pbmcs  (Thermo Fisher)


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    Structured Review

    Thermo Fisher pbmcs
    Lack of prelamin A in <t>PBMCs</t> from compliant patients receiving the 2NRTI+1PI/r regimen. ( A) Plasma concentrations of <t>lopinavir,</t> atazanavir and ritonavir from ANRS EP45 “Aging” patients reported at M0 (circles), M12 (square) and M24 (triangle). Colors indicate the durations between last drug uptake and venipuncture (Cmax, red; Cmin, green, Cintermediate, brown) and PI therapeutic concentration range (Cmax, red; Cmin, green). The majority of patients receiving lopinavir treatment were assayed at Cmin and remained within the therapeutic range. Samples from patients being treated with the atazanavir regimen were mainly assayed at mid-dose. ( B ) Representative western blots of PBMCs from patients receiving lopinavir or atazanavir treatment using three different lamin A/C-specific antibodies and one prelamin A-specific antibody. No prelamin A was detected in PBMCs from patients. Control PBMCs from healthy controls incubated with ZoPra were used as positive control cells. PI plasma concentrations in the patients shown: lopinavir 10.7 µM (M0), 12.3 µM (M12) and 9.3 µM (M24); atazanavir 1.5 µM (M0), 0.7 µM (M12) and 0 µM (M24).
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    Images

    1) Product Images from "HIV Protease Inhibitors Do Not Cause the Accumulation of Prelamin A in PBMCs from Patients Receiving First Line Therapy: The ANRS EP45 "Aging" Study"

    Article Title: HIV Protease Inhibitors Do Not Cause the Accumulation of Prelamin A in PBMCs from Patients Receiving First Line Therapy: The ANRS EP45 "Aging" Study

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0053035

    Lack of prelamin A in PBMCs from compliant patients receiving the 2NRTI+1PI/r regimen. ( A) Plasma concentrations of lopinavir, atazanavir and ritonavir from ANRS EP45 “Aging” patients reported at M0 (circles), M12 (square) and M24 (triangle). Colors indicate the durations between last drug uptake and venipuncture (Cmax, red; Cmin, green, Cintermediate, brown) and PI therapeutic concentration range (Cmax, red; Cmin, green). The majority of patients receiving lopinavir treatment were assayed at Cmin and remained within the therapeutic range. Samples from patients being treated with the atazanavir regimen were mainly assayed at mid-dose. ( B ) Representative western blots of PBMCs from patients receiving lopinavir or atazanavir treatment using three different lamin A/C-specific antibodies and one prelamin A-specific antibody. No prelamin A was detected in PBMCs from patients. Control PBMCs from healthy controls incubated with ZoPra were used as positive control cells. PI plasma concentrations in the patients shown: lopinavir 10.7 µM (M0), 12.3 µM (M12) and 9.3 µM (M24); atazanavir 1.5 µM (M0), 0.7 µM (M12) and 0 µM (M24).
    Figure Legend Snippet: Lack of prelamin A in PBMCs from compliant patients receiving the 2NRTI+1PI/r regimen. ( A) Plasma concentrations of lopinavir, atazanavir and ritonavir from ANRS EP45 “Aging” patients reported at M0 (circles), M12 (square) and M24 (triangle). Colors indicate the durations between last drug uptake and venipuncture (Cmax, red; Cmin, green, Cintermediate, brown) and PI therapeutic concentration range (Cmax, red; Cmin, green). The majority of patients receiving lopinavir treatment were assayed at Cmin and remained within the therapeutic range. Samples from patients being treated with the atazanavir regimen were mainly assayed at mid-dose. ( B ) Representative western blots of PBMCs from patients receiving lopinavir or atazanavir treatment using three different lamin A/C-specific antibodies and one prelamin A-specific antibody. No prelamin A was detected in PBMCs from patients. Control PBMCs from healthy controls incubated with ZoPra were used as positive control cells. PI plasma concentrations in the patients shown: lopinavir 10.7 µM (M0), 12.3 µM (M12) and 9.3 µM (M24); atazanavir 1.5 µM (M0), 0.7 µM (M12) and 0 µM (M24).

    Techniques Used: Concentration Assay, Western Blot, Incubation, Positive Control

    The protein concentration of PI incubation medium influences the effect of PI on prelamin A processing in PBMC. PBMC were incubated for 24 hours with increasing concentrations of lopinavir (0, 2, 20, 40, 200 µM) diluted in plasma (total protein concentration: 60–80 g/L), in RPMI culture medium supplemented with 10% FBS and 2 mM L-glutamine (total protein concentration: 4 g/L), or in the RPMI culture medium supplemented with BSA (total protein concentration: 40 g/L). ( A ) Percentage of viable cells in plasma (white), culture medium (black), and BSA supplemented culture medium (gray) (error bars = SD, n ≥3, at least 200 cells counted in each experiment). **p
    Figure Legend Snippet: The protein concentration of PI incubation medium influences the effect of PI on prelamin A processing in PBMC. PBMC were incubated for 24 hours with increasing concentrations of lopinavir (0, 2, 20, 40, 200 µM) diluted in plasma (total protein concentration: 60–80 g/L), in RPMI culture medium supplemented with 10% FBS and 2 mM L-glutamine (total protein concentration: 4 g/L), or in the RPMI culture medium supplemented with BSA (total protein concentration: 40 g/L). ( A ) Percentage of viable cells in plasma (white), culture medium (black), and BSA supplemented culture medium (gray) (error bars = SD, n ≥3, at least 200 cells counted in each experiment). **p

    Techniques Used: Protein Concentration, Incubation

    2) Product Images from "Increased Expression Levels of WAVE3 Are Associated with the Progression and Metastasis of Triple Negative Breast Cancer"

    Article Title: Increased Expression Levels of WAVE3 Are Associated with the Progression and Metastasis of Triple Negative Breast Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0042895

    WAVE3 mRNA is highly expressed in the peripheral blood of patients with metastatic breast cancer. (A) Semi-quantitative RT-PCR from total RNA extracted from a mix of 5 million cells of EBV-lin (a PBMC) and MDA-MB-231 (a highly metastatic breast cancer cell line), at the indicated ratios. MDA-MB-231 cells and EBV-Lin cells were used alone as a positive and negative controls, respectively. WAVE3 mRNA could be amplified from the positive control cells (MDA-MB-231), but not form from the white blood cells (EBV-Lin). WAVE3 mRNA could also be amplified from as few as 1 cancer cell in a million blood cells. GAPDH was used as an internal control for the integrity of the RNA and as equal loading control. (B) Semi-quantitative RT-PCR and (C) quantitative real-time RT-PCR analyses of WAVE3 mRNA expression levels in the blood of 10 patients with metastatic BC and 10 healthy controls. MCF10A was used as a control. The graphs were plotted in a logarithmic scale with the average fold change to MCF10A ± s. d. is shown under the respective bar. GAPDH was used an internal normalization control. The results are shown as the mean±s. d. of at least 3 independent assays.
    Figure Legend Snippet: WAVE3 mRNA is highly expressed in the peripheral blood of patients with metastatic breast cancer. (A) Semi-quantitative RT-PCR from total RNA extracted from a mix of 5 million cells of EBV-lin (a PBMC) and MDA-MB-231 (a highly metastatic breast cancer cell line), at the indicated ratios. MDA-MB-231 cells and EBV-Lin cells were used alone as a positive and negative controls, respectively. WAVE3 mRNA could be amplified from the positive control cells (MDA-MB-231), but not form from the white blood cells (EBV-Lin). WAVE3 mRNA could also be amplified from as few as 1 cancer cell in a million blood cells. GAPDH was used as an internal control for the integrity of the RNA and as equal loading control. (B) Semi-quantitative RT-PCR and (C) quantitative real-time RT-PCR analyses of WAVE3 mRNA expression levels in the blood of 10 patients with metastatic BC and 10 healthy controls. MCF10A was used as a control. The graphs were plotted in a logarithmic scale with the average fold change to MCF10A ± s. d. is shown under the respective bar. GAPDH was used an internal normalization control. The results are shown as the mean±s. d. of at least 3 independent assays.

    Techniques Used: Quantitative RT-PCR, Multiple Displacement Amplification, Amplification, Positive Control, Expressing

    3) Product Images from "Loss of the signaling adaptor TRAF1 causes CD8+ T cell dysregulation during human and murine chronic infection"

    Article Title: Loss of the signaling adaptor TRAF1 causes CD8+ T cell dysregulation during human and murine chronic infection

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20110675

    TRAF1 is required for HIV-specific CD8 T cell responses. (a) CD8 and CD4 T cells were separately purified from HIV + or HIV − donors. CD4 T cells were infected with a primary isolate of HIV and at 48 h, co-cultured with their autologous CD8 T cells that had been transfected with either TRAF1-specific siRNA or a control scrambled RNA (ctrl). Irradiated autologous PBMCs were added as a source of APC. The frequency of Gag + T cells was measured 5–7 d later by flow cytometry using CD3-, CD8-, and GAG-specific antibodies to assess the proportion of infected CD4 T cells. As CD4 is down-regulated on the infected cells, the absence of CD8 is used to determine the CD4 T cell population. Representative flow cytometry plots are shown in Fig. S2 . (left and middle) representative suppression curves (based on three to five replicates at each effector-to-target ratio for each donor and representative of three viral controllers and a healthy uninfected control). Statistical significance was determined by linear regression of percentage of Gag + T cells against log (effector: target ratio) using GraphPad (Prism) software. (right) Representative Western blot analysis of TRAF1 levels after knockdown, determined at 48 h after activation. (b). (left and middle) Viral suppression assay performed as in a. Bim-specific siRNA, TRAF1-specific siRNA, or both, or control scrambled RNA were used to transfect CD8 T cells from two viral controllers. CD8 T cells were plated at a ratio of 1:1 with infected CD4 T cells as in a. Open symbols on the right of each panel indicate the percentage of Gag expression in the CD4 T cells if no CD8 T cells were added at all. Cells were harvested for analysis of percentage of Gag + CD4 T cells (CD8 − T cells) after 7 d of co-culture. Statistical analysis was performed using one-way ANOVA. (right) Representative Western blot analysis of Bim levels after knockdown, determined at 48 h after activation. (c) Purified CD8 T cells from viral controllers were nucleofected with either control RNA or TRAF1 siRNA and incubated with autologous monocytes that had been pulsed with control or HIV peptide and pretreated with replication defective adenovirus expressing 4-1BBL or CD80 at a multiplicity of infection of 200, as previously described ( Bukczynski et al., 2004 ). 8 d later, the cells were harvested for FACS analysis. (top left) Representative Western blot analysis of TRAF1 levels at the end of the 8-d culture. (bottom) Representative FACS plots. (top right) Summary plot for three experiments with cells from two different donors, shown as the number of HIV-tetramer + CD8 T cells recovered in the TRAF1 siRNA-transfected population over those transfected with control RNA after stimulation with HIV peptide-pulsed 4-1BBL or CD80-expressing monocytes.
    Figure Legend Snippet: TRAF1 is required for HIV-specific CD8 T cell responses. (a) CD8 and CD4 T cells were separately purified from HIV + or HIV − donors. CD4 T cells were infected with a primary isolate of HIV and at 48 h, co-cultured with their autologous CD8 T cells that had been transfected with either TRAF1-specific siRNA or a control scrambled RNA (ctrl). Irradiated autologous PBMCs were added as a source of APC. The frequency of Gag + T cells was measured 5–7 d later by flow cytometry using CD3-, CD8-, and GAG-specific antibodies to assess the proportion of infected CD4 T cells. As CD4 is down-regulated on the infected cells, the absence of CD8 is used to determine the CD4 T cell population. Representative flow cytometry plots are shown in Fig. S2 . (left and middle) representative suppression curves (based on three to five replicates at each effector-to-target ratio for each donor and representative of three viral controllers and a healthy uninfected control). Statistical significance was determined by linear regression of percentage of Gag + T cells against log (effector: target ratio) using GraphPad (Prism) software. (right) Representative Western blot analysis of TRAF1 levels after knockdown, determined at 48 h after activation. (b). (left and middle) Viral suppression assay performed as in a. Bim-specific siRNA, TRAF1-specific siRNA, or both, or control scrambled RNA were used to transfect CD8 T cells from two viral controllers. CD8 T cells were plated at a ratio of 1:1 with infected CD4 T cells as in a. Open symbols on the right of each panel indicate the percentage of Gag expression in the CD4 T cells if no CD8 T cells were added at all. Cells were harvested for analysis of percentage of Gag + CD4 T cells (CD8 − T cells) after 7 d of co-culture. Statistical analysis was performed using one-way ANOVA. (right) Representative Western blot analysis of Bim levels after knockdown, determined at 48 h after activation. (c) Purified CD8 T cells from viral controllers were nucleofected with either control RNA or TRAF1 siRNA and incubated with autologous monocytes that had been pulsed with control or HIV peptide and pretreated with replication defective adenovirus expressing 4-1BBL or CD80 at a multiplicity of infection of 200, as previously described ( Bukczynski et al., 2004 ). 8 d later, the cells were harvested for FACS analysis. (top left) Representative Western blot analysis of TRAF1 levels at the end of the 8-d culture. (bottom) Representative FACS plots. (top right) Summary plot for three experiments with cells from two different donors, shown as the number of HIV-tetramer + CD8 T cells recovered in the TRAF1 siRNA-transfected population over those transfected with control RNA after stimulation with HIV peptide-pulsed 4-1BBL or CD80-expressing monocytes.

