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Becton Dickinson peripheral blood mononuclear cells pbmcs
Systemic immune response to <t>LCMV-GP33</t> peptide C57BL/6 mice (5 mice/group) were challenged subcutaneously with TC-1 tumor cells and subsequently treated with various combinations of cisplatin, CpG and GP33 peptide as shown. The <t>PBMCs</t> were analyzed 1 week after the last antigen delivery. The presence of GP33-specific CD8+ T cells in PBMCs was analyzed using intracellular cytokine staining for IFN-γ and CD8+ staining followed by flow cytometry analysis. A. Representative flow cytometry contour plot depicting the frequency of IFN-γ-secreting CD8+ T cells among PBMCs after being pulsed with GP33 peptide. B. Bar graph depicting the number of IFN-γ+ CD8+ T cells among PBMCs (mean ±S.E).
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1) Product Images from "Cancer immunotherapy using a potent immunodominant CTL epitope"

Article Title: Cancer immunotherapy using a potent immunodominant CTL epitope

Journal: Vaccine

doi: 10.1016/j.vaccine.2014.09.021

Systemic immune response to LCMV-GP33 peptide C57BL/6 mice (5 mice/group) were challenged subcutaneously with TC-1 tumor cells and subsequently treated with various combinations of cisplatin, CpG and GP33 peptide as shown. The PBMCs were analyzed 1 week after the last antigen delivery. The presence of GP33-specific CD8+ T cells in PBMCs was analyzed using intracellular cytokine staining for IFN-γ and CD8+ staining followed by flow cytometry analysis. A. Representative flow cytometry contour plot depicting the frequency of IFN-γ-secreting CD8+ T cells among PBMCs after being pulsed with GP33 peptide. B. Bar graph depicting the number of IFN-γ+ CD8+ T cells among PBMCs (mean ±S.E).
Figure Legend Snippet: Systemic immune response to LCMV-GP33 peptide C57BL/6 mice (5 mice/group) were challenged subcutaneously with TC-1 tumor cells and subsequently treated with various combinations of cisplatin, CpG and GP33 peptide as shown. The PBMCs were analyzed 1 week after the last antigen delivery. The presence of GP33-specific CD8+ T cells in PBMCs was analyzed using intracellular cytokine staining for IFN-γ and CD8+ staining followed by flow cytometry analysis. A. Representative flow cytometry contour plot depicting the frequency of IFN-γ-secreting CD8+ T cells among PBMCs after being pulsed with GP33 peptide. B. Bar graph depicting the number of IFN-γ+ CD8+ T cells among PBMCs (mean ±S.E).

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

2) Product Images from "Low-dose irradiation promotes tissue revascularization through VEGF release from mast cells and MMP-9-mediated progenitor cell mobilization"

Article Title: Low-dose irradiation promotes tissue revascularization through VEGF release from mast cells and MMP-9-mediated progenitor cell mobilization

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20050959

Irradiation-induced angiogenesis is dictated by MMP-9–mediated factor release from mast and stroma cells and incorporation of BM-derived cells. (A–C) HL ischemia was induced in Sl/Sl d and WBB6F1 +/+ mice, followed by 2 Gy IR or no IR ( n = 7). PBMCs were analyzed for the presence of CFU-Cs (A) and CFU-ECs (B). (C) Plasma VEGF was determined ( n = 7/group). (D) HL ischemia was induced in MMP-9 +/+ mice followed by 2 Gy IR. Mice were subdivided into groups receiving neutralizing mAbs against VEGFR-1, VEGFR-2 or a combination of both mAbs. (a) Blood flow was determined. Insert: Percentage of mice showing limb rescue after ligation. (b) Macroscopic pictures taken of ischemic and contralateral limb 7 d after ligation (*P
Figure Legend Snippet: Irradiation-induced angiogenesis is dictated by MMP-9–mediated factor release from mast and stroma cells and incorporation of BM-derived cells. (A–C) HL ischemia was induced in Sl/Sl d and WBB6F1 +/+ mice, followed by 2 Gy IR or no IR ( n = 7). PBMCs were analyzed for the presence of CFU-Cs (A) and CFU-ECs (B). (C) Plasma VEGF was determined ( n = 7/group). (D) HL ischemia was induced in MMP-9 +/+ mice followed by 2 Gy IR. Mice were subdivided into groups receiving neutralizing mAbs against VEGFR-1, VEGFR-2 or a combination of both mAbs. (a) Blood flow was determined. Insert: Percentage of mice showing limb rescue after ligation. (b) Macroscopic pictures taken of ischemic and contralateral limb 7 d after ligation (*P

