLA-DR, anti-IFNγ anti-IL-2, anti-IL-17A and anti-IL-22. Expression of cytokines was measured in the Avid - CD3 + CD4 + CD45RA - CD127 hi CD25 lo HLA-DR + and Avid - CD3 + CD4 + CD45RA - CD127 hi CD25 lo HLA-DR - Teff compartments. A representative FACS plot of PHA activated PBMC from a PTB donor with cytokine positive cells in the HLA-DR + and HLA-DR - Teff fractions is shown (A). IFNγ + , IL-2 + , IL-17A + and IL-22 + cells in response to stimulation were measured as a frequency of HLA-DR + and HLA-DR- Teff cells (B). A total of 9–11 PTB and 6 IGRA-ve donors were used. Paired Wilcoxon matched-pairs signed rank test with Bonferroni correction was used to determine P value. *p ≤ 0.013.
    Figure Legend Snippet: HLA-DR + and HLA-DR - Teff cells in PTB differ in their capacity to secrete pro-inflammatory cytokines. PBMC from PTB and IGRA-ve subjects were activated with either PHA or Mtb whole cell lysate. Brefeldin and monensin were added to cultures to prevent cytokine secretion. After 16 hrs of activation, cells were fixed, permeabilised and stained with an antibody cocktail comprising Avid, anti-CD3, anti-CD4, anti-CD45RA, anti-CD127, anti-CD25, anti-HLA-DR, anti-IFNγ anti-IL-2, anti-IL-17A and anti-IL-22. Expression of cytokines was measured in the Avid - CD3 + CD4 + CD45RA - CD127 hi CD25 lo HLA-DR + and Avid - CD3 + CD4 + CD45RA - CD127 hi CD25 lo HLA-DR - Teff compartments. A representative FACS plot of PHA activated PBMC from a PTB donor with cytokine positive cells in the HLA-DR + and HLA-DR - Teff fractions is shown (A). IFNγ + , IL-2 + , IL-17A + and IL-22 + cells in response to stimulation were measured as a frequency of HLA-DR + and HLA-DR- Teff cells (B). A total of 9–11 PTB and 6 IGRA-ve donors were used. Paired Wilcoxon matched-pairs signed rank test with Bonferroni correction was used to determine P value. *p ≤ 0.013.

    Techniques Used: Activation Assay, Staining, Expressing, FACS

    27) Product Images from "Dissecting the Immune Stimulation Promoted by CSF-470 Vaccine Plus Adjuvants in Cutaneous Melanoma Patients: Long Term Antitumor Immunity and Short Term Release of Acute Inflammatory Reactants"

    Article Title: Dissecting the Immune Stimulation Promoted by CSF-470 Vaccine Plus Adjuvants in Cutaneous Melanoma Patients: Long Term Antitumor Immunity and Short Term Release of Acute Inflammatory Reactants

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02531

    (A) Pt #006 T cell reactivity after 12 day stimulation with autologous tumor cell lysate displayed as the number of IFN-γ positive spots per 250,000 PBMCs. Representative images of spots for each mononuclear cell sample are shown. The positive control wells were restimulated with the monoclonal antibody against human CD3 OKT-3 and PHA (αCD3/PHA). The horizontal line in the graph at 350 spots indicates the highest number of spots that could be counted per well with this assay. (B) ). (C) Pt #026 T cell reactivity after 12 day stimulation with autologous tumor cell lysate displayed as the number of IFN-γ positive spots per 250,000 PBMCs. The representative images of spots for each mononuclear cell sample are shown. The positive control wells were restimulated with αCD3 and PHA. The horizontal line in the graph at 350 spots indicates the highest number of spots that could be counted per well with this assay. (D) Pt #026 serum antibodies against autologous tumor cells (1/10 serum dilution). In all of the graphs, mean and standard deviation calculated from replicates done in the same experiment, are shown for each sample.
    Figure Legend Snippet: (A) Pt #006 T cell reactivity after 12 day stimulation with autologous tumor cell lysate displayed as the number of IFN-γ positive spots per 250,000 PBMCs. Representative images of spots for each mononuclear cell sample are shown. The positive control wells were restimulated with the monoclonal antibody against human CD3 OKT-3 and PHA (αCD3/PHA). The horizontal line in the graph at 350 spots indicates the highest number of spots that could be counted per well with this assay. (B) ). (C) Pt #026 T cell reactivity after 12 day stimulation with autologous tumor cell lysate displayed as the number of IFN-γ positive spots per 250,000 PBMCs. The representative images of spots for each mononuclear cell sample are shown. The positive control wells were restimulated with αCD3 and PHA. The horizontal line in the graph at 350 spots indicates the highest number of spots that could be counted per well with this assay. (D) Pt #026 serum antibodies against autologous tumor cells (1/10 serum dilution). In all of the graphs, mean and standard deviation calculated from replicates done in the same experiment, are shown for each sample.

    Techniques Used: Positive Control, Standard Deviation

    28) Product Images from "Dissecting the Immune Stimulation Promoted by CSF-470 Vaccine Plus Adjuvants in Cutaneous Melanoma Patients: Long Term Antitumor Immunity and Short Term Release of Acute Inflammatory Reactants"

    Article Title: Dissecting the Immune Stimulation Promoted by CSF-470 Vaccine Plus Adjuvants in Cutaneous Melanoma Patients: Long Term Antitumor Immunity and Short Term Release of Acute Inflammatory Reactants

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02531

    (A) Pt #006 T cell reactivity after 12 day stimulation with autologous tumor cell lysate displayed as the number of IFN-γ positive spots per 250,000 PBMCs. Representative images of spots for each mononuclear cell sample are shown. The positive control wells were restimulated with the monoclonal antibody against human CD3 OKT-3 and PHA (αCD3/PHA). The horizontal line in the graph at 350 spots indicates the highest number of spots that could be counted per well with this assay. (B) Pt #006 serum antibodies against autologous tumor cells (1/10 serum dilution), as shown in Mordoh et al. ( 1 ). (C) Pt #026 T cell reactivity after 12 day stimulation with autologous tumor cell lysate displayed as the number of IFN-γ positive spots per 250,000 PBMCs. The representative images of spots for each mononuclear cell sample are shown. The positive control wells were restimulated with αCD3 and PHA. The horizontal line in the graph at 350 spots indicates the highest number of spots that could be counted per well with this assay. (D) Pt #026 serum antibodies against autologous tumor cells (1/10 serum dilution). In all of the graphs, mean and standard deviation calculated from replicates done in the same experiment, are shown for each sample.
    Figure Legend Snippet: (A) Pt #006 T cell reactivity after 12 day stimulation with autologous tumor cell lysate displayed as the number of IFN-γ positive spots per 250,000 PBMCs. Representative images of spots for each mononuclear cell sample are shown. The positive control wells were restimulated with the monoclonal antibody against human CD3 OKT-3 and PHA (αCD3/PHA). The horizontal line in the graph at 350 spots indicates the highest number of spots that could be counted per well with this assay. (B) Pt #006 serum antibodies against autologous tumor cells (1/10 serum dilution), as shown in Mordoh et al. ( 1 ). (C) Pt #026 T cell reactivity after 12 day stimulation with autologous tumor cell lysate displayed as the number of IFN-γ positive spots per 250,000 PBMCs. The representative images of spots for each mononuclear cell sample are shown. The positive control wells were restimulated with αCD3 and PHA. The horizontal line in the graph at 350 spots indicates the highest number of spots that could be counted per well with this assay. (D) Pt #026 serum antibodies against autologous tumor cells (1/10 serum dilution). In all of the graphs, mean and standard deviation calculated from replicates done in the same experiment, are shown for each sample.

    Techniques Used: Positive Control, Standard Deviation

    29) Product Images from "A novel primary human immunodeficiency due to deficiency in the WASP-interacting protein WIP"

    Article Title: A novel primary human immunodeficiency due to deficiency in the WASP-interacting protein WIP