    Techniques Used: Purification, Infection, Cell Culture, Transfection, Irradiation, Flow Cytometry, Cytometry, Software, Western Blot, Activation Assay, Suppression Assay, Expressing, Co-Culture Assay, Incubation, FACS

    4) Product Images from "The anti-CD74 humanized monoclonal antibody, milatuzumab, which targets the invariant chain of MHC II complexes, alters B-cell proliferation, migration, and adhesion molecule expression"

    Article Title: The anti-CD74 humanized monoclonal antibody, milatuzumab, which targets the invariant chain of MHC II complexes, alters B-cell proliferation, migration, and adhesion molecule expression

    Journal: Arthritis Research & Therapy

    doi: 10.1186/ar3767

    Effects on proliferation of CD19 + B cells and macrophage migration inhibitory factor (MIF) concentration in vitro by milatuzumab . (A) Frequency of proliferated CD19 + /CD3 - /CD14 - B cells according to their carboxyfluorescein succinimidyl ester (CFSE) fluorescence intensity. CFSE-labeled peripheral blood mononuclear cells were cultured for 7 days with or without milatuzumab or intravenous immunoglobulin (IVIG) at 37°C in 5% CO 2 and simultaneously stimulated with IL-2, IL-10, F(ab) 2 , and CpG ( n = 6). Addition of milatuzumab as well as IVIG resulted in a modest, but significant, inhibition of the proliferation (Wilcoxon test). For each condition, a representative histogram is shown. (B) The concentration of the chemokine MIF as a potential ligand of CD74 was tested in cell culture supernatants ( n = 7), as described above, and showed no significant differences between the conditions (Wilcoxon test). (C) Proportion of dead CD19 + B cells, identified as high positive staining with DAPI ( n = 3). There was no substantial influence observed by either IVIG or milatuzumab (Wilcoxon test). * P ≤ 0.05. CpG, cytosine-phosphatidyl-guanosine; DAPI, 4,6 diamidino-2-phenylindole; F(ab) 2 , protein of two antigen-binding fragments; IL, interleukin; ns, not significant.
    Figure Legend Snippet: Effects on proliferation of CD19 + B cells and macrophage migration inhibitory factor (MIF) concentration in vitro by milatuzumab . (A) Frequency of proliferated CD19 + /CD3 - /CD14 - B cells according to their carboxyfluorescein succinimidyl ester (CFSE) fluorescence intensity. CFSE-labeled peripheral blood mononuclear cells were cultured for 7 days with or without milatuzumab or intravenous immunoglobulin (IVIG) at 37°C in 5% CO 2 and simultaneously stimulated with IL-2, IL-10, F(ab) 2 , and CpG ( n = 6). Addition of milatuzumab as well as IVIG resulted in a modest, but significant, inhibition of the proliferation (Wilcoxon test). For each condition, a representative histogram is shown. (B) The concentration of the chemokine MIF as a potential ligand of CD74 was tested in cell culture supernatants ( n = 7), as described above, and showed no significant differences between the conditions (Wilcoxon test). (C) Proportion of dead CD19 + B cells, identified as high positive staining with DAPI ( n = 3). There was no substantial influence observed by either IVIG or milatuzumab (Wilcoxon test). * P ≤ 0.05. CpG, cytosine-phosphatidyl-guanosine; DAPI, 4,6 diamidino-2-phenylindole; F(ab) 2 , protein of two antigen-binding fragments; IL, interleukin; ns, not significant.

    Techniques Used: Migration, Concentration Assay, In Vitro, Fluorescence, Labeling, Cell Culture, Inhibition, Staining, Binding Assay

    5) Product Images from "Activation of peroxisome proliferator-activated receptor alpha in human peripheral blood mononuclear cells reveals an individual gene expression profile response"

    Article Title: Activation of peroxisome proliferator-activated receptor alpha in human peripheral blood mononuclear cells reveals an individual gene expression profile response

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-9-262

    Gene selection procedure after microarray analysis of PBMC of three 24 hour fasted subjects . Flow chart of the followed gene selection procedure after microarray analysis of PBMC of three 24 hour fasted subjects. PPRE; number of genes containing a peroxisome proliferator response element according to Lemay et al . Data from this fasting study was published previously [14], but has been used here after applying a different annotation procedure.
    Figure Legend Snippet: Gene selection procedure after microarray analysis of PBMC of three 24 hour fasted subjects . Flow chart of the followed gene selection procedure after microarray analysis of PBMC of three 24 hour fasted subjects. PPRE; number of genes containing a peroxisome proliferator response element according to Lemay et al . Data from this fasting study was published previously [14], but has been used here after applying a different annotation procedure.

    Techniques Used: Selection, Microarray, Flow Cytometry

    Gene selection procedure after microarray analysis of WY14,643 incubated PBMCs . Flow chart of the followed gene selection procedure after microarray analysis of WY14,643 incubated PBMCs from 6 donors. PPRE; number of genes containing a peroxisome proliferator response element according to Lemay et al .
    Figure Legend Snippet: Gene selection procedure after microarray analysis of WY14,643 incubated PBMCs . Flow chart of the followed gene selection procedure after microarray analysis of WY14,643 incubated PBMCs from 6 donors. PPRE; number of genes containing a peroxisome proliferator response element according to Lemay et al .

    Techniques Used: Selection, Microarray, Incubation, Flow Cytometry

    6) Product Images from "CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells"

    Article Title: CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20060772

    Expression of FoxP3 on different CD4 + CD127 +/− human T cell subsets. (a) PBMCs were harvested from human peripheral blood and stained with CD4, CD25, CD127 as well as intracellularly with FoxP3-specific mAbs, then analyzed on a Becton-Dickinson FACSCalibur. (b) Human PBMCs were stained for cell surface expression of CD4 and CD127. The stained cells were fixed and stained intracellularly for FoxP3. For analysis, the PBMCs were gated on lymphocytes (based on forward and side light scatter) and analyzed for CD127 and FoxP3 expression. The numbers in the dot plot indicate the percentage of gated cells expressing the relevant marker. Data are representative of > 20 independent individuals and > 10 experiments.
    Figure Legend Snippet: Expression of FoxP3 on different CD4 + CD127 +/− human T cell subsets. (a) PBMCs were harvested from human peripheral blood and stained with CD4, CD25, CD127 as well as intracellularly with FoxP3-specific mAbs, then analyzed on a Becton-Dickinson FACSCalibur. (b) Human PBMCs were stained for cell surface expression of CD4 and CD127. The stained cells were fixed and stained intracellularly for FoxP3. For analysis, the PBMCs were gated on lymphocytes (based on forward and side light scatter) and analyzed for CD127 and FoxP3 expression. The numbers in the dot plot indicate the percentage of gated cells expressing the relevant marker. Data are representative of > 20 independent individuals and > 10 experiments.

    Techniques Used: Expressing, Staining, Marker

    Suppression of allogeneic MLR by individual T cell subsets. Buffy coat samples were sorted based on CD4, CD127 and CD25 expression. 30,000 sorted cells were combined with 100,000 autologous PBMCs as responders, and 100,000 allogeneic anti-CD3–depleted, irradiated third-party PBMCs as stimulators. T cells were incubated for 7 d at 37°C in 5% CO 2 . 16 h before the end of the incubation, 1 μCi 3 H-thymidine was added to each well. Plates were harvested and data were analyzed. Data representative of nine separate experiments sorting seven different subpopulations of CD4 + cells indicated (a) CD127 + CD25 + , CD127 + CD25 − , CD127 lo/− CD25 + , CD127 lo/− CD25 − and (b) CD127 lo/− , CD25 hi , CD127 + . 100,000 responders are present in each well with decreasing numbers of sorted cells added at 1:1 ratio (30,000:100,000), 1:1/2 (15,000 sorted cells), 1:1/4 (7,500 sorted cells), 1:1/16 (1,875 sorted cells) in comparison to sorted cells alone. Results are represented as counts per minute (CPM).
    Figure Legend Snippet: Suppression of allogeneic MLR by individual T cell subsets. Buffy coat samples were sorted based on CD4, CD127 and CD25 expression. 30,000 sorted cells were combined with 100,000 autologous PBMCs as responders, and 100,000 allogeneic anti-CD3–depleted, irradiated third-party PBMCs as stimulators. T cells were incubated for 7 d at 37°C in 5% CO 2 . 16 h before the end of the incubation, 1 μCi 3 H-thymidine was added to each well. Plates were harvested and data were analyzed. Data representative of nine separate experiments sorting seven different subpopulations of CD4 + cells indicated (a) CD127 + CD25 + , CD127 + CD25 − , CD127 lo/− CD25 + , CD127 lo/− CD25 − and (b) CD127 lo/− , CD25 hi , CD127 + . 100,000 responders are present in each well with decreasing numbers of sorted cells added at 1:1 ratio (30,000:100,000), 1:1/2 (15,000 sorted cells), 1:1/4 (7,500 sorted cells), 1:1/16 (1,875 sorted cells) in comparison to sorted cells alone. Results are represented as counts per minute (CPM).

    Techniques Used: Expressing, Irradiation, Incubation

    Expression of FoxP3 on different CD4 + T cell subsets. (a) Human PBMCs were stained for cell surface expression of CD4, CD25, and CD127. The stained cells were fixed and stained intracellularly for FoxP3. For analysis, the PBMCs were gated on CD4 + lymphocytes (based on forward and side light scatter and CD4 staining) and analyzed for CD127 and FoxP3 expression. The boxes represent arbitrary designations of CD25 + versus CD25 − cells. The numbers in the histograms indicate the percentage of gated cells expressing the relevant marker. (b) Human PBMCs were stained for cell surface expression of CD4, CD25, and CD127. The stained cells were fixed and stained intracellularly for FoxP3. For analysis, the PBMCs were gated on lymphocytes (based on forward and side light scatter) and analyzed for CD4, CD25, CD127, and FoxP3 expression. The boxes represent arbitrary designations of CD127 + versus CD127 lo/− cells. The numbers in the dot plot indicate the percentage of gated cells expressing the relevant marker. (c) Similar staining and analysis was performed on whole blood obtained from 10 healthy individuals. Each symbol represents an individual person and the narrow bar represents the mean percentage of FoxP3 + T cells on either CD4 + T cells gated based on CD25 and/or CD127 expression.
    Figure Legend Snippet: Expression of FoxP3 on different CD4 + T cell subsets. (a) Human PBMCs were stained for cell surface expression of CD4, CD25, and CD127. The stained cells were fixed and stained intracellularly for FoxP3. For analysis, the PBMCs were gated on CD4 + lymphocytes (based on forward and side light scatter and CD4 staining) and analyzed for CD127 and FoxP3 expression. The boxes represent arbitrary designations of CD25 + versus CD25 − cells. The numbers in the histograms indicate the percentage of gated cells expressing the relevant marker. (b) Human PBMCs were stained for cell surface expression of CD4, CD25, and CD127. The stained cells were fixed and stained intracellularly for FoxP3. For analysis, the PBMCs were gated on lymphocytes (based on forward and side light scatter) and analyzed for CD4, CD25, CD127, and FoxP3 expression. The boxes represent arbitrary designations of CD127 + versus CD127 lo/− cells. The numbers in the dot plot indicate the percentage of gated cells expressing the relevant marker. (c) Similar staining and analysis was performed on whole blood obtained from 10 healthy individuals. Each symbol represents an individual person and the narrow bar represents the mean percentage of FoxP3 + T cells on either CD4 + T cells gated based on CD25 and/or CD127 expression.