Techniques Used: Irradiation, Derivative Assay, Mouse Assay, Flow Cytometry, Ligation

3) Product Images from "The isolation and characterization of CTC subsets related to breast cancer dormancy"

Article Title: The isolation and characterization of CTC subsets related to breast cancer dormancy

Journal: Scientific Reports

doi: 10.1038/srep17533

Embryonic stem cell gene expression profiling. PBMCs subpopulation of breast cancer patient with and without brain metastasis were sorted by FACS. uPAR +/− and int β1 +/− population were collected respectively (containing EpCAM-negative/CD45 − /CD44 + /CD24 − expression markers). RNA were extracted, amplified and real-time PCR analysis were performed using RT 2 -PCR embryonic stem cell array profiler (Qiagen). The change in mRNA expression ( > 3 fold) is shown comparing uPAR + /int β1 + population to uPAR − /int β1 − population in CTCs isolated from patients clinically diagnosed with BCBM (a) or without BCBM (b).
Figure Legend Snippet: Embryonic stem cell gene expression profiling. PBMCs subpopulation of breast cancer patient with and without brain metastasis were sorted by FACS. uPAR +/− and int β1 +/− population were collected respectively (containing EpCAM-negative/CD45 − /CD44 + /CD24 − expression markers). RNA were extracted, amplified and real-time PCR analysis were performed using RT 2 -PCR embryonic stem cell array profiler (Qiagen). The change in mRNA expression ( > 3 fold) is shown comparing uPAR + /int β1 + population to uPAR − /int β1 − population in CTCs isolated from patients clinically diagnosed with BCBM (a) or without BCBM (b).

Techniques Used: Expressing, FACS, Amplification, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Isolation

Multiparametric flow cytometry of PBMCs capturing uPAR/int β1 CTC subsets. Breast cancer PBMCs were first sorted applying gating parameters to select for DAPI − (4′, 6-diamidino-2-phenylindole)/EpCAM − /CD45 − /CD44 + /CD24 − cells. Cells were then subsequently sorted to obtain uPAR/int β1 subsets containing combinatorial expression of these markers. Antibodies used for flow cytometry and cell sorting were: anti-human CD45-APC-Cy7 (Biolegend, cat # 304015, 1:50 dilution), mouse anti-human EpCAM-PE CD326 (eBiosciences, cat # 12-9326-71, 1:40 dilution), anti-human CD24-PE ML5 (Biolegend, cat # 311106, 1:20 dilution), anti-human CD44-PE-Cy7 IM7 (Biolegend, cat # 103030, 1:20 dilution), mouse anti-human uPAR (CD87)-FITC (AbD Serotec cat # MCA2506FT, 1:10 dilution), anti-human int β1 (CD29)-ApC TS2/16 (Biolegend, cat # 3030008, 1:50 dilution). Cells were confirmed to be CTCs by performing RT-PCR, immunoflurescence and genotyping arrays. Representative images are shown.
Figure Legend Snippet: Multiparametric flow cytometry of PBMCs capturing uPAR/int β1 CTC subsets. Breast cancer PBMCs were first sorted applying gating parameters to select for DAPI − (4′, 6-diamidino-2-phenylindole)/EpCAM − /CD45 − /CD44 + /CD24 − cells. Cells were then subsequently sorted to obtain uPAR/int β1 subsets containing combinatorial expression of these markers. Antibodies used for flow cytometry and cell sorting were: anti-human CD45-APC-Cy7 (Biolegend, cat # 304015, 1:50 dilution), mouse anti-human EpCAM-PE CD326 (eBiosciences, cat # 12-9326-71, 1:40 dilution), anti-human CD24-PE ML5 (Biolegend, cat # 311106, 1:20 dilution), anti-human CD44-PE-Cy7 IM7 (Biolegend, cat # 103030, 1:20 dilution), mouse anti-human uPAR (CD87)-FITC (AbD Serotec cat # MCA2506FT, 1:10 dilution), anti-human int β1 (CD29)-ApC TS2/16 (Biolegend, cat # 3030008, 1:50 dilution). Cells were confirmed to be CTCs by performing RT-PCR, immunoflurescence and genotyping arrays. Representative images are shown.