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20110896

    Functional characterization of WIP deficiency. (A) Evaluation of the proportion of T, B, and NK cells on gated lymphocytes in whole blood (top and middle row), and of CD4- and CD8-expressing cells on the CD3 + gated cells (bottom row) of a control (Ctrl), the patient (Pt), and a WAS-null patient. Data are representative of three independent analyses. (B) T cell proliferation to PHA and anti-hCD3 with or without rIL-2, as assessed by CFSE dilution. PBMCs were used and FACS analysis was performed on CD3 + gated cells. The solid gray histogram represents exemplificative profile of unstimulated cells. An overlapping profile was obtained from unstimulated cells of control and WAS patients (not depicted). The red, gray, and blue profiles represent CFSE content in stimulated cells from patient, control, and a WAS-null patient, respectively. Data are representative of two independent experiments. (C) PBMCs were stimulated with IL-2, and gated CD3 + cells were analyzed by FACS for intracellular pSTAT5 using anti-pY 694 STAT5. The solid line represents the signal in unstimulated patient’s cells, which was comparable to that of unstimulated control and WAS-null patient cells (not depicted). The pSTAT5 signal in the rIL-2 stimulated cells from patient, control, and WAS patient are indicated in red, gray, and blue, respectively. Data are representative of three independent experiments. (D) Migration of PHA blasts from a healthy control, the patient and from a WAS-null patient toward the chemokine IP-10. The gray, red, and blue histograms represent migration of cells from control, patient, and WAS-null patient, respectively. Data are representative of two independent experiments. (E) Expression of the IP-10 receptor CXCR3 on PHA blasts from patient, control, and WAS-null patient. Isotype control staining of patient PHA blasts is shown by the filled histogram and was comparable to that of control T cells and WAS patient (not depicted). The CXCR3 signals in cells from patient, control, and WAS patient are indicated in red, gray, and blue, respectively. Data are representative of two independent experiments. (F) Expression of CD56, NKp30, NKGD2, and NKp46 on NK cells from patient (red) and control (gray). The filled histogram represents isotype control staining of patient NK cells, and was comparable to that of control NK cells (not depicted). Data are representative of two analyses. (G) Cytolytic activity by patient and control NK cell lines against the LCL 721.221 target cells, measured as the percentage of net 51 Cr release at effector (E) to target (T) ratios of 3:1 and 6:1. Data are representative of two independent experiments on the same expanded NK cells.
    Figure Legend Snippet: Functional characterization of WIP deficiency. (A) Evaluation of the proportion of T, B, and NK cells on gated lymphocytes in whole blood (top and middle row), and of CD4- and CD8-expressing cells on the CD3 + gated cells (bottom row) of a control (Ctrl), the patient (Pt), and a WAS-null patient. Data are representative of three independent analyses. (B) T cell proliferation to PHA and anti-hCD3 with or without rIL-2, as assessed by CFSE dilution. PBMCs were used and FACS analysis was performed on CD3 + gated cells. The solid gray histogram represents exemplificative profile of unstimulated cells. An overlapping profile was obtained from unstimulated cells of control and WAS patients (not depicted). The red, gray, and blue profiles represent CFSE content in stimulated cells from patient, control, and a WAS-null patient, respectively. Data are representative of two independent experiments. (C) PBMCs were stimulated with IL-2, and gated CD3 + cells were analyzed by FACS for intracellular pSTAT5 using anti-pY 694 STAT5. The solid line represents the signal in unstimulated patient’s cells, which was comparable to that of unstimulated control and WAS-null patient cells (not depicted). The pSTAT5 signal in the rIL-2 stimulated cells from patient, control, and WAS patient are indicated in red, gray, and blue, respectively. Data are representative of three independent experiments. (D) Migration of PHA blasts from a healthy control, the patient and from a WAS-null patient toward the chemokine IP-10. The gray, red, and blue histograms represent migration of cells from control, patient, and WAS-null patient, respectively. Data are representative of two independent experiments. (E) Expression of the IP-10 receptor CXCR3 on PHA blasts from patient, control, and WAS-null patient. Isotype control staining of patient PHA blasts is shown by the filled histogram and was comparable to that of control T cells and WAS patient (not depicted). The CXCR3 signals in cells from patient, control, and WAS patient are indicated in red, gray, and blue, respectively. Data are representative of two independent experiments. (F) Expression of CD56, NKp30, NKGD2, and NKp46 on NK cells from patient (red) and control (gray). The filled histogram represents isotype control staining of patient NK cells, and was comparable to that of control NK cells (not depicted). Data are representative of two analyses. (G) Cytolytic activity by patient and control NK cell lines against the LCL 721.221 target cells, measured as the percentage of net 51 Cr release at effector (E) to target (T) ratios of 3:1 and 6:1. Data are representative of two independent experiments on the same expanded NK cells.

    Techniques Used: Functional Assay, Expressing, FACS, Migration, Staining, Activity Assay

    30) Product Images from "New Genome-Wide Algorithm Identifies Novel In-Vivo Expressed Mycobacterium Tuberculosis Antigens Inducing Human T-Cell Responses with Classical and Unconventional Cytokine Profiles"

    Article Title: New Genome-Wide Algorithm Identifies Novel In-Vivo Expressed Mycobacterium Tuberculosis Antigens Inducing Human T-Cell Responses with Classical and Unconventional Cytokine Profiles

    Journal: Scientific Reports

    doi: 10.1038/srep37793

    Lymphocyte proliferative responses to IVE-TB proteins in individuals with different exposures to Mtb . ( a ) Donors ( n = 19) were divided into three groups based on their lymphocyte proliferative response to Mtb ESAT6/CFP10 (E/C) or PPD. The total number of antigens recognised (left) and the overall magnitude of responses generated (right) after stimulation are indicated per individual as a closed circle (•). ( b ) Donors were ranked according to the lymphocyte proliferation responses to PHA, PPD, E/C, the total proliferation, and the number of antigens recognised. The correlation among these variables was measured by Spearman r, and the statistical significance was expressed by two-tailed p-value.
    Figure Legend Snippet: Lymphocyte proliferative responses to IVE-TB proteins in individuals with different exposures to Mtb . ( a ) Donors ( n = 19) were divided into three groups based on their lymphocyte proliferative response to Mtb ESAT6/CFP10 (E/C) or PPD. The total number of antigens recognised (left) and the overall magnitude of responses generated (right) after stimulation are indicated per individual as a closed circle (•). ( b ) Donors were ranked according to the lymphocyte proliferation responses to PHA, PPD, E/C, the total proliferation, and the number of antigens recognised. The correlation among these variables was measured by Spearman r, and the statistical significance was expressed by two-tailed p-value.

    Techniques Used: Generated, Two Tailed Test

    Cytokine production in response to IVE-TB antigens in PBMC of individuals differently exposed to mycobacteria. PBMCs of ESAT/CFP10 (E/C) and PPD in vitro positive (+) or negative (−) donors were stimulated with each IVE-TB antigen, E/C, PPD or PHA for six days. The culture supernatants were tested for the presence of IFN-γ, IP-10, TNF-α, IL-17, IL-13, IL-10 and GM-CSF using a seven-plex assay. Results are shown for those IVE-TB antigens which elicited at least two different cytokines in ≥50% of the donors. Percentage of responders: = 100%, = 75–85% and = 50–60%.
    Figure Legend Snippet: Cytokine production in response to IVE-TB antigens in PBMC of individuals differently exposed to mycobacteria. PBMCs of ESAT/CFP10 (E/C) and PPD in vitro positive (+) or negative (−) donors were stimulated with each IVE-TB antigen, E/C, PPD or PHA for six days. The culture supernatants were tested for the presence of IFN-γ, IP-10, TNF-α, IL-17, IL-13, IL-10 and GM-CSF using a seven-plex assay. Results are shown for those IVE-TB antigens which elicited at least two different cytokines in ≥50% of the donors. Percentage of responders: = 100%, = 75–85% and = 50–60%.

    Techniques Used: In Vitro, Plex Assay

    Cytokine response profile to IVE-TB antigens in LTBI. The cytokine levels were measured in diluted whole blood supernatants of an independent cohort of LTBI ( n = 25) after 6 days stimulation with either IVE-TB antigens (10 μg/ml), ESAT6/CFP10 (E/C) (10 μg/ml), PPD (5 μg/ml), PHA (2 μg/ml) or tetanus toxoid (TT) (10 μg/ml). ( a ) Bars represent the mean of responses. Results are shown for IFN-γ, IL-17, TNF-α and IP-10 as illustrative examples. ( b ) Significance differences were evaluated between the cytokine level induced by IVE-TB antigens vs. unstimulated (Dunn’s multiple comparisons test).
    Figure Legend Snippet: Cytokine response profile to IVE-TB antigens in LTBI. The cytokine levels were measured in diluted whole blood supernatants of an independent cohort of LTBI ( n = 25) after 6 days stimulation with either IVE-TB antigens (10 μg/ml), ESAT6/CFP10 (E/C) (10 μg/ml), PPD (5 μg/ml), PHA (2 μg/ml) or tetanus toxoid (TT) (10 μg/ml). ( a ) Bars represent the mean of responses. Results are shown for IFN-γ, IL-17, TNF-α and IP-10 as illustrative examples. ( b ) Significance differences were evaluated between the cytokine level induced by IVE-TB antigens vs. unstimulated (Dunn’s multiple comparisons test).