    Techniques Used: Expressing, Staining, Marker

    FoxP3 is expressed on a significant percentage of CD4 + T cells independent of CD25 expression. Human PBMCs were cell surface stained using a combination of anti-CD4 and anti-CD25 mAbs. Once fixed, the cells were stained additionally with anti-FoxP3 mAb. Data are representative of > 20 independent individuals and > 10 experiments. The numbers in the histograms indicate the percentage of FoxP3 + cells.
    Figure Legend Snippet: FoxP3 is expressed on a significant percentage of CD4 + T cells independent of CD25 expression. Human PBMCs were cell surface stained using a combination of anti-CD4 and anti-CD25 mAbs. Once fixed, the cells were stained additionally with anti-FoxP3 mAb. Data are representative of > 20 independent individuals and > 10 experiments. The numbers in the histograms indicate the percentage of FoxP3 + cells.

    Techniques Used: Expressing, Staining

    Proliferative response of isolated T cell subsets. Buffy coat samples were sorted based on CD4, CD127, and CD25 expression. 30,000 sorted cells were put into culture with allogeneic anti-CD3–depleted, irradiated, third-party PBMCs as stimulators. T cells were incubated for 7 d at 37°C in 5% CO 2 . 16 h before the end of the incubation, 1 μCi 3 H-thymidine was added to each well. Plates were harvested and data were analyzed. Data are representative of nine separate experiments.
    Figure Legend Snippet: Proliferative response of isolated T cell subsets. Buffy coat samples were sorted based on CD4, CD127, and CD25 expression. 30,000 sorted cells were put into culture with allogeneic anti-CD3–depleted, irradiated, third-party PBMCs as stimulators. T cells were incubated for 7 d at 37°C in 5% CO 2 . 16 h before the end of the incubation, 1 μCi 3 H-thymidine was added to each well. Plates were harvested and data were analyzed. Data are representative of nine separate experiments.

    Techniques Used: Isolation, Expressing, Irradiation, Incubation

    7) Product Images from "The Effect of Single Nucleotide Polymorphisms in the Tumor Necrosis Factor-? Gene on Reproductive Performance and Immune Function in Dairy Cattle"

    Article Title: The Effect of Single Nucleotide Polymorphisms in the Tumor Necrosis Factor-? Gene on Reproductive Performance and Immune Function in Dairy Cattle

    Journal: The Journal of Reproduction and Development

    doi: 10.1262/jrd.2013-140

    Association of the TNF-α promoter genotype and IL-8 mRNA expression levels in PMNs (a) and PBMCs (b) before culturing. The data are expressed as means ± SEM (n = 4, n = 7 and n = 3 for A/A, A/G and G/G in PMNs; n = 4, n = 8 and n = 6 for A/A, A/G and G/G in PBMCs). The asterisk denotes significantly different values (P
    Figure Legend Snippet: Association of the TNF-α promoter genotype and IL-8 mRNA expression levels in PMNs (a) and PBMCs (b) before culturing. The data are expressed as means ± SEM (n = 4, n = 7 and n = 3 for A/A, A/G and G/G in PMNs; n = 4, n = 8 and n = 6 for A/A, A/G and G/G in PBMCs). The asterisk denotes significantly different values (P

    Techniques Used: Expressing

    8) Product Images from "Regulatory activity of azabisphosphonate-capped dendrimers on human CD4+ T cell proliferation enhances ex-vivo expansion of NK cells from PBMCs for immunotherapy"

    Article Title: Regulatory activity of azabisphosphonate-capped dendrimers on human CD4+ T cell proliferation enhances ex-vivo expansion of NK cells from PBMCs for immunotherapy

    Journal: Journal of Translational Medicine

    doi: 10.1186/1479-5876-7-82

    Specific and competitive interaction of azabisphonate dendrimers with pure CD4 + T cells . a) Equilibrium binding curve (dots) and equation of the two-component binding interaction after software modelling (Values of the constants are detailed on the graph). b) Competition with 20 μM 3a-G1 increases Kd showing that both dendrimers are competing for same binding sites. c) Equilibrium binding curve of Julo- 3a-G1 , Kd and Bmax, comparing CD4, CD8 T cells and NK cells using monocyte depleted PBMCs.
    Figure Legend Snippet: Specific and competitive interaction of azabisphonate dendrimers with pure CD4 + T cells . a) Equilibrium binding curve (dots) and equation of the two-component binding interaction after software modelling (Values of the constants are detailed on the graph). b) Competition with 20 μM 3a-G1 increases Kd showing that both dendrimers are competing for same binding sites. c) Equilibrium binding curve of Julo- 3a-G1 , Kd and Bmax, comparing CD4, CD8 T cells and NK cells using monocyte depleted PBMCs.

    Techniques Used: Binding Assay, Software

    3a-G1 treated PBMCs show progressive enrichment in NK cells at CD4 + T cell expense during the second week of culture . a) CD25 expression gated on CD4 + T cells (left graphs) and NK cell versus CD4 + T cell proportion at days 5, 7, 9 and 12 of culture (right graph). b) Amplification factor (left) and proportion (right) of NK and CD4 + T cell populations among PBMCs from eleven different donors after 12 to 14 days treatment with 3a-G1 . Histograms indicate the means of the data collected from the eleven donors (Wilcoxon signed rank t test, *: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).
    Figure Legend Snippet: 3a-G1 treated PBMCs show progressive enrichment in NK cells at CD4 + T cell expense during the second week of culture . a) CD25 expression gated on CD4 + T cells (left graphs) and NK cell versus CD4 + T cell proportion at days 5, 7, 9 and 12 of culture (right graph). b) Amplification factor (left) and proportion (right) of NK and CD4 + T cell populations among PBMCs from eleven different donors after 12 to 14 days treatment with 3a-G1 . Histograms indicate the means of the data collected from the eleven donors (Wilcoxon signed rank t test, *: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).

    Techniques Used: Expressing, Amplification

    Regulatory activity of 3a-G1 upon CD4 + T cell proliferation is direct and T cell restricted . a) CFSE dilution of CD4 + T cells within IL-2 treated CFSE labelled PBMCs or depleted of monocytes (Right), CFSE dilution of CFSE labelled purified CD4 + T cells ± 3a-G1 ± autologous monocytes (Ratio 5:1). b) Regulatory activity of dendrimers is not mediated by autologous monocytes as 3a-G1 also inhibits CFSE dilution of purified CD4 + T cells stimulated with anti-CD3/CD28 coated beads. c) Regulatory activity of 3a-G1 is restricted to T cells as under the same conditions IL-2 stimulated proliferation of autologous NK cells is not affected. Cell cycle analysis shows that the decrease of proliferation of 3a-G1 treated CD4 + T cells correlates with a reduction of mitotic events.
    Figure Legend Snippet: Regulatory activity of 3a-G1 upon CD4 + T cell proliferation is direct and T cell restricted . a) CFSE dilution of CD4 + T cells within IL-2 treated CFSE labelled PBMCs or depleted of monocytes (Right), CFSE dilution of CFSE labelled purified CD4 + T cells ± 3a-G1 ± autologous monocytes (Ratio 5:1). b) Regulatory activity of dendrimers is not mediated by autologous monocytes as 3a-G1 also inhibits CFSE dilution of purified CD4 + T cells stimulated with anti-CD3/CD28 coated beads. c) Regulatory activity of 3a-G1 is restricted to T cells as under the same conditions IL-2 stimulated proliferation of autologous NK cells is not affected. Cell cycle analysis shows that the decrease of proliferation of 3a-G1 treated CD4 + T cells correlates with a reduction of mitotic events.

    Techniques Used: Activity Assay, Purification, Cell Cycle Assay

    3a-G1 prevents IL-2 driven expansion of CD4 + /Foxp3 high regulatory T cells . a) Expanded CD4 + /CD25 + T cells among IL-2 treated PBMCs present characteristics of regulatory T cells, e.g. CD127 -/low and FoxP3 high . 3a-G1 interferes with the expansion of these cells. Markers analysed in upper quadrants are obtained after gating on lived cells based on Forward/Side scatter signal. Insert of CD25 staining is gated on FoxP3 + cells overlaid with isotypic control antibody staining. CD25/CD127 quadrant is gated on CD4 + T cells. Percentage of cells from parental gate is indicated in each quadrant. b) Increased ratio of NK:FoxP3 high T cells during 3a-G1 driven expansion of human NK cells from PBMCs.
    Figure Legend Snippet: 3a-G1 prevents IL-2 driven expansion of CD4 + /Foxp3 high regulatory T cells . a) Expanded CD4 + /CD25 + T cells among IL-2 treated PBMCs present characteristics of regulatory T cells, e.g. CD127 -/low and FoxP3 high . 3a-G1 interferes with the expansion of these cells. Markers analysed in upper quadrants are obtained after gating on lived cells based on Forward/Side scatter signal. Insert of CD25 staining is gated on FoxP3 + cells overlaid with isotypic control antibody staining. CD25/CD127 quadrant is gated on CD4 + T cells. Percentage of cells from parental gate is indicated in each quadrant. b) Increased ratio of NK:FoxP3 high T cells during 3a-G1 driven expansion of human NK cells from PBMCs.

    Techniques Used: Staining

    Dendrimer 3a-G1 selectively inhibits CD4 + T cell proliferation among IL-2 cultured PBMCs during the first week of culture . a) Among PBMCs, NK and CD4 + T cells are the two major cell populations which spontaneously proliferate in response to IL-2 during the first week of culture. 3a-G1 not only enhances the proliferation of NK cells but it also affects the capacity of the CD4 + T cell population to proliferate. b) Average NK cell proliferation increased 29.4% ± 12.1% while CD4 + T cell proliferation decreased 66.1% ± 7.03% in 3a-G1 treated cultures compared to those containing only IL-2 (Day 7, n = 4). c) Impaired proliferation of CD4 + T cells in the presence of 3a-G1 is not rescued by higher IL-2 concentration after a week of culture. Results representative of two independent experiments performed on two individual donors. d) CD8 + T cell proliferation was induced adding anti-CD3/CD28 coated beads on IL-2 cultured PBMCs. The percentages indicated are expressed after gating on the relevant CD4 + or CD8 + T cell population. Addition of 3a-G1 in these conditions affected CD4 + as well as CD8 + T cell proliferation.
    Figure Legend Snippet: Dendrimer 3a-G1 selectively inhibits CD4 + T cell proliferation among IL-2 cultured PBMCs during the first week of culture . a) Among PBMCs, NK and CD4 + T cells are the two major cell populations which spontaneously proliferate in response to IL-2 during the first week of culture. 3a-G1 not only enhances the proliferation of NK cells but it also affects the capacity of the CD4 + T cell population to proliferate. b) Average NK cell proliferation increased 29.4% ± 12.1% while CD4 + T cell proliferation decreased 66.1% ± 7.03% in 3a-G1 treated cultures compared to those containing only IL-2 (Day 7, n = 4). c) Impaired proliferation of CD4 + T cells in the presence of 3a-G1 is not rescued by higher IL-2 concentration after a week of culture. Results representative of two independent experiments performed on two individual donors. d) CD8 + T cell proliferation was induced adding anti-CD3/CD28 coated beads on IL-2 cultured PBMCs. The percentages indicated are expressed after gating on the relevant CD4 + or CD8 + T cell population. Addition of 3a-G1 in these conditions affected CD4 + as well as CD8 + T cell proliferation.