Techniques Used: Flow Cytometry, Cytometry, Expressing, FACS, Reverse Transcription Polymerase Chain Reaction

4) Product Images from "Therapeutic effects of anti-CD154 antibody in cynomolgus monkeys with advanced rheumatoid arthritis"

Article Title: Therapeutic effects of anti-CD154 antibody in cynomolgus monkeys with advanced rheumatoid arthritis

Journal: Scientific Reports

doi: 10.1038/s41598-018-20566-z

Comparison of B-cell subsets. Flow cytometric analysis for B-cell subsets was performed using blood samples collected before treatment and at 2, 3, 5, 6, 7, and 8 weeks after treatment. PBMCs were stained with antibodies against CD45, CD3, CD20, CD27, CD21, IgD, and IgM. Data are expressed as the mean ± SEM (n = 4 per group). The means of groups at each time points were compared using student t-tests. *Significant (p
Figure Legend Snippet: Comparison of B-cell subsets. Flow cytometric analysis for B-cell subsets was performed using blood samples collected before treatment and at 2, 3, 5, 6, 7, and 8 weeks after treatment. PBMCs were stained with antibodies against CD45, CD3, CD20, CD27, CD21, IgD, and IgM. Data are expressed as the mean ± SEM (n = 4 per group). The means of groups at each time points were compared using student t-tests. *Significant (p

Techniques Used: Flow Cytometry, Staining

Comparison of T-cell subsets. Flow cytometric analysis of T-cell subset was performed using blood samples collected before treatment and at 2, 3, 5, 6, 7, and 8 weeks after treatment. For analysis of T-cell populations peripheral blood mononuclear cells (PBMCs) were stained with antibodies against CD45, CD3, CD4, CD8, CD28, and CD95. For analysis of regulatory T cells, PBMCs were stained with antibodies against CD45, CD4, CD25, and FoxP3. Data are expressed as the mean ± SEM (n = 4 per group). The means of groups at each time points were compared using student t-tests. * Significant ( p
Figure Legend Snippet: Comparison of T-cell subsets. Flow cytometric analysis of T-cell subset was performed using blood samples collected before treatment and at 2, 3, 5, 6, 7, and 8 weeks after treatment. For analysis of T-cell populations peripheral blood mononuclear cells (PBMCs) were stained with antibodies against CD45, CD3, CD4, CD8, CD28, and CD95. For analysis of regulatory T cells, PBMCs were stained with antibodies against CD45, CD4, CD25, and FoxP3. Data are expressed as the mean ± SEM (n = 4 per group). The means of groups at each time points were compared using student t-tests. * Significant ( p

Techniques Used: Flow Cytometry, Staining

5) Product Images from "Circulating Tumor Cells from Prostate Cancer Patients Interact with E-Selectin under Physiologic Blood Flow"

Article Title: Circulating Tumor Cells from Prostate Cancer Patients Interact with E-Selectin under Physiologic Blood Flow