    Techniques Used:

    31) Product Images from "Primary T-lymphocytes rescue the replication of HIV-1 DIS RNA mutants in part by facilitating reverse transcription"

    Article Title: Primary T-lymphocytes rescue the replication of HIV-1 DIS RNA mutants in part by facilitating reverse transcription

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm1149

    PBMCs but not T-cell lines can support the replication of HIV-1 DIS RNA mutants. WT HIV-1 and five HIV-1 DIS mutants (NL ACGCGT , NL GCGCGC , NL Palindrome neg , NL Ext Stem and NL Ext Loop ) were normalized for viral RT activity and used to infect PHA-stimulated freshly isolated PBMC ( A ) and a number of T-cell lines ( B – F ). Supernatants were collected 3, 7, 10 and 14 days postinfection and RT activity in each sample was measured. Results represent means and standard deviations of triplicate samples and are representative of six different PBMC donors and either four or six sets of independent experiments for the T-cells lines.
    Figure Legend Snippet: PBMCs but not T-cell lines can support the replication of HIV-1 DIS RNA mutants. WT HIV-1 and five HIV-1 DIS mutants (NL ACGCGT , NL GCGCGC , NL Palindrome neg , NL Ext Stem and NL Ext Loop ) were normalized for viral RT activity and used to infect PHA-stimulated freshly isolated PBMC ( A ) and a number of T-cell lines ( B – F ). Supernatants were collected 3, 7, 10 and 14 days postinfection and RT activity in each sample was measured. Results represent means and standard deviations of triplicate samples and are representative of six different PBMC donors and either four or six sets of independent experiments for the T-cells lines.

    Techniques Used: Activity Assay, Isolation

    Production of HIV-1 DIS RNA mutants from PBMCs partially rescues defects in viral cDNA synthesis. WT and DIS mutant HIV-1 viruses were produced either by transfection of 293T cells or infection of PHA-stimulated freshly isolated PBMCs. Equivalent amounts of DNase treated virus were used to infect either SupT1 cells ( A ) or PBMCs ( B ). Cells were lysed 24 h postinfection and HIV-1 cDNA was measured by real-time PCR. HIV-1 DNA copies were normalized by CCR5. Results are reported as a percentage of the WT control and represent means (±SE) of separate experiments.
    Figure Legend Snippet: Production of HIV-1 DIS RNA mutants from PBMCs partially rescues defects in viral cDNA synthesis. WT and DIS mutant HIV-1 viruses were produced either by transfection of 293T cells or infection of PHA-stimulated freshly isolated PBMCs. Equivalent amounts of DNase treated virus were used to infect either SupT1 cells ( A ) or PBMCs ( B ). Cells were lysed 24 h postinfection and HIV-1 cDNA was measured by real-time PCR. HIV-1 DNA copies were normalized by CCR5. Results are reported as a percentage of the WT control and represent means (±SE) of separate experiments.

    Techniques Used: Mutagenesis, Produced, Transfection, Infection, Isolation, Real-time Polymerase Chain Reaction

    32) Product Images from "Identification of Human T Cell Antigens for the Development of Vaccines Against Mycobacterium Tuberculosis"

    Article Title: Identification of Human T Cell Antigens for the Development of Vaccines Against Mycobacterium Tuberculosis

    Journal:

    doi:

    Levels of IFN-γ released by antigen-stimulated human PBMC were measured in vitro. PPD + and PPD - PBMC were incubated for 72 h in medium, PHA (10 μg/ml), Mtb lysate (10 μg/ml), or Mtb recombinant proteins (50 μg/ml). Antigens
    Figure Legend Snippet: Levels of IFN-γ released by antigen-stimulated human PBMC were measured in vitro. PPD + and PPD - PBMC were incubated for 72 h in medium, PHA (10 μg/ml), Mtb lysate (10 μg/ml), or Mtb recombinant proteins (50 μg/ml). Antigens

    Techniques Used: In Vitro, Incubation, Recombinant

    33) Product Images from "The Pathway To Establishing HIV Latency Is Critical to How Latency Is Maintained and Reversed"

    Article Title: The Pathway To Establishing HIV Latency Is Critical to How Latency Is Maintained and Reversed

    Journal: Journal of Virology

    doi: 10.1128/JVI.02225-17

    HIV infection and virus reactivation in pre- and postactivation latency. (A) Flow cytometry gating and analysis used to measure EGFP expression in CD4 + T cells using the in vitro latency models. Live cells were defined by forward scatter (FSC) versus side scatter (SSC). Uninfected T cells were used to detect any background EGFP expression in our system. EGFP expression was measured against the PE channel. Plots show EGFP expression in mock-infected T cells (uninfected resting CD4 + ) with chemokine treatment (+CCL19) or mock-activated T cells (uninfected activated CD4 + ) and infected T cells (activated CD4 + ). (B) Plots representing EGFP expression following activation. Data are shown as percentages of EGFP in each plot in preactivation (+CCL19 or untreated CD4 + T cells) or postactivation latency. Data are representative of results for a matched sample in one experiment. (C) Frequency of productive infection in infected CD4 + T cells measured by EGFP expression at 5 days postinfection in the preactivation models (CCL19 treated [+] [red open circles] and untreated [−] [blue open circles]) and at day 7 in the postactivation model (black open circles). (D to F) Induced EGFP expression was measured following stimulation of sorted EGFP − cells in preactivation latency with CCL19 (D) or without CCL19 (E) and in postactivation latency (F), using monocytes (mono), monocytes and anti-CD3 (20 μg/ml) (mono+aCD3), PHA (10 μg/ml) and PMA (50 ng/ml) (PMA/PHA), or PMA and ionomycin (500 ng/ml) (PMA/Iono) as well as plate-bound anti-CD3 (20 μg/ml) and anti-CD28 (3.6 μg/ml) (aCD3/aCD28) stimulation or culturing with antiretrovirals only (Unstim). EGFP expression was measured 72 h after coculture. Each dot represents data for a single donor, and the box plots show 25th and 75th percentiles, medians, and ranges. *, P ≤ 0.05; **, P ≤ 0.01 (as determined by a Wilcoxon matched-pairs signed-rank test).
    Figure Legend Snippet: HIV infection and virus reactivation in pre- and postactivation latency. (A) Flow cytometry gating and analysis used to measure EGFP expression in CD4 + T cells using the in vitro latency models. Live cells were defined by forward scatter (FSC) versus side scatter (SSC). Uninfected T cells were used to detect any background EGFP expression in our system. EGFP expression was measured against the PE channel. Plots show EGFP expression in mock-infected T cells (uninfected resting CD4 + ) with chemokine treatment (+CCL19) or mock-activated T cells (uninfected activated CD4 + ) and infected T cells (activated CD4 + ). (B) Plots representing EGFP expression following activation. Data are shown as percentages of EGFP in each plot in preactivation (+CCL19 or untreated CD4 + T cells) or postactivation latency. Data are representative of results for a matched sample in one experiment. (C) Frequency of productive infection in infected CD4 + T cells measured by EGFP expression at 5 days postinfection in the preactivation models (CCL19 treated [+] [red open circles] and untreated [−] [blue open circles]) and at day 7 in the postactivation model (black open circles). (D to F) Induced EGFP expression was measured following stimulation of sorted EGFP − cells in preactivation latency with CCL19 (D) or without CCL19 (E) and in postactivation latency (F), using monocytes (mono), monocytes and anti-CD3 (20 μg/ml) (mono+aCD3), PHA (10 μg/ml) and PMA (50 ng/ml) (PMA/PHA), or PMA and ionomycin (500 ng/ml) (PMA/Iono) as well as plate-bound anti-CD3 (20 μg/ml) and anti-CD28 (3.6 μg/ml) (aCD3/aCD28) stimulation or culturing with antiretrovirals only (Unstim). EGFP expression was measured 72 h after coculture. Each dot represents data for a single donor, and the box plots show 25th and 75th percentiles, medians, and ranges. *, P ≤ 0.05; **, P ≤ 0.01 (as determined by a Wilcoxon matched-pairs signed-rank test).

    Techniques Used: Infection, Flow Cytometry, Cytometry, Expressing, In Vitro, Activation Assay

    Cellular proliferation and virus expression from sorted EGFP − cells following stimulation. (A) Levels of cell proliferation following treatment with different stimuli. The sorted EGFP − T cells in the preactivation models (CCL19 treated [+] [open red circles] and untreated [−] [open blue circles]) and postactivation models were stained with proliferation dye (eFluor670) and then cocultured with allogeneic monocytes, with and without anti-CD3, plate-bound anti-CD/anti-CD28, or PMA-PHA and PMA-ionomycin, or with an antiretroviral alone (unstim). At 72 h postactivation, cellular proliferation was measured by FCM. Each point represents data for an individual donor; the box plots show 25th and 75th percentiles, medians, and ranges. *, P ≤ 0.05 (as determined by a Wilcoxon matched-pairs signed-rank test). (B) EGFP expression in nonproliferating (eFluor670 hi ) and proliferating (eFluor670 lo ) cells under each stimulation condition. Each point represents data for a single donor. *, P ≤ 0.05; ***, P ≤ 0.001 (as determined by a Wilcoxon matched-pairs signed-rank test). The median is shown as a blue line. (C) Correlation between EGFP expression and cell proliferation in pre- and postactivation latency, determined by using Spearman's rank test. (D) Correlation between the frequency of EGFP expression in nonproliferating (eFluor670 hi ) and proliferating (eFluor670 lo ) cells in both models. Data from all cultures were pooled, and the level of EGFP expression in nonproliferating (eFlour670 hi ) cells was plotted against the level of EGFP expression in proliferating (eFluor670 lo ) cells in both models.
    Figure Legend Snippet: Cellular proliferation and virus expression from sorted EGFP − cells following stimulation. (A) Levels of cell proliferation following treatment with different stimuli. The sorted EGFP − T cells in the preactivation models (CCL19 treated [+] [open red circles] and untreated [−] [open blue circles]) and postactivation models were stained with proliferation dye (eFluor670) and then cocultured with allogeneic monocytes, with and without anti-CD3, plate-bound anti-CD/anti-CD28, or PMA-PHA and PMA-ionomycin, or with an antiretroviral alone (unstim). At 72 h postactivation, cellular proliferation was measured by FCM. Each point represents data for an individual donor; the box plots show 25th and 75th percentiles, medians, and ranges. *, P ≤ 0.05 (as determined by a Wilcoxon matched-pairs signed-rank test). (B) EGFP expression in nonproliferating (eFluor670 hi ) and proliferating (eFluor670 lo ) cells under each stimulation condition. Each point represents data for a single donor. *, P ≤ 0.05; ***, P ≤ 0.001 (as determined by a Wilcoxon matched-pairs signed-rank test). The median is shown as a blue line. (C) Correlation between EGFP expression and cell proliferation in pre- and postactivation latency, determined by using Spearman's rank test. (D) Correlation between the frequency of EGFP expression in nonproliferating (eFluor670 hi ) and proliferating (eFluor670 lo ) cells in both models. Data from all cultures were pooled, and the level of EGFP expression in nonproliferating (eFlour670 hi ) cells was plotted against the level of EGFP expression in proliferating (eFluor670 lo ) cells in both models.