    Techniques Used: Cell Culture, Concentration Assay

    9) Product Images from "Impact of Lentiviral Vector-Mediated Transduction on the Tightness of a Polarized Model of Airway Epithelium and Effect of Cationic Polymer Polyethylenimine"

    Article Title: Impact of Lentiviral Vector-Mediated Transduction on the Tightness of a Polarized Model of Airway Epithelium and Effect of Cationic Polymer Polyethylenimine

    Journal: Journal of Biomedicine and Biotechnology

    doi: 10.1155/2010/103976

    Effect of LV transduction on occludin localization . Expression of occludin mRNA by RT-PCR (a). Note that occludin (OCLN) transcript is detected in 16HBE14o- cells (lane 1) but not in PBMCs (lane 2). β -actin (ACT) transcripts were detected in 16HBE14o- and PMBCs (lanes 4 and 5, respectively). In lane 3, λ HindIII marker. Polarized 16HBE41o- cells were incubated with 50 (C), 500 (D) or 2000 (E) MOIs and, 24 hours later, occludin localization was evaluated by immunofluorescence and confocal microscopy. Controls included mock cells (B) and cells treated with 12 mM EGTA for 30 minutes (F). En-face micrographs are shown. Occludin-specific signal is in green. A peripheral chicken-wire pattern of occludin localization is noted in B and C. In D and E, white arrows point out to lack of occludin staining at cell borders and between cells. In F, occludin staining is lost from cell borders and distributed intracellularly. Note that some intracellular signal is visible also in B-E. Bar: 10 μ m.
    Figure Legend Snippet: Effect of LV transduction on occludin localization . Expression of occludin mRNA by RT-PCR (a). Note that occludin (OCLN) transcript is detected in 16HBE14o- cells (lane 1) but not in PBMCs (lane 2). β -actin (ACT) transcripts were detected in 16HBE14o- and PMBCs (lanes 4 and 5, respectively). In lane 3, λ HindIII marker. Polarized 16HBE41o- cells were incubated with 50 (C), 500 (D) or 2000 (E) MOIs and, 24 hours later, occludin localization was evaluated by immunofluorescence and confocal microscopy. Controls included mock cells (B) and cells treated with 12 mM EGTA for 30 minutes (F). En-face micrographs are shown. Occludin-specific signal is in green. A peripheral chicken-wire pattern of occludin localization is noted in B and C. In D and E, white arrows point out to lack of occludin staining at cell borders and between cells. In F, occludin staining is lost from cell borders and distributed intracellularly. Note that some intracellular signal is visible also in B-E. Bar: 10 μ m.

    Techniques Used: Transduction, Expressing, Reverse Transcription Polymerase Chain Reaction, Activated Clotting Time Assay, Marker, Incubation, Immunofluorescence, Confocal Microscopy, Staining

    10) Product Images from "Mucosal delivery of a multistage subunit vaccine promotes development of lung-resident memory T cells and affords interleukin-17-dependent protection against pulmonary tuberculosis"

    Article Title: Mucosal delivery of a multistage subunit vaccine promotes development of lung-resident memory T cells and affords interleukin-17-dependent protection against pulmonary tuberculosis

    Journal: NPJ Vaccines

    doi: 10.1038/s41541-020-00255-7

    Pulmonary vaccination with CysVac2/AdvaxCpG demonstrates improved protection against M. tuberculosis infection compared to parenteral administration. C57BL/6 mice ( n = 5–6) were vaccinated by either the i.m. or i.t. route with CysVac2(CV2)/Advax CpG (three times, 2 weeks apart). One week after last vaccination mice were bled for vaccine immunogenicity assessment. Six weeks after last immunization mice were challenged with H37Rv by aerosol (~100 CFU) and 4 weeks later culled to enumerate the bacterial burden and the T cell phenotype in the lung ( a ). PBMCs from tail blood of vaccinated mice ( b – d ) or cells from lung of infected mice ( e – g ) were restimulated ex vivo with CysVac2, and the production cytokines (IFN-γ, IL-2, IL-17, TNF), or transcription factors (TF; T-bet, RORγT) by CD4 + T cells was determined by flow cytometry using the gating strategy described in Supplementary Fig. 1. Data are represented as the percentage of cytokine-producing or transcription factor-positive CD4 + T cells ± SEM. Bacterial load was assessed in the lungs ( h ) and in the spleen ( i ) and presented as log 10 of the mean CFU ± SEM. Data are pooled from three independent experiments. The significance of differences between the groups was determined by ANOVA (* p
    Figure Legend Snippet: Pulmonary vaccination with CysVac2/AdvaxCpG demonstrates improved protection against M. tuberculosis infection compared to parenteral administration. C57BL/6 mice ( n = 5–6) were vaccinated by either the i.m. or i.t. route with CysVac2(CV2)/Advax CpG (three times, 2 weeks apart). One week after last vaccination mice were bled for vaccine immunogenicity assessment. Six weeks after last immunization mice were challenged with H37Rv by aerosol (~100 CFU) and 4 weeks later culled to enumerate the bacterial burden and the T cell phenotype in the lung ( a ). PBMCs from tail blood of vaccinated mice ( b – d ) or cells from lung of infected mice ( e – g ) were restimulated ex vivo with CysVac2, and the production cytokines (IFN-γ, IL-2, IL-17, TNF), or transcription factors (TF; T-bet, RORγT) by CD4 + T cells was determined by flow cytometry using the gating strategy described in Supplementary Fig. 1. Data are represented as the percentage of cytokine-producing or transcription factor-positive CD4 + T cells ± SEM. Bacterial load was assessed in the lungs ( h ) and in the spleen ( i ) and presented as log 10 of the mean CFU ± SEM. Data are pooled from three independent experiments. The significance of differences between the groups was determined by ANOVA (* p

    Techniques Used: Infection, Mouse Assay, Ex Vivo, Flow Cytometry

    11) Product Images from "CD4+ T Cell Defects in a Mulibrey Patient With Specific TRIM37 Mutations"

    Article Title: CD4+ T Cell Defects in a Mulibrey Patient With Specific TRIM37 Mutations

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.01742

    CD4 + T cells from the MUL child are reduced in number and expressed low levels of intracellular TRIM37. (A) Flow cytometry plots show CD4 + and CD8 + cell frequency in peripheral blood mononuclear cells (PBMCs) from the MUL child and matched healthy control. Numbers in plots indicate positive cells. Data are from one representative experiment out of six. (B) Flow cytometry plots show the Annexin V and Propidium Iodide (PI) staining in CD4 + (upper panels) and CD8 + (lower panels) T cells from the MUL and healthy child. Numbers in plots indicate positive cells. Data are from one representative experiment out of three. (C) Flow cytometry plots show Annexin V and PI in 48 h TCR-stimulated CD4 + (upper panels) and CD8 + (lower panels) T cells from the MUL and healthy child. Numbers in plots indicate positive cells. Data are from one representative experiment out of two. (D) The upper panel shows representative immunoblot for TRIM37 and actin proteins in unstimulated and 30 min TCR-stimulated CD4 + T cells from the MUL and healthy child. The lower panel shows the relative densitometric quantitation of the TRIM37 protein normalized on actin in CD4 + T cells from the MUL and healthy child, in the aforementioned experimental conditions. Data are shown as mean ± SEM ( n = 4 densitometric quantitation derived from four films with different exposure timing). ** P
    Figure Legend Snippet: CD4 + T cells from the MUL child are reduced in number and expressed low levels of intracellular TRIM37. (A) Flow cytometry plots show CD4 + and CD8 + cell frequency in peripheral blood mononuclear cells (PBMCs) from the MUL child and matched healthy control. Numbers in plots indicate positive cells. Data are from one representative experiment out of six. (B) Flow cytometry plots show the Annexin V and Propidium Iodide (PI) staining in CD4 + (upper panels) and CD8 + (lower panels) T cells from the MUL and healthy child. Numbers in plots indicate positive cells. Data are from one representative experiment out of three. (C) Flow cytometry plots show Annexin V and PI in 48 h TCR-stimulated CD4 + (upper panels) and CD8 + (lower panels) T cells from the MUL and healthy child. Numbers in plots indicate positive cells. Data are from one representative experiment out of two. (D) The upper panel shows representative immunoblot for TRIM37 and actin proteins in unstimulated and 30 min TCR-stimulated CD4 + T cells from the MUL and healthy child. The lower panel shows the relative densitometric quantitation of the TRIM37 protein normalized on actin in CD4 + T cells from the MUL and healthy child, in the aforementioned experimental conditions. Data are shown as mean ± SEM ( n = 4 densitometric quantitation derived from four films with different exposure timing). ** P

    Techniques Used: Flow Cytometry, Staining, Quantitation Assay, Derivative Assay

    12) Product Images from "Long non-coding RNA expression profiles identify lncRNA-XLOC_I2_006631 as a potential novel blood biomarker for Hashimoto's thyroiditis"

    Article Title: Long non-coding RNA expression profiles identify lncRNA-XLOC_I2_006631 as a potential novel blood biomarker for Hashimoto's thyroiditis

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2020.4755

    Validation of selected lncRNAs via RT-qPCR. PBMCs were obtained from 28 patients with HT and 27 healthy controls. The transcript levels of (A) lncRNA-LOC729737, (B) lncRNA-XLOC_I2_006631, (C) lncRNA-AL137655_2, (D) lncRNA-BC041964, (E) lncRNA-LOC100288778 and (F) lncRNA-EPT1 in PBMCs from patients with HT and healthy controls were determined via RT-qPCR. (G) Fold changes of six selected lncRNAs expression between RT-qPCR and sequencing were determined. Horizontal lines show the mean. * P
    Figure Legend Snippet: Validation of selected lncRNAs via RT-qPCR. PBMCs were obtained from 28 patients with HT and 27 healthy controls. The transcript levels of (A) lncRNA-LOC729737, (B) lncRNA-XLOC_I2_006631, (C) lncRNA-AL137655_2, (D) lncRNA-BC041964, (E) lncRNA-LOC100288778 and (F) lncRNA-EPT1 in PBMCs from patients with HT and healthy controls were determined via RT-qPCR. (G) Fold changes of six selected lncRNAs expression between RT-qPCR and sequencing were determined. Horizontal lines show the mean. * P