Journal: PLoS ONE

doi: 10.1371/journal.pone.0085143

Isolation of prostate CTCs from metastatic PCa patients using anti-CD45 immunomagnetic depletion. 2.5 ml blood from three metastatic PCa patients ( > 50 CTCs/ 2.5 ml blood) was processed via ficoll density centrifugation and the PBMC fraction was collected. Immunomagnetic anti-CD45 depletion was performed on the obtained PBMCs and the remaining cells were washed, cytospunned onto the slides. Slides were stained for PSMA, EpCAM, sLe x , and CXCR4 using the protocol as described in Figure S1 . MDA, PC3, and KG1 cells were simultaneously stained as a control for the following markers: PSMA= Magenta, EpCAM= Yellow, HECA-452= Green, CXCR4= Red. All prostate CTCs expressed CXCR4, while, sLe x expression was variable. The analysis of sLe x intensity is shown in Figure 4 .
Figure Legend Snippet: Isolation of prostate CTCs from metastatic PCa patients using anti-CD45 immunomagnetic depletion. 2.5 ml blood from three metastatic PCa patients ( > 50 CTCs/ 2.5 ml blood) was processed via ficoll density centrifugation and the PBMC fraction was collected. Immunomagnetic anti-CD45 depletion was performed on the obtained PBMCs and the remaining cells were washed, cytospunned onto the slides. Slides were stained for PSMA, EpCAM, sLe x , and CXCR4 using the protocol as described in Figure S1 . MDA, PC3, and KG1 cells were simultaneously stained as a control for the following markers: PSMA= Magenta, EpCAM= Yellow, HECA-452= Green, CXCR4= Red. All prostate CTCs expressed CXCR4, while, sLe x expression was variable. The analysis of sLe x intensity is shown in Figure 4 .

Techniques Used: Isolation, Centrifugation, Staining, Multiple Displacement Amplification, Expressing

6) Product Images from "Control of Simian Immunodeficiency Virus Replication by Vaccine-Induced Gag- and Vif-Specific CD8+ T Cells"

Article Title: Control of Simian Immunodeficiency Virus Replication by Vaccine-Induced Gag- and Vif-Specific CD8+ T Cells

Journal: Journal of Virology

doi: 10.1128/JVI.02634-13

Viral loads and percentages of CD4 in Vif/Nef-vaccinated animals after SIVmac239 challenge. (A) Protocol of Vif/Nef vaccination and SIVmac239 challenge; (B) plasma viral loads; (C) percentages of CD4 + T cells in PBMCs. In panels B and C, data on unvaccinated animals are shown by dotted lines for comparison.
Figure Legend Snippet: Viral loads and percentages of CD4 in Vif/Nef-vaccinated animals after SIVmac239 challenge. (A) Protocol of Vif/Nef vaccination and SIVmac239 challenge; (B) plasma viral loads; (C) percentages of CD4 + T cells in PBMCs. In panels B and C, data on unvaccinated animals are shown by dotted lines for comparison.

Techniques Used:

7) Product Images from "Selective DNA Methylation of BDNF Promoter in Bipolar Disorder: Differences Among Patients with BDI and BDII"

Article Title: Selective DNA Methylation of BDNF Promoter in Bipolar Disorder: Differences Among Patients with BDI and BDII

Journal: Neuropsychopharmacology

doi: 10.1038/npp.2012.10

Levels of brain-derived neurotrophic factor (BDNF) mRNA in peripheral blood mononuclear cells from patients diagnosed with bipolar disorders type 1 (BD I;  n =16) and BD type 2 (BD II;  n =16). Box plots with whiskers from minimum to maximum represent 2 −DDCt
Figure Legend Snippet: Levels of brain-derived neurotrophic factor (BDNF) mRNA in peripheral blood mononuclear cells from patients diagnosed with bipolar disorders type 1 (BD I; n =16) and BD type 2 (BD II; n =16). Box plots with whiskers from minimum to maximum represent 2 −DDCt

Techniques Used: Derivative Assay

Amount of methylated DNA in the promoter region of brain-derived neurotrophic factor (BDNF) in peripheral blood mononuclear cells from patients diagnosed with bipolar disorders type 1 (BD I) or BD type 2 (BD II) in therapy with (+) or without
Figure Legend Snippet: Amount of methylated DNA in the promoter region of brain-derived neurotrophic factor (BDNF) in peripheral blood mononuclear cells from patients diagnosed with bipolar disorders type 1 (BD I) or BD type 2 (BD II) in therapy with (+) or without