    Techniques Used: Expressing, Staining

    Postsort spontaneous EGFP expression is associated with higher-level productive infection. (A) The level of spontaneous EGFP expression from infected cells was measured 72 h after coculture of sorted EGFP − cells with antiretrovirals only. Each point represents data for an individual donor; the box plots show 25th and 75th percentiles, medians, and ranges. ****, P ≤ 0.0001 (as determined by a Mann-Whitney test). (B) Correlation between EGFP + productively infected T cells and EGFP expression from sorted EGFP − cells (spontaneous expression) 72 h after stimulation with monocytes, monocytes–anti-CD3 (20 μg/ml), PHA (10 μg/ml)–PMA (50 ng/ml), PMA–ionomycin (500 ng/ml), plate-coated anti-CD3 (20 μg/ml)/anti-CD28 (3.6 μg/ml), or antiretrovirals only. (C) Comparison of induced EGFP expression after stimulation and spontaneous expression (unstimulated expression). (D) Correlation between EGFP + productively infected cells and induced EGFP expression. Each point represents data for a single donor; correlations were determined by Spearman's rank test.
    Figure Legend Snippet: Postsort spontaneous EGFP expression is associated with higher-level productive infection. (A) The level of spontaneous EGFP expression from infected cells was measured 72 h after coculture of sorted EGFP − cells with antiretrovirals only. Each point represents data for an individual donor; the box plots show 25th and 75th percentiles, medians, and ranges. ****, P ≤ 0.0001 (as determined by a Mann-Whitney test). (B) Correlation between EGFP + productively infected T cells and EGFP expression from sorted EGFP − cells (spontaneous expression) 72 h after stimulation with monocytes, monocytes–anti-CD3 (20 μg/ml), PHA (10 μg/ml)–PMA (50 ng/ml), PMA–ionomycin (500 ng/ml), plate-coated anti-CD3 (20 μg/ml)/anti-CD28 (3.6 μg/ml), or antiretrovirals only. (C) Comparison of induced EGFP expression after stimulation and spontaneous expression (unstimulated expression). (D) Correlation between EGFP + productively infected cells and induced EGFP expression. Each point represents data for a single donor; correlations were determined by Spearman's rank test.

    Techniques Used: Expressing, Infection, MANN-WHITNEY

    34) Product Images from "Identification of a human CD8+ regulatory T cell subset that mediates suppression through the chemokine CC chemokine ligand 4"

    Article Title: Identification of a human CD8+ regulatory T cell subset that mediates suppression through the chemokine CC chemokine ligand 4

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

    doi: 10.1073/pnas.0702257104

    In vitro stimulation of PBMCs of primed donors induces T cells with suppressive capacities. PBMCs from PPD responder ( a–c , e , and f ) and nonresponder ( d ) donors were stimulated with live bacillus Calmette–Guérin ( c , d , and f ) or TT ( e ), or left unstimulated ( b and e ); on day 6, IL-2 was added, and on day 13, suppression was tested. Cells were added to autologous PBMCs in the presence of PHA (white bars) or PPD (black bars). After 5 days, proliferation was measured by [ 3 H]TdR incorporation. Stimulation indices (S.I.s) were calculated by dividing PHA- or PPD-induced proliferation (CPM, counts per minute) by unstimulated values. Bars represent the mean + standard error of triplicate wells. Data are shown for one representative donor (of six tested). PBMCs stimulated with TT and live bacillus Calmette–Guérin, and antigen-unstimulated, IL-2-activated cells, were tested to inhibit proliferation of a CD4 + T cell clone stimulated with its cognate peptide. [ 3 H]TdR incorporation was measured at day 3. ( e ) Suppression as measured by CFSE dilution. PKH-26-labeled Tregs were excluded to analyze CFSE dilution of responder cells on day 6. Data are expressed as the geometric mean of the total minus the geometric mean of the undivided population. Data from PPD- or PHA-stimulated samples are divided by those from unstimulated samples to obtain an S.I. Data are shown of one representative donor of three tested.
    Figure Legend Snippet: In vitro stimulation of PBMCs of primed donors induces T cells with suppressive capacities. PBMCs from PPD responder ( a–c , e , and f ) and nonresponder ( d ) donors were stimulated with live bacillus Calmette–Guérin ( c , d , and f ) or TT ( e ), or left unstimulated ( b and e ); on day 6, IL-2 was added, and on day 13, suppression was tested. Cells were added to autologous PBMCs in the presence of PHA (white bars) or PPD (black bars). After 5 days, proliferation was measured by [ 3 H]TdR incorporation. Stimulation indices (S.I.s) were calculated by dividing PHA- or PPD-induced proliferation (CPM, counts per minute) by unstimulated values. Bars represent the mean + standard error of triplicate wells. Data are shown for one representative donor (of six tested). PBMCs stimulated with TT and live bacillus Calmette–Guérin, and antigen-unstimulated, IL-2-activated cells, were tested to inhibit proliferation of a CD4 + T cell clone stimulated with its cognate peptide. [ 3 H]TdR incorporation was measured at day 3. ( e ) Suppression as measured by CFSE dilution. PKH-26-labeled Tregs were excluded to analyze CFSE dilution of responder cells on day 6. Data are expressed as the geometric mean of the total minus the geometric mean of the undivided population. Data from PPD- or PHA-stimulated samples are divided by those from unstimulated samples to obtain an S.I. Data are shown of one representative donor of three tested.

    Techniques Used: In Vitro, Labeling

    CD8 + LAG-3 + T cells inhibit proliferation, and supernatants from bacillus Calmette–Guérin-stimulated cells from responders can transfer suppression. ( a ) PBMCs were stimulated with live bacillus Calmette–Guérin; on day 6, CD4 + and CD8 + cells were separated, stained for LAG-3, and FACS sorted; CD8 + LAG-3 + cells were added to autologous PBMCs in the presence of PHA or PPD. Proliferation was measured 5 days later by [ 3 H]TdR incorporation. ( b ) PBMCs from both PPD responder and nonresponder healthy donors were stimulated with live bacillus Calmette–Guérin or left unstimulated. At day 6, the capacity of supernatants to suppress the CD4 + T cell clone was assessed, by the addition of 50 μl of supernatant in a total of 200 μl, and after 3 days [ 3 H]TdR incorporation was measured. For comparisons, a suppressor ratio was calculated by dividing the response of bacillus Calmette–Guérin supernatants by the response of unstimulated supernatants. A ratio
    Figure Legend Snippet: CD8 + LAG-3 + T cells inhibit proliferation, and supernatants from bacillus Calmette–Guérin-stimulated cells from responders can transfer suppression. ( a ) PBMCs were stimulated with live bacillus Calmette–Guérin; on day 6, CD4 + and CD8 + cells were separated, stained for LAG-3, and FACS sorted; CD8 + LAG-3 + cells were added to autologous PBMCs in the presence of PHA or PPD. Proliferation was measured 5 days later by [ 3 H]TdR incorporation. ( b ) PBMCs from both PPD responder and nonresponder healthy donors were stimulated with live bacillus Calmette–Guérin or left unstimulated. At day 6, the capacity of supernatants to suppress the CD4 + T cell clone was assessed, by the addition of 50 μl of supernatant in a total of 200 μl, and after 3 days [ 3 H]TdR incorporation was measured. For comparisons, a suppressor ratio was calculated by dividing the response of bacillus Calmette–Guérin supernatants by the response of unstimulated supernatants. A ratio