    Techniques Used: Quantitative RT-PCR, Expressing, Sequencing

    Potential prediction of lncRNAs target genes and the effect of lncRNA-XLOC_I2_006631 on the transcription of MECP2. (A) Relative expression of MECP2 mRNA in PBMCs from patients with HT and healthy controls was detected via RT-qPCR. (B) Correlation between the transcript levels of MECP2 and lncRNA-XLOC_I2_006631 in patients with HT. The correlations between the transcript levels of MECP2 and the serum concentrations of (C) TgAb and (D) TPOAb. (E) Purified PBMCs was transfected with lncRNA-XLOC_I2_006631-specific siRNAs and NC (100 nM) in the presence of functional anti-human CD3 mAb and anti-human CD28 mAb. (F) Transcript levels of lncRNA-XLOC_I2_006631 were detected via RT-qPCR after transfection with lncRNA-XLOC_I2_006631-siRNA1-3 and NC. (G) Transcript level of MECP2 was detected after transfection. Each data point represents an individual subject, horizontal lines represent the mean. * P
    Figure Legend Snippet: Potential prediction of lncRNAs target genes and the effect of lncRNA-XLOC_I2_006631 on the transcription of MECP2. (A) Relative expression of MECP2 mRNA in PBMCs from patients with HT and healthy controls was detected via RT-qPCR. (B) Correlation between the transcript levels of MECP2 and lncRNA-XLOC_I2_006631 in patients with HT. The correlations between the transcript levels of MECP2 and the serum concentrations of (C) TgAb and (D) TPOAb. (E) Purified PBMCs was transfected with lncRNA-XLOC_I2_006631-specific siRNAs and NC (100 nM) in the presence of functional anti-human CD3 mAb and anti-human CD28 mAb. (F) Transcript levels of lncRNA-XLOC_I2_006631 were detected via RT-qPCR after transfection with lncRNA-XLOC_I2_006631-siRNA1-3 and NC. (G) Transcript level of MECP2 was detected after transfection. Each data point represents an individual subject, horizontal lines represent the mean. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Purification, Transfection, Functional Assay

    lncRNA expression profiles in patients with HT. PBMCs of five patients with HT and five healthy volunteers (controls) were enrolled into next-generation high-throughput sequencing. (A) Hierarchical clustering analysis of differentially expressed lncRNAs. Red and green represented upregulated and downregulated lncRNAs, respectively. (B) Volcano plots and (C) scatter plots were used to distinguish differentially expressed lncRNAs. The red squares of volcano plots represented statistically significant dysregulated lncRNAs. The red dots above the diagonal line in the middle of the scatter plot indicated significantly upregulated lncRNAs, and the green dots below represented significantly downregulated lncRNAs. (D) A total of 97,286 known differentially expressed lncRNAs, including 69,544 upregulated and 27,742 downregulated lncRNAs, were identified. Moreover, 218 novel lncRNAs were identified, 94 lncRNAs were significantly upregulated and 124 lncRNAs were significantly downregulated. (E) Number of upregulated (red) and downregulated (green) lncRNAs according to the categories of formation mode. HT, Hashimoto's thyroiditis; lncRNA, long non-coding RNA.
    Figure Legend Snippet: lncRNA expression profiles in patients with HT. PBMCs of five patients with HT and five healthy volunteers (controls) were enrolled into next-generation high-throughput sequencing. (A) Hierarchical clustering analysis of differentially expressed lncRNAs. Red and green represented upregulated and downregulated lncRNAs, respectively. (B) Volcano plots and (C) scatter plots were used to distinguish differentially expressed lncRNAs. The red squares of volcano plots represented statistically significant dysregulated lncRNAs. The red dots above the diagonal line in the middle of the scatter plot indicated significantly upregulated lncRNAs, and the green dots below represented significantly downregulated lncRNAs. (D) A total of 97,286 known differentially expressed lncRNAs, including 69,544 upregulated and 27,742 downregulated lncRNAs, were identified. Moreover, 218 novel lncRNAs were identified, 94 lncRNAs were significantly upregulated and 124 lncRNAs were significantly downregulated. (E) Number of upregulated (red) and downregulated (green) lncRNAs according to the categories of formation mode. HT, Hashimoto's thyroiditis; lncRNA, long non-coding RNA.

    Techniques Used: Expressing, Next-Generation Sequencing

    13) Product Images from "Induction of Cell Death in the Human Acute Lymphoblastic Leukemia Cell Line Reh by Infection with Rotavirus Isolate Wt1-5"

    Article Title: Induction of Cell Death in the Human Acute Lymphoblastic Leukemia Cell Line Reh by Infection with Rotavirus Isolate Wt1-5

    Journal: Biomedicines

    doi: 10.3390/biomedicines8080242

    Effects of Wt1-5 infection of Reh cells and PBMCs on cell viability and cell membrane permeability. Reh cells at a logarithmic growth phase and PBMCs were inoculated with trypsin-activated Wt1-5 (MOI 1 to 6) in RPMI culture medium without FBS. Non-infected cells, UV-inactivated virus-infected cells at MOI 2, and non-infected and H 2 O 2 -treated cells were used as a control. Infected Reh cells ( A ) and PBMCs ( B ) were harvested at the indicated post-infection times (h.p.i.) and subjected to the trypan exclusion test. Reh Cells ( C ) and PBMCs ( D ) were collected at the post-infection times indicated and the viable cells counted in a Neubauer chamber and expressed as cells/mL. Cell proliferation analysis of Reh cells ( E ) and PBMCs ( F ) infected or not infected with Wt1-5 (MOIs 1 to 6), using CellTracker™ Blue CMAC Dye. Quantification is expressed in terms of arbitrary fluorescence units (AUF) through the post-infection period indicated. Cell membrane permeability of Reh Cells ( G ) and PBMCs ( H ) was determined at the indicated post-infection times by measuring lactate dehydrogenase (LDH) activity in the 700 g supernatant of infected, non-infected and UV-inactivated virus-infected cells cultured in RPMI culture medium without FBS. Data are shown as mean SD of three independent experiments performed in duplicate.
    Figure Legend Snippet: Effects of Wt1-5 infection of Reh cells and PBMCs on cell viability and cell membrane permeability. Reh cells at a logarithmic growth phase and PBMCs were inoculated with trypsin-activated Wt1-5 (MOI 1 to 6) in RPMI culture medium without FBS. Non-infected cells, UV-inactivated virus-infected cells at MOI 2, and non-infected and H 2 O 2 -treated cells were used as a control. Infected Reh cells ( A ) and PBMCs ( B ) were harvested at the indicated post-infection times (h.p.i.) and subjected to the trypan exclusion test. Reh Cells ( C ) and PBMCs ( D ) were collected at the post-infection times indicated and the viable cells counted in a Neubauer chamber and expressed as cells/mL. Cell proliferation analysis of Reh cells ( E ) and PBMCs ( F ) infected or not infected with Wt1-5 (MOIs 1 to 6), using CellTracker™ Blue CMAC Dye. Quantification is expressed in terms of arbitrary fluorescence units (AUF) through the post-infection period indicated. Cell membrane permeability of Reh Cells ( G ) and PBMCs ( H ) was determined at the indicated post-infection times by measuring lactate dehydrogenase (LDH) activity in the 700 g supernatant of infected, non-infected and UV-inactivated virus-infected cells cultured in RPMI culture medium without FBS. Data are shown as mean SD of three independent experiments performed in duplicate.

    Techniques Used: Infection, Permeability, Fluorescence, Activity Assay, Cell Culture

    14) Product Images from "Visfatin Enhances Breast Cancer Progression through CXCL1 Induction in Tumor-Associated Macrophages"

    Article Title: Visfatin Enhances Breast Cancer Progression through CXCL1 Induction in Tumor-Associated Macrophages

    Journal: Cancers

    doi: 10.3390/cancers12123526

    Breast cancer cell-derived visfatin induced THP-1 and PBMCs differentiation. ( a ) Breast cancer cells were harvested after culture for 48 h, and intracellular visfatin expression was analyzed by Western blot and extracellular visfatin level by ELISA. Breast cancer cells included triple-negative breast cancer (TNBC) cells (MDA-MB-231, Hs578T, and MDA-MB-486) and lumina l breast cancer cells (MCF-7, T47D, and ZR75-1). ( b ) Correlation between intra/extracellular visfatin was analyzed, with each point representing one breast cancer cell line. ( c ) Conditioned medium (CM) refers to the medium collected after breast cancer cell culture. Human monocytic cell line THP-1 was treated with CM from various breast cancer cell lines for six days, and M2 markers CD163 and CD206 were analyzed by flow cytometry. ( d ) THP-1 cells were treated with visfatin at different doses (0, 100, 200, 300 ng/mL) for six days, and M2 markers CD163 and CD206 were detected in attached cells by immunofluorescence staining. Giemsa stain was used to identify macrophages. (40×) ( e ) PBMCs were treated with visfatin at different doses (0, 100, 200, 300 ng/mL) for six days, and M2 markers CD163 and CD206 were detected in attached cells by immunofluorescence staining, with CD11b as a macrophage marker. Statistical analysis was performed using the t -test and the correlation analysis by linear regression (40×). p -value ≤ 0.05 marked as *; p -value ≤ 0.01 marked as **; p -value ≤ 0.001 marked as ***.
    Figure Legend Snippet: Breast cancer cell-derived visfatin induced THP-1 and PBMCs differentiation. ( a ) Breast cancer cells were harvested after culture for 48 h, and intracellular visfatin expression was analyzed by Western blot and extracellular visfatin level by ELISA. Breast cancer cells included triple-negative breast cancer (TNBC) cells (MDA-MB-231, Hs578T, and MDA-MB-486) and lumina l breast cancer cells (MCF-7, T47D, and ZR75-1). ( b ) Correlation between intra/extracellular visfatin was analyzed, with each point representing one breast cancer cell line. ( c ) Conditioned medium (CM) refers to the medium collected after breast cancer cell culture. Human monocytic cell line THP-1 was treated with CM from various breast cancer cell lines for six days, and M2 markers CD163 and CD206 were analyzed by flow cytometry. ( d ) THP-1 cells were treated with visfatin at different doses (0, 100, 200, 300 ng/mL) for six days, and M2 markers CD163 and CD206 were detected in attached cells by immunofluorescence staining. Giemsa stain was used to identify macrophages. (40×) ( e ) PBMCs were treated with visfatin at different doses (0, 100, 200, 300 ng/mL) for six days, and M2 markers CD163 and CD206 were detected in attached cells by immunofluorescence staining, with CD11b as a macrophage marker. Statistical analysis was performed using the t -test and the correlation analysis by linear regression (40×). p -value ≤ 0.05 marked as *; p -value ≤ 0.01 marked as **; p -value ≤ 0.001 marked as ***.

    Techniques Used: Derivative Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Multiple Displacement Amplification, Cell Culture, Flow Cytometry, Immunofluorescence, Staining, Giemsa Stain, Marker

    15) Product Images from "Tick saliva-induced programmed death-1 and PD-ligand 1 and its related host immunosuppression"

    Article Title: Tick saliva-induced programmed death-1 and PD-ligand 1 and its related host immunosuppression

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-80251-y

    Upregulation of PD-1 and PD-L1 expression by Rhipicephalus microplus saliva (Rm-saliva). ( a – f ) PBMCs were cultured with Rm-saliva. ( a ) Gating strategy for PD-1 staining. ( b , c ) PD-1 expression in CD4 + ( b ) and CD8 + ( c ) T cells was measured by flow cytometry. ( d ) Gating strategy for PD-L1 staining. ( e , f ) PD-L1 expression in CD14 + ( e ) and CD11c + ( f ) cells was measured by flow cytometry. ( a – f ) Statistical difference was identified by the Wilcoxon signed-rank test. MFI mean fluorescence intensity.
    Figure Legend Snippet: Upregulation of PD-1 and PD-L1 expression by Rhipicephalus microplus saliva (Rm-saliva). ( a – f ) PBMCs were cultured with Rm-saliva. ( a ) Gating strategy for PD-1 staining. ( b , c ) PD-1 expression in CD4 + ( b ) and CD8 + ( c ) T cells was measured by flow cytometry. ( d ) Gating strategy for PD-L1 staining. ( e , f ) PD-L1 expression in CD14 + ( e ) and CD11c + ( f ) cells was measured by flow cytometry. ( a – f ) Statistical difference was identified by the Wilcoxon signed-rank test. MFI mean fluorescence intensity.

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

    Inhibition of Th1 responses by Rm-saliva. ( a – e ) PBMCs were cultured with Rm-saliva in the presence of anti-CD3 and anti-CD28 mAbs. ( a ) Gating strategy for CD69 expression. ( b , c ) CD69 expression in CD4 + ( b ) and CD8 + ( c ) cells was measured by flow cytometry. ( d , e ) IFN-γ ( d ) and TNF-α ( e ) concentrations in culture supernatants were determined by ELISA. ( f ) PBMCs were cultured with anti-PD-L1 Ab (Boch4G12) in the presence of Rm-saliva. Bovine IgG was used as a control Ab. PBMCs were stimulated by anti-CD3 and anti-CD28 mAbs. IFN-γ concentration was determined by ELISA. ( a – f ) Statistical difference was identified by the Wilcoxon signed-rank test.
    Figure Legend Snippet: Inhibition of Th1 responses by Rm-saliva. ( a – e ) PBMCs were cultured with Rm-saliva in the presence of anti-CD3 and anti-CD28 mAbs. ( a ) Gating strategy for CD69 expression. ( b , c ) CD69 expression in CD4 + ( b ) and CD8 + ( c ) cells was measured by flow cytometry. ( d , e ) IFN-γ ( d ) and TNF-α ( e ) concentrations in culture supernatants were determined by ELISA. ( f ) PBMCs were cultured with anti-PD-L1 Ab (Boch4G12) in the presence of Rm-saliva. Bovine IgG was used as a control Ab. PBMCs were stimulated by anti-CD3 and anti-CD28 mAbs. IFN-γ concentration was determined by ELISA. ( a – f ) Statistical difference was identified by the Wilcoxon signed-rank test.