Techniques Used: Methylation, Derivative Assay

Amount of methylated DNA in the promoter region of brain-derived neurotrophic factor (BDNF) in peripheral blood mononuclear cells from patients diagnosed with bipolar disorders type 1 (BD I) + BDs type 2 (BD II) in therapy with (+) or
Figure Legend Snippet: Amount of methylated DNA in the promoter region of brain-derived neurotrophic factor (BDNF) in peripheral blood mononuclear cells from patients diagnosed with bipolar disorders type 1 (BD I) + BDs type 2 (BD II) in therapy with (+) or

Techniques Used: Methylation, Derivative Assay

8) Product Images from "Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels"

Article Title: Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels

Journal: PLoS ONE

doi: 10.1371/journal.pone.0035461

Usage of four alternative poly(A) sites for 3′-end cleavage and polyadenylation of human HFE transcripts. (A) The diagram represents the human HFE 3′ untranslated region comprising exons (Ex) six and seven. The length of the exons is shown in base pairs (bp). The position of native translation termination codon (STOP) is represented. Arrows above the diagram indicate the relative position of the four different forward primers (EX6F; EX6G; EX7G and EX7H) used in 3′-RACE experiments. The vertical black bars represent the poly(A) signals (numbered from 1 to 4) that were found to be used in the HFE mRNA 3′-end processing. Their sequence is also shown. Below, the thin lines represent the 3′-RACE products obtained by each primer (indicated on the left). The correspondence between each 3′-RACE product, its polyadenylation site and its length in bp is also represented. (B) Representative agarose gel electrophoresis showing 3′-RACE products from human liver total RNA, obtained by nested PCR using forward primers specified above each lane, the universal primer and the master mix provided by the BD SMART RACE cDNA Amplification Kit (BD Biosciences Clontech). The molecular weight marker (M) is the 1 kb DNA ladder (Invitrogen). (C) Schematic representation of the four human HFE 3′ untranslated regions identified and characterized by cloning and sequencing of the 3′-RACE products obtained from total RNA, isolated from duodenum, liver, heart, peripheral blood mononuclear cells (PBMCs), kidney, testis, spleen, small intestine and ovary. Again, vertical black bars represent the polyadenylation signal that is used in each isoform, with the corresponding sequence depicted and the distance from the poly(A) signal to the cleavage site given in nucleotides (nts). The size of each 3′ untranslated region is shown in nts, below each diagram. Each isoform is numbered from 1 to 4 according to the usage of the corresponding poly(A) signal, as depict above in A . (A) n represents the poly(A) tail. The table on the right shows the presence (✓) or absence (−) of each HFE alternative polyadenylation isoform in each tissue analyzed.
Figure Legend Snippet: Usage of four alternative poly(A) sites for 3′-end cleavage and polyadenylation of human HFE transcripts. (A) The diagram represents the human HFE 3′ untranslated region comprising exons (Ex) six and seven. The length of the exons is shown in base pairs (bp). The position of native translation termination codon (STOP) is represented. Arrows above the diagram indicate the relative position of the four different forward primers (EX6F; EX6G; EX7G and EX7H) used in 3′-RACE experiments. The vertical black bars represent the poly(A) signals (numbered from 1 to 4) that were found to be used in the HFE mRNA 3′-end processing. Their sequence is also shown. Below, the thin lines represent the 3′-RACE products obtained by each primer (indicated on the left). The correspondence between each 3′-RACE product, its polyadenylation site and its length in bp is also represented. (B) Representative agarose gel electrophoresis showing 3′-RACE products from human liver total RNA, obtained by nested PCR using forward primers specified above each lane, the universal primer and the master mix provided by the BD SMART RACE cDNA Amplification Kit (BD Biosciences Clontech). The molecular weight marker (M) is the 1 kb DNA ladder (Invitrogen). (C) Schematic representation of the four human HFE 3′ untranslated regions identified and characterized by cloning and sequencing of the 3′-RACE products obtained from total RNA, isolated from duodenum, liver, heart, peripheral blood mononuclear cells (PBMCs), kidney, testis, spleen, small intestine and ovary. Again, vertical black bars represent the polyadenylation signal that is used in each isoform, with the corresponding sequence depicted and the distance from the poly(A) signal to the cleavage site given in nucleotides (nts). The size of each 3′ untranslated region is shown in nts, below each diagram. Each isoform is numbered from 1 to 4 according to the usage of the corresponding poly(A) signal, as depict above in A . (A) n represents the poly(A) tail. The table on the right shows the presence (✓) or absence (−) of each HFE alternative polyadenylation isoform in each tissue analyzed.