    Techniques Used: Staining, FACS

    Both exogenous and endogenous CCL4 can inhibit T cell proliferation. ( a ) PBMCs were stimulated in the presence of CCL4, and proliferation was measured by [ 3 H]TdR incorporation on day 5. Data are shown of one representative donor (of three tested). ( Right ) The effect of CCL4 on a CD4 + T cell clone stimulated with peptide, representative of three T cell clones. Data from stimulated samples are divided by those from unstimulated samples to obtain a stimulation index (S.I.). Bars represent the mean + standard error of triplicate wells. ( b ) PBMCs of PPD responders were stimulated with live bacillus Calmette–Guérin for 13 days and a suppression assay was performed on PHA-stimulated autologous PBMCs. Blocking CCL3, CCL4 antibodies or isotype controls were added. Proliferation was measured by [ 3 H]TdR incorporation at day 5. PHA-induced proliferation, corrected for unstimulated values, was set at 100%. The percentage blocking was calculated after a natural logarithmic transformation of the stimulation index (S.I.), and inhibition of proliferation in the presence and absence of antibodies was calculated and expressed as percentage. Blocking by CCL4 antibodies was 39–59% for 1:12–1:3 Tregs, respectively. Data are shown of one representative donor of three donors tested. In all individual experiments and in the combined results, significant, although not complete, inhibition with anti-CCL4 was observed ( P = 0.008, Wilcoxon signed ranks test). Bars represent the mean + standard error of triplicate wells.
    Figure Legend Snippet: Both exogenous and endogenous CCL4 can inhibit T cell proliferation. ( a ) PBMCs were stimulated in the presence of CCL4, and proliferation was measured by [ 3 H]TdR incorporation on day 5. Data are shown of one representative donor (of three tested). ( Right ) The effect of CCL4 on a CD4 + T cell clone stimulated with peptide, representative of three T cell clones. Data from stimulated samples are divided by those from unstimulated samples to obtain a stimulation index (S.I.). Bars represent the mean + standard error of triplicate wells. ( b ) PBMCs of PPD responders were stimulated with live bacillus Calmette–Guérin for 13 days and a suppression assay was performed on PHA-stimulated autologous PBMCs. Blocking CCL3, CCL4 antibodies or isotype controls were added. Proliferation was measured by [ 3 H]TdR incorporation at day 5. PHA-induced proliferation, corrected for unstimulated values, was set at 100%. The percentage blocking was calculated after a natural logarithmic transformation of the stimulation index (S.I.), and inhibition of proliferation in the presence and absence of antibodies was calculated and expressed as percentage. Blocking by CCL4 antibodies was 39–59% for 1:12–1:3 Tregs, respectively. Data are shown of one representative donor of three donors tested. In all individual experiments and in the combined results, significant, although not complete, inhibition with anti-CCL4 was observed ( P = 0.008, Wilcoxon signed ranks test). Bars represent the mean + standard error of triplicate wells.

    Techniques Used: Clone Assay, Suppression Assay, Blocking Assay, Transformation Assay, Inhibition

    35) Product Images from "A Novel Neuraminidase Virus-Like Particle Vaccine Offers Protection Against Heterologous H3N2 Influenza Virus Infection in the Porcine Model"

    Article Title: A Novel Neuraminidase Virus-Like Particle Vaccine Offers Protection Against Heterologous H3N2 Influenza Virus Infection in the Porcine Model

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2022.915364

    Vaccination with NA2 VLPS decreases TNF-α secreting T cells compared to mock vaccination following infection with A/swine/NC/KH1552516/2016 virus. PBMCs from pigs vaccinated in a prime-boost regimen 21 days apart with a QWIV, NA2 VLPs, or mock vaccinated with water-in-oil-in-water adjuvant were collected at 21 and 35 days and re-stimulated with A/swine/NC/KH1552516/2016 virus or PHA for enumeration of cytokine secreting cells. They were tested for IFN-γ secreting cells following (A) virus re-stimulation. (B) PHA re-stimulation. (C) no stimulation (D) ratio of virus vs PHA stimulated cells They were tested for TNF-α secreting cells following (E) virus re-stimulation. (F) PHA re-stimulation. (G) no stimulation (H) ratio of virus vs PHA stimulated cells. Pink squares represent NA VLP vaccinated pigs, cyan triangkes represent commercial quadrivalent whole inactivated virus vaccinated pigs (QWIV), and black circles represent mock vaccinated pigs. Horizontal lines represent the mean for each group. Statistical analysis was performed by two-way ANOVA test. Error bars represent SEM. ** = p
    Figure Legend Snippet: Vaccination with NA2 VLPS decreases TNF-α secreting T cells compared to mock vaccination following infection with A/swine/NC/KH1552516/2016 virus. PBMCs from pigs vaccinated in a prime-boost regimen 21 days apart with a QWIV, NA2 VLPs, or mock vaccinated with water-in-oil-in-water adjuvant were collected at 21 and 35 days and re-stimulated with A/swine/NC/KH1552516/2016 virus or PHA for enumeration of cytokine secreting cells. They were tested for IFN-γ secreting cells following (A) virus re-stimulation. (B) PHA re-stimulation. (C) no stimulation (D) ratio of virus vs PHA stimulated cells They were tested for TNF-α secreting cells following (E) virus re-stimulation. (F) PHA re-stimulation. (G) no stimulation (H) ratio of virus vs PHA stimulated cells. Pink squares represent NA VLP vaccinated pigs, cyan triangkes represent commercial quadrivalent whole inactivated virus vaccinated pigs (QWIV), and black circles represent mock vaccinated pigs. Horizontal lines represent the mean for each group. Statistical analysis was performed by two-way ANOVA test. Error bars represent SEM. ** = p

    Techniques Used: Infection

    36) Product Images from "Immunity to the melanoma inhibitor of apoptosis protein (ML-IAP; livin) in patients with malignant melanoma"

    Article Title: Immunity to the melanoma inhibitor of apoptosis protein (ML-IAP; livin) in patients with malignant melanoma

    Journal: Cancer Immunology, Immunotherapy

    doi: 10.1007/s00262-011-1124-1

    Generation of CTL to ML-IAP HLA-A*0201 epitopes. PBMC from HLA-A*0201 donors underwent multiple rounds of stimulation with CD40L-activated B cells pulsed with IAP237 peptide. CD8 + cells were further enriched by IFN-γ selection and the cells expanded in the presence of irradiated PBMC, PHA, and IL-2. a ELISPOT assay for γ-IFN production proved specific IAP237 CD8 + CTL ( left upper panel ). Specificity to HLA-A*0201 activity was further demonstrated by the ability to block activity with the addition of the anti-class I antibody W6/32 ( left lower panel ). Functional cytotoxicity of manufactured cells was further revealed by CTL assay utilizing T2 cells in the presence of 10 μg/ml IAP237 peptide ( right panel ). The effector to target ratio (E/T) is represented on the x -axis and percent cytotoxicity is represented on the y -axis. b The cytolytic activity of IAP237 CTL were further analyzed against HLA-A2 expressing melanoma cell lines in the absence ( left panel ) or presence of 10 μg/ml IAP237 peptide ( right panel ). Melanoma cell line K029 expresses ML-IAP while cell lines K008 and K028 have very low level of ML-IAP expression. Cell line K008 has lost surface HLA-A2 expression as determined by flow cytometry
    Figure Legend Snippet: Generation of CTL to ML-IAP HLA-A*0201 epitopes. PBMC from HLA-A*0201 donors underwent multiple rounds of stimulation with CD40L-activated B cells pulsed with IAP237 peptide. CD8 + cells were further enriched by IFN-γ selection and the cells expanded in the presence of irradiated PBMC, PHA, and IL-2. a ELISPOT assay for γ-IFN production proved specific IAP237 CD8 + CTL ( left upper panel ). Specificity to HLA-A*0201 activity was further demonstrated by the ability to block activity with the addition of the anti-class I antibody W6/32 ( left lower panel ). Functional cytotoxicity of manufactured cells was further revealed by CTL assay utilizing T2 cells in the presence of 10 μg/ml IAP237 peptide ( right panel ). The effector to target ratio (E/T) is represented on the x -axis and percent cytotoxicity is represented on the y -axis. b The cytolytic activity of IAP237 CTL were further analyzed against HLA-A2 expressing melanoma cell lines in the absence ( left panel ) or presence of 10 μg/ml IAP237 peptide ( right panel ). Melanoma cell line K029 expresses ML-IAP while cell lines K008 and K028 have very low level of ML-IAP expression. Cell line K008 has lost surface HLA-A2 expression as determined by flow cytometry

    Techniques Used: Selection, Irradiation, Enzyme-linked Immunospot, Activity Assay, Blocking Assay, Functional Assay, CTL Assay, Expressing, Flow Cytometry