    Techniques Used: Inhibition, Cell Culture, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Concentration Assay

    16) Product Images from "MicroRNA-21-5p protects melanocytes via targeting STAT3 and modulating Treg/Teff balance to alleviate vitiligo"

    Article Title: MicroRNA-21-5p protects melanocytes via targeting STAT3 and modulating Treg/Teff balance to alleviate vitiligo

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2020.11689

    Imbalance of T effector/Treg cells and dysregulation of miR-21-5p in patients with VIT. Percentages of (A) Th1, (B) Th17, (C) Th22 and (D) Treg cells in CD4 + T cells of peripheral blood mononuclear cells in patients with VIT and healthy controls (n=15 for each group) were measured via flow cytometry. Protein levels of (E) IFN-γ, (F) IL-17A and (G) IL-22 in the peripheral blood of patients with VIT and healthy controls were measured via enzyme-linked immunosorbent assay. Relative expression levels of (H) Foxp3 and (I) miR-21-5p in the peripheral blood of patients with VIT and healthy controls were measured via reverse transcription-quantitative PCR. GAPDH and U6 were used as the internal controls, respectively. **P
    Figure Legend Snippet: Imbalance of T effector/Treg cells and dysregulation of miR-21-5p in patients with VIT. Percentages of (A) Th1, (B) Th17, (C) Th22 and (D) Treg cells in CD4 + T cells of peripheral blood mononuclear cells in patients with VIT and healthy controls (n=15 for each group) were measured via flow cytometry. Protein levels of (E) IFN-γ, (F) IL-17A and (G) IL-22 in the peripheral blood of patients with VIT and healthy controls were measured via enzyme-linked immunosorbent assay. Relative expression levels of (H) Foxp3 and (I) miR-21-5p in the peripheral blood of patients with VIT and healthy controls were measured via reverse transcription-quantitative PCR. GAPDH and U6 were used as the internal controls, respectively. **P

    Techniques Used: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction

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    Isolation:

    Article Title: Curcumin modulation of the activation of PYK2 in peripheral blood mononuclear cells from patients with lupus nephritis
    Article Snippet: .. Isolation and culture of PBMCs PBMCs from LN patients and healthy controls were purified from heparinised peripheral venous blood using Lymphoprep density gradient centrifugation (Invitrogen). .. The isolated PBMCs were suspended in buffered RPMI-1640 medium and then were divided into four groups: the first group (1 × 106 /ml) was cultured with 5 μl phosphate-buffered saline (PBS) for 48 h, as control, the second group (1 × 106 /ml) was cultured with 5 μl (0.1 μg/ml) PMA (Sigma) for 48 h, and the third (1 × 106 /ml) and the fourth group (1 × 106 /ml) were, respectively, pretreated with 1 μl (0.1 μg/ml) TyrA9 (Calbiochem) or 1 μl (0.1 μg/ml) curcumin (Shanghai Jinma Biotechnology, Shanghai, China) for one hour and then was continually cultured with 5 μl PMA for 48 h. These four groups were incubated in 24-well culture dishes in 5% CO2 at 37°C.

    Article Title: Activation of nuclear factor kappa B in peripheral blood mononuclear cells from malaria patients
    Article Snippet: .. Preparation of PBMCs PBMCs were isolated from freshly heparinized blood by gradient centrifugation, using Isoprep® separation medium (Robbins Scientific, CA, USA), according to the manufacturer’s instructions. ..

    Purification:

    Article Title: Curcumin modulation of the activation of PYK2 in peripheral blood mononuclear cells from patients with lupus nephritis
    Article Snippet: .. Isolation and culture of PBMCs PBMCs from LN patients and healthy controls were purified from heparinised peripheral venous blood using Lymphoprep density gradient centrifugation (Invitrogen). .. The isolated PBMCs were suspended in buffered RPMI-1640 medium and then were divided into four groups: the first group (1 × 106 /ml) was cultured with 5 μl phosphate-buffered saline (PBS) for 48 h, as control, the second group (1 × 106 /ml) was cultured with 5 μl (0.1 μg/ml) PMA (Sigma) for 48 h, and the third (1 × 106 /ml) and the fourth group (1 × 106 /ml) were, respectively, pretreated with 1 μl (0.1 μg/ml) TyrA9 (Calbiochem) or 1 μl (0.1 μg/ml) curcumin (Shanghai Jinma Biotechnology, Shanghai, China) for one hour and then was continually cultured with 5 μl PMA for 48 h. These four groups were incubated in 24-well culture dishes in 5% CO2 at 37°C.

    Gradient Centrifugation:

    Article Title: Curcumin modulation of the activation of PYK2 in peripheral blood mononuclear cells from patients with lupus nephritis
    Article Snippet: .. Isolation and culture of PBMCs PBMCs from LN patients and healthy controls were purified from heparinised peripheral venous blood using Lymphoprep density gradient centrifugation (Invitrogen). .. The isolated PBMCs were suspended in buffered RPMI-1640 medium and then were divided into four groups: the first group (1 × 106 /ml) was cultured with 5 μl phosphate-buffered saline (PBS) for 48 h, as control, the second group (1 × 106 /ml) was cultured with 5 μl (0.1 μg/ml) PMA (Sigma) for 48 h, and the third (1 × 106 /ml) and the fourth group (1 × 106 /ml) were, respectively, pretreated with 1 μl (0.1 μg/ml) TyrA9 (Calbiochem) or 1 μl (0.1 μg/ml) curcumin (Shanghai Jinma Biotechnology, Shanghai, China) for one hour and then was continually cultured with 5 μl PMA for 48 h. These four groups were incubated in 24-well culture dishes in 5% CO2 at 37°C.

    Article Title: Activation of nuclear factor kappa B in peripheral blood mononuclear cells from malaria patients
    Article Snippet: .. Preparation of PBMCs PBMCs were isolated from freshly heparinized blood by gradient centrifugation, using Isoprep® separation medium (Robbins Scientific, CA, USA), according to the manufacturer’s instructions. ..

    PBMC Assay:

    Article Title: Age-related waning of in vitro Interferon-? levels against r32kDaBCG in BCG vaccinated children
    Article Snippet: After the supernatant was discarded and the cells resuspended in 500ul of PBS, the samples were acquired within 2 hrs after staining and anlysed by using CellQuest Pro software (BD Biosciences, San Jose, CA, USA). .. PBMC assay For assessing T cell proliferation, blood was drawn in Heparin (5000 I.U/5 ml; Biological E limited, Hyderabad, AP, India), diluted with equal volume of RPMI-1640 medium (Invitrogen corporation, Grand Island, N.Y. USA) without AB serum, layered on Histopaque (Sigma, St Louis, MO, USA) gradient in 1:3 proportion and centrifuged at 200–350 g (1500–2000 rpm) for 30 minutes. .. After the peripheral blood mononuclear cells (PBMC) were isolated, washed twice to remove the cell debris and platelets each at 90 g (1000 rpm) for 10 minutes, the cell concentration was adjusted to 1 million cells/ml.

    Incubation:

    Article Title: Activation of TAFI on the Surface of Streptococcus pyogenes Evokes Inflammatory Reactions by Modulating the Kallikrein/Kinin System
    Article Snippet: Human peripheral blood mononuclear cells (PBMCs) were isolated as previously described [ ]. .. Stimulation of PBMCs PBMCs were incubated with 1% (v/v) S. pyogenes (M41) supernatants (obtained from overnight cultures of single colonies in 40 ml TH medium) in RPMI 1640 medium (Invitrogen, Paisley, UK) in the presence of 2 mM l-glutamine for 24 h at 37°C. .. Cells were pelleted by centrifugation and the supernatant was assayed for IL-1β content by ELISA (Quantikine immunoassay kit; R & D Systems, Minneapolis, Minn., USA).

    Article Title: Induction of Human T-cell and Cytokine Responses Following Vaccination with a Novel Influenza Vaccine
    Article Snippet: .. Flow cytometry analysis of PBMCs PBMCs were incubated with LIVE/DEAD® Fixable Aqua stain (ThermoFisher Scientific) followed by anti-BDCA-1 APC/Cy7 (L161; BioLegend). anti-BDCA2 PE (AC144; Miltenyi Biotec), anti-CD3 BUV395 (UCHT1; Becton Dickinson), anti-CD4 BUV737 (SK3; Becton Dickinson), anti-CD8 PE/Cy7 (53–6.7; Becton Dickinson), anti-CD11c BV421 (B-ly6; Becton Dickinson), anti-CD14 BV711 (MΦP9; Becton Dickinson), anti-CD16 PE/Cy5 (3G8; BioLegend), anti-CD19 BV786 (SJ25C1; Becton Dickinson), anti-CD56 PE/CF594 (B159; Becton Dickinson) and anti-HLA-DR APC (L243; Becton Dickinson). .. Cells were fixed and permeabilised using Foxp3/Transcription Factor Staining Buffer set (eBioscience) and DDX17 detected in a two step process using rabbit anti-DDX17 primary (EPR13807[B]; Abcam) and by polyclonal secondary donkey anti-rabbit DyLight®488 (Abcam).

    Staining:

    Article Title: Oncolytic measles virus therapy enhances tumor antigen-specific T-cell responses in patients with multiple myeloma
    Article Snippet: The cells were then collected and re-stimulated with neoantigen pools in the IFNγ coated ELISPOT plate. .. Immunophenotyping of PBMCs PBMCs (5 × 105) were resuspended in 100 μL 1X phosphate buffered saline and stained with eFluor 455UV fixable live-dead stain (Thermo Fisher Scientific, Carlsbad, CA), to stain nonviable cells. .. The cells then were surface stained with anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, anti-CCR7, anti-CD127, anti-CD25, and anti-PD1 fluorochrome conjugated antibodies (BioLegend, San Diego, CA) at room temperature for 30 min to identify the T-cell population and their memory or effector phenotypes.

    Article Title: Induction of Human T-cell and Cytokine Responses Following Vaccination with a Novel Influenza Vaccine
    Article Snippet: .. Flow cytometry analysis of PBMCs PBMCs were incubated with LIVE/DEAD® Fixable Aqua stain (ThermoFisher Scientific) followed by anti-BDCA-1 APC/Cy7 (L161; BioLegend). anti-BDCA2 PE (AC144; Miltenyi Biotec), anti-CD3 BUV395 (UCHT1; Becton Dickinson), anti-CD4 BUV737 (SK3; Becton Dickinson), anti-CD8 PE/Cy7 (53–6.7; Becton Dickinson), anti-CD11c BV421 (B-ly6; Becton Dickinson), anti-CD14 BV711 (MΦP9; Becton Dickinson), anti-CD16 PE/Cy5 (3G8; BioLegend), anti-CD19 BV786 (SJ25C1; Becton Dickinson), anti-CD56 PE/CF594 (B159; Becton Dickinson) and anti-HLA-DR APC (L243; Becton Dickinson). .. Cells were fixed and permeabilised using Foxp3/Transcription Factor Staining Buffer set (eBioscience) and DDX17 detected in a two step process using rabbit anti-DDX17 primary (EPR13807[B]; Abcam) and by polyclonal secondary donkey anti-rabbit DyLight®488 (Abcam).