Techniques Used: Sequencing, Agarose Gel Electrophoresis, Nested PCR, Amplification, Molecular Weight, Marker, Clone Assay, Isolation

9) Product Images from "Toll-Like Receptor 2-Mediated Innate Immune Responses against Junín Virus in Mice Lead to Antiviral Adaptive Immune Responses during Systemic Infection and Do Not Affect Viral Replication in the Brain"

Article Title: Toll-Like Receptor 2-Mediated Innate Immune Responses against Junín Virus in Mice Lead to Antiviral Adaptive Immune Responses during Systemic Infection and Do Not Affect Viral Replication in the Brain

Journal: Journal of Virology

doi: 10.1128/JVI.00050-14

Increased levels of Junín virus-specific CD8 + T cells but not humoral responses require TLR2 signaling. Mice were infected with JUNV C1 or LCMV. At 1 week postinfection, mouse PBMCs were collected and stained for NP-specific CD8 + T cells. (A and
Figure Legend Snippet: Increased levels of Junín virus-specific CD8 + T cells but not humoral responses require TLR2 signaling. Mice were infected with JUNV C1 or LCMV. At 1 week postinfection, mouse PBMCs were collected and stained for NP-specific CD8 + T cells. (A and

Techniques Used: Mouse Assay, Infection, Staining

10) Product Images from "Therapeutic effects of anti-CD154 antibody in cynomolgus monkeys with advanced rheumatoid arthritis"

Article Title: Therapeutic effects of anti-CD154 antibody in cynomolgus monkeys with advanced rheumatoid arthritis

Journal: Scientific Reports

doi: 10.1038/s41598-018-20566-z

Comparison of B-cell subsets. Flow cytometric analysis for B-cell subsets was performed using blood samples collected before treatment and at 2, 3, 5, 6, 7, and 8 weeks after treatment. PBMCs were stained with antibodies against CD45, CD3, CD20, CD27, CD21, IgD, and IgM. Data are expressed as the mean ± SEM (n = 4 per group). The means of groups at each time points were compared using student t-tests. *Significant (p
Figure Legend Snippet: Comparison of B-cell subsets. Flow cytometric analysis for B-cell subsets was performed using blood samples collected before treatment and at 2, 3, 5, 6, 7, and 8 weeks after treatment. PBMCs were stained with antibodies against CD45, CD3, CD20, CD27, CD21, IgD, and IgM. Data are expressed as the mean ± SEM (n = 4 per group). The means of groups at each time points were compared using student t-tests. *Significant (p

Techniques Used: Flow Cytometry, Staining

Comparison of T-cell subsets. Flow cytometric analysis of T-cell subset was performed using blood samples collected before treatment and at 2, 3, 5, 6, 7, and 8 weeks after treatment. For analysis of T-cell populations peripheral blood mononuclear cells (PBMCs) were stained with antibodies against CD45, CD3, CD4, CD8, CD28, and CD95. For analysis of regulatory T cells, PBMCs were stained with antibodies against CD45, CD4, CD25, and FoxP3. Data are expressed as the mean ± SEM (n = 4 per group). The means of groups at each time points were compared using student t-tests. * Significant ( p
Figure Legend Snippet: Comparison of T-cell subsets. Flow cytometric analysis of T-cell subset was performed using blood samples collected before treatment and at 2, 3, 5, 6, 7, and 8 weeks after treatment. For analysis of T-cell populations peripheral blood mononuclear cells (PBMCs) were stained with antibodies against CD45, CD3, CD4, CD8, CD28, and CD95. For analysis of regulatory T cells, PBMCs were stained with antibodies against CD45, CD4, CD25, and FoxP3. Data are expressed as the mean ± SEM (n = 4 per group). The means of groups at each time points were compared using student t-tests. * Significant ( p

Techniques Used: Flow Cytometry, Staining

11) Product Images from "Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels"