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    Thermo Fisher pha
    WJ-MSC inhibit CD3 + CD4 + and CD3 + CD8 + T cell proliferation. WJ-MSC were seeded, after 24 hours. <t>PBMC</t> was added and stimulated with <t>PHA</t> for 3 days in the presence of WJ-MSC; the ratio used was 1 : 10 (WJ-MSC : PBMC). The WJ-MSC passages used in experiments were either P5 or P6. T cells were collected, stained with anti-CD3, anti-CD4, and anti-CD8 antibodies, and proliferation was measured by flow cytometry using the Click It Kit. (a) % proliferation of CD3 + CD4 + Click It + T cell and (b) % proliferation of CD3 + CD8 + Click It + T cell. The percentage of inhibition (Inhib) was calculated using the percentage of proliferation of PBMC + PHA + WJ-MSC compared to the control PBMC + PHA. Experiments were performed in triplicate. Results are represented by mean ± SD. Statistically significant differences are shown as ∗∗∗ p
    Pha, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 2 article reviews
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    Thermo Fisher pha l
    Multi-dimensional experiment combining in a rat dual-anterograde tracing with <t>PHA-L</t> (blue) and BDA (red) with retrograde tracing using rabies virus (RV; green). Here, we wanted to evaluate whether corticostriatal and thalamostriatal projections converge onto striatofugal neurons. PHA-L and BDA were iontophoretically delivered into the primary motor cortex and parafascicular thalamic nucleus, respectively. RV was pressure-delivered into either the external globus pallidus or in the substantia nigra pars compacta. a Striatopallidal; B striatonigral neuron. The insets show details. Detection of RV was carried out with an antibody against a soluble rabies phosphoprotein, resulting in Golgi-silver impregnation like labeling of dendrites, with details like dendritic spines
    Pha L, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    WJ-MSC inhibit CD3 + CD4 + and CD3 + CD8 + T cell proliferation. WJ-MSC were seeded, after 24 hours. PBMC was added and stimulated with PHA for 3 days in the presence of WJ-MSC; the ratio used was 1 : 10 (WJ-MSC : PBMC). The WJ-MSC passages used in experiments were either P5 or P6. T cells were collected, stained with anti-CD3, anti-CD4, and anti-CD8 antibodies, and proliferation was measured by flow cytometry using the Click It Kit. (a) % proliferation of CD3 + CD4 + Click It + T cell and (b) % proliferation of CD3 + CD8 + Click It + T cell. The percentage of inhibition (Inhib) was calculated using the percentage of proliferation of PBMC + PHA + WJ-MSC compared to the control PBMC + PHA. Experiments were performed in triplicate. Results are represented by mean ± SD. Statistically significant differences are shown as ∗∗∗ p

    Journal: Stem Cells International

    Article Title: Intrinsic Variability Present in Wharton's Jelly Mesenchymal Stem Cells and T Cell Responses May Impact Cell Therapy

    doi: 10.1155/2017/8492797

    Figure Lengend Snippet: WJ-MSC inhibit CD3 + CD4 + and CD3 + CD8 + T cell proliferation. WJ-MSC were seeded, after 24 hours. PBMC was added and stimulated with PHA for 3 days in the presence of WJ-MSC; the ratio used was 1 : 10 (WJ-MSC : PBMC). The WJ-MSC passages used in experiments were either P5 or P6. T cells were collected, stained with anti-CD3, anti-CD4, and anti-CD8 antibodies, and proliferation was measured by flow cytometry using the Click It Kit. (a) % proliferation of CD3 + CD4 + Click It + T cell and (b) % proliferation of CD3 + CD8 + Click It + T cell. The percentage of inhibition (Inhib) was calculated using the percentage of proliferation of PBMC + PHA + WJ-MSC compared to the control PBMC + PHA. Experiments were performed in triplicate. Results are represented by mean ± SD. Statistically significant differences are shown as ∗∗∗ p

    Article Snippet: Cells were then incubated for 72 h with 3×105 PBMC (ratio 1 : 10) in the absence or presence of PHA (Gibco, Carlsbad, CA).

    Techniques: Staining, Flow Cytometry, Cytometry, Inhibition

    WJ-MSC licensed with IFN- γ inhibit CD3 + T cell proliferation. WJ-MSC were seeded, and IFN- γ was added for 24 hours. PBMC were stimulated with PHA for 3 days in the presence of WJ-MSC; the ratio used was 1 : 10 (WJ-MSC : PBMC). The WJ-MSC passages used in the experiments were P5 and P10. T cells were collected, stained with anti-CD3, anti-CD4, and anti-CD8 antibodies, and proliferation was measured by flow cytometry using the Click It Kit. The percentage of inhibition (Inhib) was calculated using the percentage of proliferation of PBMC + PHA + WJ-MSC compared to the control PBMC + PHA. Experiments were performed in triplicate. Results are represented by mean ± SD. Statistically significant differences are shown as ∗ p

    Journal: Stem Cells International

    Article Title: Intrinsic Variability Present in Wharton's Jelly Mesenchymal Stem Cells and T Cell Responses May Impact Cell Therapy

    doi: 10.1155/2017/8492797

    Figure Lengend Snippet: WJ-MSC licensed with IFN- γ inhibit CD3 + T cell proliferation. WJ-MSC were seeded, and IFN- γ was added for 24 hours. PBMC were stimulated with PHA for 3 days in the presence of WJ-MSC; the ratio used was 1 : 10 (WJ-MSC : PBMC). The WJ-MSC passages used in the experiments were P5 and P10. T cells were collected, stained with anti-CD3, anti-CD4, and anti-CD8 antibodies, and proliferation was measured by flow cytometry using the Click It Kit. The percentage of inhibition (Inhib) was calculated using the percentage of proliferation of PBMC + PHA + WJ-MSC compared to the control PBMC + PHA. Experiments were performed in triplicate. Results are represented by mean ± SD. Statistically significant differences are shown as ∗ p

    Article Snippet: Cells were then incubated for 72 h with 3×105 PBMC (ratio 1 : 10) in the absence or presence of PHA (Gibco, Carlsbad, CA).

    Techniques: Staining, Flow Cytometry, Cytometry, Inhibition

    Gene expression of IL-10 and IDO is increased in WJ-MSC after coculture with PBMC. WJ-MSC were seeded, after 24 hours. PBMC was added and stimulated with PHA for 3 days in the presence of MSC; the ratio used was 1 : 10 (MSC : PBMC). The WJ-MSC passages used in the experiments were P5 and P10. After 3 days, WJ-MSC were lysed, total RNA extracted, and real-time PCR performed. (a) IDO and (b) IL-10 gene expression of WJ-MSC1, WJ-MSC2, and WJ-MSC3. Gene expression was normalized by GAPDH and expressed as fold change compared to the control WJ-MSC. Experiments were performed in triplicate. Results are represented as mean ± SD. Statistically significant differences are shown as ∗ p

    Journal: Stem Cells International

    Article Title: Intrinsic Variability Present in Wharton's Jelly Mesenchymal Stem Cells and T Cell Responses May Impact Cell Therapy

    doi: 10.1155/2017/8492797

    Figure Lengend Snippet: Gene expression of IL-10 and IDO is increased in WJ-MSC after coculture with PBMC. WJ-MSC were seeded, after 24 hours. PBMC was added and stimulated with PHA for 3 days in the presence of MSC; the ratio used was 1 : 10 (MSC : PBMC). The WJ-MSC passages used in the experiments were P5 and P10. After 3 days, WJ-MSC were lysed, total RNA extracted, and real-time PCR performed. (a) IDO and (b) IL-10 gene expression of WJ-MSC1, WJ-MSC2, and WJ-MSC3. Gene expression was normalized by GAPDH and expressed as fold change compared to the control WJ-MSC. Experiments were performed in triplicate. Results are represented as mean ± SD. Statistically significant differences are shown as ∗ p

    Article Snippet: Cells were then incubated for 72 h with 3×105 PBMC (ratio 1 : 10) in the absence or presence of PHA (Gibco, Carlsbad, CA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    WJ-MSC inhibit CD3 + T cell proliferation. WJ-MSC were seeded, after 24 hours. PBMC were added and stimulated with PHA for 3 days in the presence of MSC; the ratio used was 1 : 10 (WJ-MSC : PBMC). The WJ-MSC passages used in experiments were either P5 or P6. T cells were collected and stained with anti-CD3 antibody and proliferation measured by flow cytometry using the Click It Kit. The percentage of inhibition (Inhib) was calculated using the percentage of proliferation of PBMC + PHA + WJ-MSC compared to the control PBMC + PHA. Experiments were performed in triplicate. Results are represented by mean ± SD. Statistically significant differences are shown as ∗∗∗ p

    Journal: Stem Cells International

    Article Title: Intrinsic Variability Present in Wharton's Jelly Mesenchymal Stem Cells and T Cell Responses May Impact Cell Therapy

    doi: 10.1155/2017/8492797

    Figure Lengend Snippet: WJ-MSC inhibit CD3 + T cell proliferation. WJ-MSC were seeded, after 24 hours. PBMC were added and stimulated with PHA for 3 days in the presence of MSC; the ratio used was 1 : 10 (WJ-MSC : PBMC). The WJ-MSC passages used in experiments were either P5 or P6. T cells were collected and stained with anti-CD3 antibody and proliferation measured by flow cytometry using the Click It Kit. The percentage of inhibition (Inhib) was calculated using the percentage of proliferation of PBMC + PHA + WJ-MSC compared to the control PBMC + PHA. Experiments were performed in triplicate. Results are represented by mean ± SD. Statistically significant differences are shown as ∗∗∗ p

    Article Snippet: Cells were then incubated for 72 h with 3×105 PBMC (ratio 1 : 10) in the absence or presence of PHA (Gibco, Carlsbad, CA).