    Flow Cytometry:

    Article Title: Induction of Human T-cell and Cytokine Responses Following Vaccination with a Novel Influenza Vaccine
    Article Snippet: .. Flow cytometry analysis of PBMCs PBMCs were incubated with LIVE/DEAD® Fixable Aqua stain (ThermoFisher Scientific) followed by anti-BDCA-1 APC/Cy7 (L161; BioLegend). anti-BDCA2 PE (AC144; Miltenyi Biotec), anti-CD3 BUV395 (UCHT1; Becton Dickinson), anti-CD4 BUV737 (SK3; Becton Dickinson), anti-CD8 PE/Cy7 (53–6.7; Becton Dickinson), anti-CD11c BV421 (B-ly6; Becton Dickinson), anti-CD14 BV711 (MΦP9; Becton Dickinson), anti-CD16 PE/Cy5 (3G8; BioLegend), anti-CD19 BV786 (SJ25C1; Becton Dickinson), anti-CD56 PE/CF594 (B159; Becton Dickinson) and anti-HLA-DR APC (L243; Becton Dickinson). .. Cells were fixed and permeabilised using Foxp3/Transcription Factor Staining Buffer set (eBioscience) and DDX17 detected in a two step process using rabbit anti-DDX17 primary (EPR13807[B]; Abcam) and by polyclonal secondary donkey anti-rabbit DyLight®488 (Abcam).

    Cytometry:

    Article Title: Induction of Human T-cell and Cytokine Responses Following Vaccination with a Novel Influenza Vaccine
    Article Snippet: .. Flow cytometry analysis of PBMCs PBMCs were incubated with LIVE/DEAD® Fixable Aqua stain (ThermoFisher Scientific) followed by anti-BDCA-1 APC/Cy7 (L161; BioLegend). anti-BDCA2 PE (AC144; Miltenyi Biotec), anti-CD3 BUV395 (UCHT1; Becton Dickinson), anti-CD4 BUV737 (SK3; Becton Dickinson), anti-CD8 PE/Cy7 (53–6.7; Becton Dickinson), anti-CD11c BV421 (B-ly6; Becton Dickinson), anti-CD14 BV711 (MΦP9; Becton Dickinson), anti-CD16 PE/Cy5 (3G8; BioLegend), anti-CD19 BV786 (SJ25C1; Becton Dickinson), anti-CD56 PE/CF594 (B159; Becton Dickinson) and anti-HLA-DR APC (L243; Becton Dickinson). .. Cells were fixed and permeabilised using Foxp3/Transcription Factor Staining Buffer set (eBioscience) and DDX17 detected in a two step process using rabbit anti-DDX17 primary (EPR13807[B]; Abcam) and by polyclonal secondary donkey anti-rabbit DyLight®488 (Abcam).

    Labeling:

    Article Title: Immunomodulatory effect of canine periodontal ligament stem cells on allogenic and xenogenic peripheral blood mononuclear cells
    Article Snippet: The absorbance at 490 nm was read using an ELISA plate reader (Thermomax, Molecular Devices Co., Menlo Park, CA, USA), and the proliferation indexes were calculated relative to the OD value of the positive control. .. CFSE assays for cell division of PBMCs PBMCs were labeled with 2 µM carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen, Eugene, OR, USA) before being added to the mixed cell culture [ ]. .. After five days in mixed cell culture, PBMCs were analyzed using flow cytometry (FACSCalibur, Becton Dickinson, San Diego, CA, USA) to detect green fluorescence (CFSE).

    Cell Culture:

    Article Title: Immunomodulatory effect of canine periodontal ligament stem cells on allogenic and xenogenic peripheral blood mononuclear cells
    Article Snippet: The absorbance at 490 nm was read using an ELISA plate reader (Thermomax, Molecular Devices Co., Menlo Park, CA, USA), and the proliferation indexes were calculated relative to the OD value of the positive control. .. CFSE assays for cell division of PBMCs PBMCs were labeled with 2 µM carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen, Eugene, OR, USA) before being added to the mixed cell culture [ ]. .. After five days in mixed cell culture, PBMCs were analyzed using flow cytometry (FACSCalibur, Becton Dickinson, San Diego, CA, USA) to detect green fluorescence (CFSE).

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  • 99
    Thermo Fisher flow fish method pbmcs
    Combined detection of IFNγ protein and mRNA with ImageStream (a) <t>PBMCs</t> from an HIV-negative subject were left unstimulated or stimulated with SEB for 12 hours. At the end of this incubation time, a 45-minute capture assay was performed for detection of IFNγ-protein, followed by IFN-γmRNA staining with the <t>flow-FISH</t> assay. CD4 T cells were then acquired on an ImageStream instrument. Images illustrate expression of IFN-γ protein, IFN-γ mRNA, CD4 and CD3 in the lymphocyte gate in unstimulated cells (left panels) and in stimulated cells (right panels).( b–c ) Co-localization analysis for CD3/CD4 in black and IFNg protein/mRNA in white using two different analytical tools ( b ) Similarity score (the log transformed Pearson’s correlation coefficient, measuring the spatial linear correlation between two total stains within a cell mask) and ( c ) Bright Detail Similarity score (the log transformed Pearson’s correlation coefficient, measuring the spatial linear correlation between the only the bright regions of two stains within a cell mask). We found a low correlation between secreted IFNg protein and internal mRNA using both measures (median score ~1). In comparison, surface stained CD3 and CD4 were well correlated (median score ~2).
    Flow Fish Method Pbmcs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher cd34 enriched pbmcs
    <t>CD34</t> cells represent a small fraction of <t>PBMCs.</t> Left scale represent the blood volume taken per donors. Paired T-test was used to analyse statistical significance between ‘after blood prep group’ and ‘after red blood cell (RBC) lysis group’ (n=10). Paired Pearson correlation analysis was performed between the blood volume taken and the final number of sorted CD34 cells and no correlation was found (p= 0.115, n=14, Pearson r=0.441).
    Cd34 Enriched Pbmcs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher pbmcs
    Effect of FlaB-treated DCs on intracellular IL-4 and IL-17 production of α-GalCer-stimulated <t>iNKT</t> cells following FlaB treatment of <t>PBMCs</t> from asthma patients. CD14 + monocyte-derived DCs were generated and treated for 48 hours. (A) IL-10 and IL-12 production from FlaB-treated DCs in asthma patients (n=4) and normal controls (n=3) measured by ELISA. In HDM-sensitive asthma patients ( Table 2 ), FlaB-treated DCs and autologous CD3 + T cells were co-cultured with IL-10R or IL-12 mAbs in the presence of HDM extracts for 6 days. Intracellular cytokines of α-GalCer-stimulated iNKT cells were determined by flow cytometry. (B) Characteristics of each experimental group in the co-cultures. (C) IL-10-dependent inhibition by FlaB-treated DCs on IL-4 + iNKT cells (n=3). (D) IL-10-dependent inhibition by FlaB-treated DCs on IL-17 + iNKT cells (n=3). A Wilcoxon test was performed. Data represent the mean±SEM. For Fig. 6C and D, * P
    Pbmcs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Combined detection of IFNγ protein and mRNA with ImageStream (a) PBMCs from an HIV-negative subject were left unstimulated or stimulated with SEB for 12 hours. At the end of this incubation time, a 45-minute capture assay was performed for detection of IFNγ-protein, followed by IFN-γmRNA staining with the flow-FISH assay. CD4 T cells were then acquired on an ImageStream instrument. Images illustrate expression of IFN-γ protein, IFN-γ mRNA, CD4 and CD3 in the lymphocyte gate in unstimulated cells (left panels) and in stimulated cells (right panels).( b–c ) Co-localization analysis for CD3/CD4 in black and IFNg protein/mRNA in white using two different analytical tools ( b ) Similarity score (the log transformed Pearson’s correlation coefficient, measuring the spatial linear correlation between two total stains within a cell mask) and ( c ) Bright Detail Similarity score (the log transformed Pearson’s correlation coefficient, measuring the spatial linear correlation between the only the bright regions of two stains within a cell mask). We found a low correlation between secreted IFNg protein and internal mRNA using both measures (median score ~1). In comparison, surface stained CD3 and CD4 were well correlated (median score ~2).

    Journal: Nature communications

    Article Title: High throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry

    doi: 10.1038/ncomms6641

    Figure Lengend Snippet: Combined detection of IFNγ protein and mRNA with ImageStream (a) PBMCs from an HIV-negative subject were left unstimulated or stimulated with SEB for 12 hours. At the end of this incubation time, a 45-minute capture assay was performed for detection of IFNγ-protein, followed by IFN-γmRNA staining with the flow-FISH assay. CD4 T cells were then acquired on an ImageStream instrument. Images illustrate expression of IFN-γ protein, IFN-γ mRNA, CD4 and CD3 in the lymphocyte gate in unstimulated cells (left panels) and in stimulated cells (right panels).( b–c ) Co-localization analysis for CD3/CD4 in black and IFNg protein/mRNA in white using two different analytical tools ( b ) Similarity score (the log transformed Pearson’s correlation coefficient, measuring the spatial linear correlation between two total stains within a cell mask) and ( c ) Bright Detail Similarity score (the log transformed Pearson’s correlation coefficient, measuring the spatial linear correlation between the only the bright regions of two stains within a cell mask). We found a low correlation between secreted IFNg protein and internal mRNA using both measures (median score ~1). In comparison, surface stained CD3 and CD4 were well correlated (median score ~2).

    Article Snippet: Flow-FISH method PBMCs were stained with viability dye (Invitrogen LIVE/DEAD) for 30 minutes at room temperature.

    Techniques: Incubation, Staining, Flow Cytometry, Fluorescence In Situ Hybridization, Expressing, Transformation Assay

    CD34 cells represent a small fraction of PBMCs. Left scale represent the blood volume taken per donors. Paired T-test was used to analyse statistical significance between ‘after blood prep group’ and ‘after red blood cell (RBC) lysis group’ (n=10). Paired Pearson correlation analysis was performed between the blood volume taken and the final number of sorted CD34 cells and no correlation was found (p= 0.115, n=14, Pearson r=0.441).

    Journal: F1000Research

    Article Title: A method for transplantation of human HSCs into zebrafish, to replace humanised murine transplantation models

    doi: 10.12688/f1000research.14507.1

    Figure Lengend Snippet: CD34 cells represent a small fraction of PBMCs. Left scale represent the blood volume taken per donors. Paired T-test was used to analyse statistical significance between ‘after blood prep group’ and ‘after red blood cell (RBC) lysis group’ (n=10). Paired Pearson correlation analysis was performed between the blood volume taken and the final number of sorted CD34 cells and no correlation was found (p= 0.115, n=14, Pearson r=0.441).

    Article Snippet: CD34 enriched PBMCs were labelled with anti-Human CD34-eFluor450 antibody (eBioscience- RRID:AB_10734946) and positive cells were sorted using Fluorescence Activated Cell Sorting (FACS).

    Techniques: Lysis

    Diagram of our protocol: How to purify human CD34 cells from whole blood and transplant into zebrafish. Whole blood preparation by Percoll gradient allowed us to separate peripheral blood mononuclear cells (PBMCs) from neutrophils and red blood cells (RBCs). PBMCs were enriched for CD34 cells using a positive selection magnetic column. A pure CD34 cell population was sorted using a human anti-CD34eFLuor450 antibody by FACS. CD34 cells were labelled using fluorescein and injected into the Duct of Cuvier of 52 hour post fertilisation zebrafish larvae. Animal with human cells in their CHT were selected for further high-resolution imaging.

    Journal: F1000Research

    Article Title: A method for transplantation of human HSCs into zebrafish, to replace humanised murine transplantation models

    doi: 10.12688/f1000research.14507.1

    Figure Lengend Snippet: Diagram of our protocol: How to purify human CD34 cells from whole blood and transplant into zebrafish. Whole blood preparation by Percoll gradient allowed us to separate peripheral blood mononuclear cells (PBMCs) from neutrophils and red blood cells (RBCs). PBMCs were enriched for CD34 cells using a positive selection magnetic column. A pure CD34 cell population was sorted using a human anti-CD34eFLuor450 antibody by FACS. CD34 cells were labelled using fluorescein and injected into the Duct of Cuvier of 52 hour post fertilisation zebrafish larvae. Animal with human cells in their CHT were selected for further high-resolution imaging.