Article Title: Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels

Journal: PLoS ONE

doi: 10.1371/journal.pone.0035461

Usage of four alternative poly(A) sites for 3′-end cleavage and polyadenylation of human HFE transcripts. (A) The diagram represents the human HFE 3′ untranslated region comprising exons (Ex) six and seven. The length of the exons is shown in base pairs (bp). The position of native translation termination codon (STOP) is represented. Arrows above the diagram indicate the relative position of the four different forward primers (EX6F; EX6G; EX7G and EX7H) used in 3′-RACE experiments. The vertical black bars represent the poly(A) signals (numbered from 1 to 4) that were found to be used in the HFE mRNA 3′-end processing. Their sequence is also shown. Below, the thin lines represent the 3′-RACE products obtained by each primer (indicated on the left). The correspondence between each 3′-RACE product, its polyadenylation site and its length in bp is also represented. (B) Representative agarose gel electrophoresis showing 3′-RACE products from human liver total RNA, obtained by nested PCR using forward primers specified above each lane, the universal primer and the master mix provided by the BD SMART RACE cDNA Amplification Kit (BD Biosciences Clontech). The molecular weight marker (M) is the 1 kb DNA ladder (Invitrogen). (C) Schematic representation of the four human HFE 3′ untranslated regions identified and characterized by cloning and sequencing of the 3′-RACE products obtained from total RNA, isolated from duodenum, liver, heart, peripheral blood mononuclear cells (PBMCs), kidney, testis, spleen, small intestine and ovary. Again, vertical black bars represent the polyadenylation signal that is used in each isoform, with the corresponding sequence depicted and the distance from the poly(A) signal to the cleavage site given in nucleotides (nts). The size of each 3′ untranslated region is shown in nts, below each diagram. Each isoform is numbered from 1 to 4 according to the usage of the corresponding poly(A) signal, as depict above in A . (A) n represents the poly(A) tail. The table on the right shows the presence (✓) or absence (−) of each HFE alternative polyadenylation isoform in each tissue analyzed.
Figure Legend Snippet: Usage of four alternative poly(A) sites for 3′-end cleavage and polyadenylation of human HFE transcripts. (A) The diagram represents the human HFE 3′ untranslated region comprising exons (Ex) six and seven. The length of the exons is shown in base pairs (bp). The position of native translation termination codon (STOP) is represented. Arrows above the diagram indicate the relative position of the four different forward primers (EX6F; EX6G; EX7G and EX7H) used in 3′-RACE experiments. The vertical black bars represent the poly(A) signals (numbered from 1 to 4) that were found to be used in the HFE mRNA 3′-end processing. Their sequence is also shown. Below, the thin lines represent the 3′-RACE products obtained by each primer (indicated on the left). The correspondence between each 3′-RACE product, its polyadenylation site and its length in bp is also represented. (B) Representative agarose gel electrophoresis showing 3′-RACE products from human liver total RNA, obtained by nested PCR using forward primers specified above each lane, the universal primer and the master mix provided by the BD SMART RACE cDNA Amplification Kit (BD Biosciences Clontech). The molecular weight marker (M) is the 1 kb DNA ladder (Invitrogen). (C) Schematic representation of the four human HFE 3′ untranslated regions identified and characterized by cloning and sequencing of the 3′-RACE products obtained from total RNA, isolated from duodenum, liver, heart, peripheral blood mononuclear cells (PBMCs), kidney, testis, spleen, small intestine and ovary. Again, vertical black bars represent the polyadenylation signal that is used in each isoform, with the corresponding sequence depicted and the distance from the poly(A) signal to the cleavage site given in nucleotides (nts). The size of each 3′ untranslated region is shown in nts, below each diagram. Each isoform is numbered from 1 to 4 according to the usage of the corresponding poly(A) signal, as depict above in A . (A) n represents the poly(A) tail. The table on the right shows the presence (✓) or absence (−) of each HFE alternative polyadenylation isoform in each tissue analyzed.