    Techniques: Staining, Flow Cytometry, Cytometry, Inhibition

    Inhibitory effect of human mesenchymal stem cells (hMSCs) on the proliferation of phytohemagglutinin (PHA)-CD3+ T cells as detected by carboxyfluorescein succinimidyl ester (CFSE). (A) Representative analysis of the altered rate of PHA-CD3+ T cells in the mixed lymphocyte reactions (MLR) and transwell experiments when co-cultured with hMSCs was performed by CFSE labeling at days 1 and 4. Solid purple histogram represents data of T cell-only culture, while green and pink peaks show the result of direct (MLR) and indirect (transwell) culture, respectively. (B) Comparative analyses of three cell cultures indicate that hBM-MSCs are the most effective cell type in the repression of induced T-cell proliferation. (C) Graphical representation of the cell proliferation in different cell cultures supports the results of previous data (n=3, mean ± standard deviation; Δ p

    Journal: Turkish Journal of Hematology

    Article Title: Comparative Analyses of Immunosuppressive Characteristics of Bone-Marrow, Wharton’s Jelly, and Adipose Tissue-Derived Human Mesenchymal Stem Cells

    doi: 10.4274/tjh.2016.0171

    Figure Lengend Snippet: Inhibitory effect of human mesenchymal stem cells (hMSCs) on the proliferation of phytohemagglutinin (PHA)-CD3+ T cells as detected by carboxyfluorescein succinimidyl ester (CFSE). (A) Representative analysis of the altered rate of PHA-CD3+ T cells in the mixed lymphocyte reactions (MLR) and transwell experiments when co-cultured with hMSCs was performed by CFSE labeling at days 1 and 4. Solid purple histogram represents data of T cell-only culture, while green and pink peaks show the result of direct (MLR) and indirect (transwell) culture, respectively. (B) Comparative analyses of three cell cultures indicate that hBM-MSCs are the most effective cell type in the repression of induced T-cell proliferation. (C) Graphical representation of the cell proliferation in different cell cultures supports the results of previous data (n=3, mean ± standard deviation; Δ p

    Article Snippet: CD3+ T cells were stimulated with 10 µg/mL mitogen PHA (Invitrogen/GIBCO) for 24 h in RPMI-1640 medium (Invitrogen/GIBCO) containing 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 200 mM glutamax (Invitrogen/GIBCO).

    Techniques: Cell Culture, Labeling, Standard Deviation

    Representative panels of immunofluorescence in phytohemagglutinin (PHA)-CD3+ T cells. Fluorescence microscopy analysis of the expression of cell surface markers staining for CD3 (A), CD5 (B), and CD8 (C) was positive whereas it was negative for CD23 (D), CD29 (E), and CD105 (F) in PHA-CD3+ T cells (scale bars=50 µm).

    Journal: Turkish Journal of Hematology

    Article Title: Comparative Analyses of Immunosuppressive Characteristics of Bone-Marrow, Wharton’s Jelly, and Adipose Tissue-Derived Human Mesenchymal Stem Cells

    doi: 10.4274/tjh.2016.0171

    Figure Lengend Snippet: Representative panels of immunofluorescence in phytohemagglutinin (PHA)-CD3+ T cells. Fluorescence microscopy analysis of the expression of cell surface markers staining for CD3 (A), CD5 (B), and CD8 (C) was positive whereas it was negative for CD23 (D), CD29 (E), and CD105 (F) in PHA-CD3+ T cells (scale bars=50 µm).

    Article Snippet: CD3+ T cells were stimulated with 10 µg/mL mitogen PHA (Invitrogen/GIBCO) for 24 h in RPMI-1640 medium (Invitrogen/GIBCO) containing 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 200 mM glutamax (Invitrogen/GIBCO).

    Techniques: Immunofluorescence, Fluorescence, Microscopy, Expressing, Staining

    Alterations of cytokine levels in mesenchymal stem cells (MSCs) and phytohemagglutinin (PHA)-CD3+ T cells after co-culture supernatants were analyzed by ELISA. Pro-inflammatory cytokines interferon-γ, tumor necrosis factor-α, and interleukin-2 (IL-2) were inhibited significantly by MSCs in mixed lymphocyte reactions and transwell experiments on day 4. Transforming growth factor-β and IL-6 were significantly increased in both experiments on day 4 after co-culture with PHA-CD3+ T cells (n=3, mean ± standard deviation; # p

    Journal: Turkish Journal of Hematology

    Article Title: Comparative Analyses of Immunosuppressive Characteristics of Bone-Marrow, Wharton’s Jelly, and Adipose Tissue-Derived Human Mesenchymal Stem Cells

    doi: 10.4274/tjh.2016.0171

    Figure Lengend Snippet: Alterations of cytokine levels in mesenchymal stem cells (MSCs) and phytohemagglutinin (PHA)-CD3+ T cells after co-culture supernatants were analyzed by ELISA. Pro-inflammatory cytokines interferon-γ, tumor necrosis factor-α, and interleukin-2 (IL-2) were inhibited significantly by MSCs in mixed lymphocyte reactions and transwell experiments on day 4. Transforming growth factor-β and IL-6 were significantly increased in both experiments on day 4 after co-culture with PHA-CD3+ T cells (n=3, mean ± standard deviation; # p

    Article Snippet: CD3+ T cells were stimulated with 10 µg/mL mitogen PHA (Invitrogen/GIBCO) for 24 h in RPMI-1640 medium (Invitrogen/GIBCO) containing 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 200 mM glutamax (Invitrogen/GIBCO).

    Techniques: Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Activation of CD3+ T cells by phytohemagglutinin (PHA). Phase-contrast microscopy showing that as CD3+ T cells (A) were activated by PHA-M for 24 h, the morphologies and shapes of the cells changed, and they presented extensions (B, C). Activation of CD3+ T cells (by PHA) was determined by WST-1 (D). Increased levels of proliferation (of PHA-CD3+ T cells) were determined by WST-1 (n=3, mean ± SE, p

    Journal: Turkish Journal of Hematology

    Article Title: Comparative Analyses of Immunosuppressive Characteristics of Bone-Marrow, Wharton’s Jelly, and Adipose Tissue-Derived Human Mesenchymal Stem Cells

    doi: 10.4274/tjh.2016.0171

    Figure Lengend Snippet: Activation of CD3+ T cells by phytohemagglutinin (PHA). Phase-contrast microscopy showing that as CD3+ T cells (A) were activated by PHA-M for 24 h, the morphologies and shapes of the cells changed, and they presented extensions (B, C). Activation of CD3+ T cells (by PHA) was determined by WST-1 (D). Increased levels of proliferation (of PHA-CD3+ T cells) were determined by WST-1 (n=3, mean ± SE, p

    Article Snippet: CD3+ T cells were stimulated with 10 µg/mL mitogen PHA (Invitrogen/GIBCO) for 24 h in RPMI-1640 medium (Invitrogen/GIBCO) containing 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 200 mM glutamax (Invitrogen/GIBCO).

    Techniques: Activation Assay, Microscopy

    T cell proliferation of US and anti-CD3 PA PBMC in response to anti-CD3, IL-2 and other mitogens. Proliferative response of US and PA PBMC to anti-CD3, IL-2 + anti-CD3, PHA or Con A. [ 3 H]-thymidine uptake is given in cpm (* P

    Journal: Clinical and Experimental Immunology

    Article Title: Functional and molecular aspects of transient T cell unresponsiveness: role of selective interleukin-2 deficiency

    doi: 10.1046/j.1365-2249.2003.02150.x

    Figure Lengend Snippet: T cell proliferation of US and anti-CD3 PA PBMC in response to anti-CD3, IL-2 and other mitogens. Proliferative response of US and PA PBMC to anti-CD3, IL-2 + anti-CD3, PHA or Con A. [ 3 H]-thymidine uptake is given in cpm (* P

    Article Snippet: Cells were either cultured with medium alone or restimulated with anti-CD3 (10 ng/ml), IL-2 (40 U/ml; Genzyme, Cambridge, MA, USA), anti-CD3+ IL-2, PHA (6·25 µg/ml, GIBCO ) or Con A (10 ng/ml; ICN Pharmaceuticals, Costa Mesa, CA, USA).