    Article Snippet: CD34 enriched PBMCs were labelled with anti-Human CD34-eFluor450 antibody (eBioscience- RRID:AB_10734946) and positive cells were sorted using Fluorescence Activated Cell Sorting (FACS).

    Techniques: Selection, FACS, Injection, Imaging

    Effect of FlaB-treated DCs on intracellular IL-4 and IL-17 production of α-GalCer-stimulated iNKT cells following FlaB treatment of PBMCs from asthma patients. CD14 + monocyte-derived DCs were generated and treated for 48 hours. (A) IL-10 and IL-12 production from FlaB-treated DCs in asthma patients (n=4) and normal controls (n=3) measured by ELISA. In HDM-sensitive asthma patients ( Table 2 ), FlaB-treated DCs and autologous CD3 + T cells were co-cultured with IL-10R or IL-12 mAbs in the presence of HDM extracts for 6 days. Intracellular cytokines of α-GalCer-stimulated iNKT cells were determined by flow cytometry. (B) Characteristics of each experimental group in the co-cultures. (C) IL-10-dependent inhibition by FlaB-treated DCs on IL-4 + iNKT cells (n=3). (D) IL-10-dependent inhibition by FlaB-treated DCs on IL-17 + iNKT cells (n=3). A Wilcoxon test was performed. Data represent the mean±SEM. For Fig. 6C and D, * P

    Journal: Allergy, Asthma & Immunology Research

    Article Title: Flagellin Modulates the Function of Invariant NKT Cells From Patients With Asthma via Dendritic Cells

    doi: 10.4168/aair.2016.8.3.206

    Figure Lengend Snippet: Effect of FlaB-treated DCs on intracellular IL-4 and IL-17 production of α-GalCer-stimulated iNKT cells following FlaB treatment of PBMCs from asthma patients. CD14 + monocyte-derived DCs were generated and treated for 48 hours. (A) IL-10 and IL-12 production from FlaB-treated DCs in asthma patients (n=4) and normal controls (n=3) measured by ELISA. In HDM-sensitive asthma patients ( Table 2 ), FlaB-treated DCs and autologous CD3 + T cells were co-cultured with IL-10R or IL-12 mAbs in the presence of HDM extracts for 6 days. Intracellular cytokines of α-GalCer-stimulated iNKT cells were determined by flow cytometry. (B) Characteristics of each experimental group in the co-cultures. (C) IL-10-dependent inhibition by FlaB-treated DCs on IL-4 + iNKT cells (n=3). (D) IL-10-dependent inhibition by FlaB-treated DCs on IL-17 + iNKT cells (n=3). A Wilcoxon test was performed. Data represent the mean±SEM. For Fig. 6C and D, * P

    Article Snippet: Measurement of Foxp3+ iNKT cells To quantify Foxp3+ iNKT cells, PBMCs were permeabilized using a Foxp3 staining buffer kit (eBioscience) and stained with FITC-TCRvα24 (Beckman Coulter), APC-TCRvβ11 (BD Biosciences), and PE-Foxp3 (eBioscience) mAbs.

    Techniques: Derivative Assay, Generated, Enzyme-linked Immunosorbent Assay, Cell Culture, Flow Cytometry, Cytometry, Inhibition

    Protocol for the measurement of cytokines of α-GalCer-stimulated iNKT cells in FlaB-treated PBMC cultures. PBMCs were treated with FlaB for 48 hours. To stimulate iNKT cells, α-GalCer was added 24 hours before cytokine measurements. Secreted and intracellular cytokines were measured using ELISA and flow cytometry, respectively. In some experiments, IL-10R or IL-12 mAbs were added 6 hours before FlaB treatment to determine IL-10- or IL-12-dependent effect of FlaB on iNKT cell function.

    Journal: Allergy, Asthma & Immunology Research

    Article Title: Flagellin Modulates the Function of Invariant NKT Cells From Patients With Asthma via Dendritic Cells

    doi: 10.4168/aair.2016.8.3.206

    Figure Lengend Snippet: Protocol for the measurement of cytokines of α-GalCer-stimulated iNKT cells in FlaB-treated PBMC cultures. PBMCs were treated with FlaB for 48 hours. To stimulate iNKT cells, α-GalCer was added 24 hours before cytokine measurements. Secreted and intracellular cytokines were measured using ELISA and flow cytometry, respectively. In some experiments, IL-10R or IL-12 mAbs were added 6 hours before FlaB treatment to determine IL-10- or IL-12-dependent effect of FlaB on iNKT cell function.

    Article Snippet: Measurement of Foxp3+ iNKT cells To quantify Foxp3+ iNKT cells, PBMCs were permeabilized using a Foxp3 staining buffer kit (eBioscience) and stained with FITC-TCRvα24 (Beckman Coulter), APC-TCRvβ11 (BD Biosciences), and PE-Foxp3 (eBioscience) mAbs.

    Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Cell Function Assay

    Representative examples of flow cytometric analysis. (A) Intracellular cytokines of iNKT cells in PBMCs. Lymphocytes and Vα24 + and Vβ11 + iNKT cells were gated. IL-4 + , IFN-γ + or IL-17 + cells were measured. Histograms show cytokine-positive iNKT cells (colored) and isotype antibodies (blank). (B) Appropriate measurement time point for each intracellular cytokine following α-GalCer stimulation in PBMCs. IL-4 + , IFN-γ + , or IL-17 + iNKT cells were determined at baseline and 6, 12, and 24 hours after α-GalCer stimulation. Histograms show cytokine-positive iNKT cells (colored) and isotype antibodies (blank).

    Journal: Allergy, Asthma & Immunology Research

    Article Title: Flagellin Modulates the Function of Invariant NKT Cells From Patients With Asthma via Dendritic Cells

    doi: 10.4168/aair.2016.8.3.206

    Figure Lengend Snippet: Representative examples of flow cytometric analysis. (A) Intracellular cytokines of iNKT cells in PBMCs. Lymphocytes and Vα24 + and Vβ11 + iNKT cells were gated. IL-4 + , IFN-γ + or IL-17 + cells were measured. Histograms show cytokine-positive iNKT cells (colored) and isotype antibodies (blank). (B) Appropriate measurement time point for each intracellular cytokine following α-GalCer stimulation in PBMCs. IL-4 + , IFN-γ + , or IL-17 + iNKT cells were determined at baseline and 6, 12, and 24 hours after α-GalCer stimulation. Histograms show cytokine-positive iNKT cells (colored) and isotype antibodies (blank).

    Article Snippet: Measurement of Foxp3+ iNKT cells To quantify Foxp3+ iNKT cells, PBMCs were permeabilized using a Foxp3 staining buffer kit (eBioscience) and stained with FITC-TCRvα24 (Beckman Coulter), APC-TCRvβ11 (BD Biosciences), and PE-Foxp3 (eBioscience) mAbs.

    Techniques: Flow Cytometry

    IL-4, IFN-γ, and IL-17 production from α-GalCer-stimulated iNKT cells following FlaB treatment of PBMC cultures from asthma patients and healthy controls. FlaB treatment and α-GalCer stimulation were performed as described in Fig. 1 . Cytokines in supernatants were measured by using ELISA. (A) Cytokine production in cells from asthma patients (n=10). (B) Cytokine production in cells from healthy controls (n=10). A Wilcoxon test was performed. Horizontal bars represent the median.

    Journal: Allergy, Asthma & Immunology Research

    Article Title: Flagellin Modulates the Function of Invariant NKT Cells From Patients With Asthma via Dendritic Cells

    doi: 10.4168/aair.2016.8.3.206

    Figure Lengend Snippet: IL-4, IFN-γ, and IL-17 production from α-GalCer-stimulated iNKT cells following FlaB treatment of PBMC cultures from asthma patients and healthy controls. FlaB treatment and α-GalCer stimulation were performed as described in Fig. 1 . Cytokines in supernatants were measured by using ELISA. (A) Cytokine production in cells from asthma patients (n=10). (B) Cytokine production in cells from healthy controls (n=10). A Wilcoxon test was performed. Horizontal bars represent the median.

    Article Snippet: Measurement of Foxp3+ iNKT cells To quantify Foxp3+ iNKT cells, PBMCs were permeabilized using a Foxp3 staining buffer kit (eBioscience) and stained with FITC-TCRvα24 (Beckman Coulter), APC-TCRvβ11 (BD Biosciences), and PE-Foxp3 (eBioscience) mAbs.

    Techniques: Enzyme-linked Immunosorbent Assay

    Intracellular IL-4, IFN-γ, IL-17, and IL-10 production of α-GalCer-stimulated iNKT cells following FlaB treatment in PBMC cultures from asthma patients and healthy controls. FlaB treatment and α-GalCer stimulation were performed as described in Fig. 1 . Intracellular cytokines were measured using flow cytometry. (A) Intracellular cytokine production in cells from asthma patients (n=10). (B) Intracellular cytokine production in cells from healthy controls (n=10). A Wilcoxon test was performed. Horizontal bars represent the median. NS, not significant.

    Journal: Allergy, Asthma & Immunology Research

    Article Title: Flagellin Modulates the Function of Invariant NKT Cells From Patients With Asthma via Dendritic Cells

    doi: 10.4168/aair.2016.8.3.206

    Figure Lengend Snippet: Intracellular IL-4, IFN-γ, IL-17, and IL-10 production of α-GalCer-stimulated iNKT cells following FlaB treatment in PBMC cultures from asthma patients and healthy controls. FlaB treatment and α-GalCer stimulation were performed as described in Fig. 1 . Intracellular cytokines were measured using flow cytometry. (A) Intracellular cytokine production in cells from asthma patients (n=10). (B) Intracellular cytokine production in cells from healthy controls (n=10). A Wilcoxon test was performed. Horizontal bars represent the median. NS, not significant.

    Article Snippet: Measurement of Foxp3+ iNKT cells To quantify Foxp3+ iNKT cells, PBMCs were permeabilized using a Foxp3 staining buffer kit (eBioscience) and stained with FITC-TCRvα24 (Beckman Coulter), APC-TCRvβ11 (BD Biosciences), and PE-Foxp3 (eBioscience) mAbs.

    Techniques: Flow Cytometry, Cytometry

    IL-10-dependent induction of Foxp3 + iNKT cells following FlaB treatment of PBMCs from asthma patients. FlaB treatment and α-GalCer stimulation were performed as described in Fig. 1 . IL-10R mAb was added before FlaB treatment, and intracellular cytokines were measured by flow cytometry. (A) A representative example for analyzing Foxp3 + iNKT cells. (B) IL-10-dependent induction of Foxp3 + iNKT cells (n=3). A Wilcoxon test was performed. Data represent the mean±SEM.

    Journal: Allergy, Asthma & Immunology Research

    Article Title: Flagellin Modulates the Function of Invariant NKT Cells From Patients With Asthma via Dendritic Cells

    doi: 10.4168/aair.2016.8.3.206

    Figure Lengend Snippet: IL-10-dependent induction of Foxp3 + iNKT cells following FlaB treatment of PBMCs from asthma patients. FlaB treatment and α-GalCer stimulation were performed as described in Fig. 1 . IL-10R mAb was added before FlaB treatment, and intracellular cytokines were measured by flow cytometry. (A) A representative example for analyzing Foxp3 + iNKT cells. (B) IL-10-dependent induction of Foxp3 + iNKT cells (n=3). A Wilcoxon test was performed. Data represent the mean±SEM.

    Article Snippet: Measurement of Foxp3+ iNKT cells To quantify Foxp3+ iNKT cells, PBMCs were permeabilized using a Foxp3 staining buffer kit (eBioscience) and stained with FITC-TCRvα24 (Beckman Coulter), APC-TCRvβ11 (BD Biosciences), and PE-Foxp3 (eBioscience) mAbs.

    Techniques: Flow Cytometry, Cytometry