    Techniques:

    Multi-dimensional experiment combining in a rat dual-anterograde tracing with PHA-L (blue) and BDA (red) with retrograde tracing using rabies virus (RV; green). Here, we wanted to evaluate whether corticostriatal and thalamostriatal projections converge onto striatofugal neurons. PHA-L and BDA were iontophoretically delivered into the primary motor cortex and parafascicular thalamic nucleus, respectively. RV was pressure-delivered into either the external globus pallidus or in the substantia nigra pars compacta. a Striatopallidal; B striatonigral neuron. The insets show details. Detection of RV was carried out with an antibody against a soluble rabies phosphoprotein, resulting in Golgi-silver impregnation like labeling of dendrites, with details like dendritic spines

    Journal: Brain Structure & Function

    Article Title: Neuroanatomical tract-tracing techniques that did go viral

    doi: 10.1007/s00429-020-02041-6

    Figure Lengend Snippet: Multi-dimensional experiment combining in a rat dual-anterograde tracing with PHA-L (blue) and BDA (red) with retrograde tracing using rabies virus (RV; green). Here, we wanted to evaluate whether corticostriatal and thalamostriatal projections converge onto striatofugal neurons. PHA-L and BDA were iontophoretically delivered into the primary motor cortex and parafascicular thalamic nucleus, respectively. RV was pressure-delivered into either the external globus pallidus or in the substantia nigra pars compacta. a Striatopallidal; B striatonigral neuron. The insets show details. Detection of RV was carried out with an antibody against a soluble rabies phosphoprotein, resulting in Golgi-silver impregnation like labeling of dendrites, with details like dendritic spines

    Article Snippet: The perforant pathway was labeled in rats via injection of PHA-L in layers II–III of medial parahippocampal cortex, while the hippocampal CA1 neurons were intracellularly injected with Alexa Fluor® 555 (AF555) (Molecular Probes, Eugene, OR).

    Techniques: Anterograde Tracing, Retrograde Tracing, Labeling

    One-dimensional neuroanatomical tracing: a anterograde tracing. Tracers such as PHA-L, BDA, RDA, Biocytin, or WGA-HRP injected in nucleus ‘A’ label complete efferent fiber trajectories including axon terminals in nucleus ‘B’. Neurons in nucleus ‘A’ are labeled irrespective of their neurochemical phenotype. In the injection site projection, neurons and interneurons both become labeled and are indistinguishable from each other. WGA-HRP and BDA are to some degree retrogradely transported (indicated here as granules in the first-order neurons in nucleus ‘B’). b Retrograde tracing. Compounds such as Fluoro-Gold, CTB, RV, and PRV label projection neurons irrespective of their neurochemical phenotype

    Journal: Brain Structure & Function

    Article Title: Neuroanatomical tract-tracing techniques that did go viral

    doi: 10.1007/s00429-020-02041-6

    Figure Lengend Snippet: One-dimensional neuroanatomical tracing: a anterograde tracing. Tracers such as PHA-L, BDA, RDA, Biocytin, or WGA-HRP injected in nucleus ‘A’ label complete efferent fiber trajectories including axon terminals in nucleus ‘B’. Neurons in nucleus ‘A’ are labeled irrespective of their neurochemical phenotype. In the injection site projection, neurons and interneurons both become labeled and are indistinguishable from each other. WGA-HRP and BDA are to some degree retrogradely transported (indicated here as granules in the first-order neurons in nucleus ‘B’). b Retrograde tracing. Compounds such as Fluoro-Gold, CTB, RV, and PRV label projection neurons irrespective of their neurochemical phenotype

    Article Snippet: The perforant pathway was labeled in rats via injection of PHA-L in layers II–III of medial parahippocampal cortex, while the hippocampal CA1 neurons were intracellularly injected with Alexa Fluor® 555 (AF555) (Molecular Probes, Eugene, OR).

    Techniques: Anterograde Tracing, Whole Genome Amplification, Injection, Labeling, Retrograde Tracing, CtB Assay

    Two-dimensional neuroanatomical tracing. High-power confocal laser scanning images of the two-dimensional experiments illustrated in Fig. 3 d, e. a Neurobiotin-labeled dendrite imaged in the green channel. b PHA-L labeled fiber (simultaneously) imaged in the infrared channel. c Merge. Encircled: an ending of a PHA-L labeled fiber (red) contacts a neurobiotin-labeled dendrite (green). d PHA-L-labeled perforant pathway fibers in stratum lacunosum moleculare imaged in the green channel, e AF555-labeled dendrite simultaneously imaged in the red channel. f Merge image; the circles indicate sites where boutons of PHA-L labeled fibers are in contact with the AF555 labeled dendrite. Images a – f are Z-projections. g 3D computer reconstruction made on basis of the images in ( a , b ). H, 3D computer reconstruction made on basis of the images in ( d , e ). Contacts are encircled

    Journal: Brain Structure & Function

    Article Title: Neuroanatomical tract-tracing techniques that did go viral

    doi: 10.1007/s00429-020-02041-6

    Figure Lengend Snippet: Two-dimensional neuroanatomical tracing. High-power confocal laser scanning images of the two-dimensional experiments illustrated in Fig. 3 d, e. a Neurobiotin-labeled dendrite imaged in the green channel. b PHA-L labeled fiber (simultaneously) imaged in the infrared channel. c Merge. Encircled: an ending of a PHA-L labeled fiber (red) contacts a neurobiotin-labeled dendrite (green). d PHA-L-labeled perforant pathway fibers in stratum lacunosum moleculare imaged in the green channel, e AF555-labeled dendrite simultaneously imaged in the red channel. f Merge image; the circles indicate sites where boutons of PHA-L labeled fibers are in contact with the AF555 labeled dendrite. Images a – f are Z-projections. g 3D computer reconstruction made on basis of the images in ( a , b ). H, 3D computer reconstruction made on basis of the images in ( d , e ). Contacts are encircled

    Article Snippet: The perforant pathway was labeled in rats via injection of PHA-L in layers II–III of medial parahippocampal cortex, while the hippocampal CA1 neurons were intracellularly injected with Alexa Fluor® 555 (AF555) (Molecular Probes, Eugene, OR).

    Techniques: Labeling

    Anterograde tracing: injection sites and labeling. a , b Case 2012-6 (rat). In the same surgical session, we injected PHA-L in the central caudate putamen (CPu) of the left (L) cerebral hemisphere, while contralaterally (R), we injected BDA in the caudal CPu. Note significant retrograde transport of BDA-to-cerebral cortical pyramidal neurons ipsilateral to the injection site (dashed ellipse). Injection sites measure approximately 1 mm in diameter. c Low-power fluorescence montage in case FS-95166: injection site of PHA-L in the central nucleus (CE) of the amygdaloid complex. Bmg magnocellular basal amygdaloid nucleus. d Low-magnification montage of a combination of PHA-L tracing and neurobiotin injection. PHA-L-labeled fibers (red; Cy3) in layers II–III of medial parahippocampal cortex in contact with apical dendrites of neurobiotin-labeled pyramidal cells (green) located deep (layer V). LD lamina dissecans. e Low magnification: combination of PHA-L tracing and AF555 intracellular injection. An apical dendrite of an intracellularly AF555 injected hippocampal CA1 pyramidal neuron (red) penetrating stratum lacunosum moleculare (LM) The latter contains a terminal field of PHA-L labeled fibers (Alexa Fluor ® 488; green) belonging to the perforant pathway (PHA-L injected in medial parahippocampal cortex). SR stratum radiatum

    Journal: Brain Structure & Function

    Article Title: Neuroanatomical tract-tracing techniques that did go viral

    doi: 10.1007/s00429-020-02041-6

    Figure Lengend Snippet: Anterograde tracing: injection sites and labeling. a , b Case 2012-6 (rat). In the same surgical session, we injected PHA-L in the central caudate putamen (CPu) of the left (L) cerebral hemisphere, while contralaterally (R), we injected BDA in the caudal CPu. Note significant retrograde transport of BDA-to-cerebral cortical pyramidal neurons ipsilateral to the injection site (dashed ellipse). Injection sites measure approximately 1 mm in diameter. c Low-power fluorescence montage in case FS-95166: injection site of PHA-L in the central nucleus (CE) of the amygdaloid complex. Bmg magnocellular basal amygdaloid nucleus. d Low-magnification montage of a combination of PHA-L tracing and neurobiotin injection. PHA-L-labeled fibers (red; Cy3) in layers II–III of medial parahippocampal cortex in contact with apical dendrites of neurobiotin-labeled pyramidal cells (green) located deep (layer V). LD lamina dissecans. e Low magnification: combination of PHA-L tracing and AF555 intracellular injection. An apical dendrite of an intracellularly AF555 injected hippocampal CA1 pyramidal neuron (red) penetrating stratum lacunosum moleculare (LM) The latter contains a terminal field of PHA-L labeled fibers (Alexa Fluor ® 488; green) belonging to the perforant pathway (PHA-L injected in medial parahippocampal cortex). SR stratum radiatum

    Article Snippet: The perforant pathway was labeled in rats via injection of PHA-L in layers II–III of medial parahippocampal cortex, while the hippocampal CA1 neurons were intracellularly injected with Alexa Fluor® 555 (AF555) (Molecular Probes, Eugene, OR).

    Techniques: Anterograde Tracing, Injection, Labeling, Fluorescence