Techniques Used: Sequencing, Agarose Gel Electrophoresis, Nested PCR, Amplification, Molecular Weight, Marker, Clone Assay, Isolation

12) Product Images from "The Herpes Simplex Virus Latency-Associated Transcript Gene Is Associated with a Broader Repertoire of Virus-Specific Exhausted CD8+ T Cells Retained within the Trigeminal Ganglia of Latently Infected HLA Transgenic Rabbits"

Article Title: The Herpes Simplex Virus Latency-Associated Transcript Gene Is Associated with a Broader Repertoire of Virus-Specific Exhausted CD8+ T Cells Retained within the Trigeminal Ganglia of Latently Infected HLA Transgenic Rabbits

Journal: Journal of Virology

doi: 10.1128/JVI.02450-15

HLA Tg rabbits infected with LAT + virus have increased spontaneous virus shedding in tears and express high levels of HLA-A2 molecules in TG compared to HLA Tg rabbits infected with LAT – virus. (A) Peripheral blood mononuclear cells (PBMCs) from either HLA transgenic rabbits (HLA Tg rabbits) or from wild-type nontransgenic rabbit controls (WT rabbits) were stained with PE-conjugated anti-HLA-A2 MAb (clone BB7.2) and analyzed by flow cytometry for the relative expression of HLA-A2 molecules (top two panels) and for the percentage of cells expressing HLA-A2 molecules (bottom two panels). Rabbits with the highest levels of HLA-A2 molecules and with the highest percentages of cells expressing HLA-A2 molecules were selected for the remainder of the study. (B) LAT + TG have increased levels of HLA-A2 molecules compared to LAT – TG. TG and spleens (control) from HLA Tg rabbits infected with either LAT + or LAT – virus were removed on day 35 postocular infection. (C) Representative data of the percentages of total TG-derived cells expressing HLA-A2 molecules detected by FACS were compared in LAT + TG versus LAT – TG. (D) Each bar represents the means ± the SD of the fluorescence intensity from two independent experiments from the spleen (control) and from TG harvested from five HLA Tg rabbits at 35 days postinfection. *, P
Figure Legend Snippet: HLA Tg rabbits infected with LAT + virus have increased spontaneous virus shedding in tears and express high levels of HLA-A2 molecules in TG compared to HLA Tg rabbits infected with LAT – virus. (A) Peripheral blood mononuclear cells (PBMCs) from either HLA transgenic rabbits (HLA Tg rabbits) or from wild-type nontransgenic rabbit controls (WT rabbits) were stained with PE-conjugated anti-HLA-A2 MAb (clone BB7.2) and analyzed by flow cytometry for the relative expression of HLA-A2 molecules (top two panels) and for the percentage of cells expressing HLA-A2 molecules (bottom two panels). Rabbits with the highest levels of HLA-A2 molecules and with the highest percentages of cells expressing HLA-A2 molecules were selected for the remainder of the study. (B) LAT + TG have increased levels of HLA-A2 molecules compared to LAT – TG. TG and spleens (control) from HLA Tg rabbits infected with either LAT + or LAT – virus were removed on day 35 postocular infection. (C) Representative data of the percentages of total TG-derived cells expressing HLA-A2 molecules detected by FACS were compared in LAT + TG versus LAT – TG. (D) Each bar represents the means ± the SD of the fluorescence intensity from two independent experiments from the spleen (control) and from TG harvested from five HLA Tg rabbits at 35 days postinfection. *, P

Techniques Used: Infection, Transgenic Assay, Staining, Flow Cytometry, Cytometry, Expressing, Derivative Assay, FACS, Fluorescence

13) Product Images from "Aging and human CD4+ regulatory T cells"

Article Title: Aging and human CD4+ regulatory T cells

Journal: Mechanisms of ageing and development

doi: 10.1016/j.mad.2009.06.003

Measuring CTLA-4 and Fas expression by CD4 + ,FOXP3 + T cells as well as apoptosis of CD4 + ,CD25 bright and CD25 − T cells in young and elderly humans. (A – D) PBMCs from young (n = 15) and elderly (n = 15) individuals were stained with Abs
Figure Legend Snippet: Measuring CTLA-4 and Fas expression by CD4 + ,FOXP3 + T cells as well as apoptosis of CD4 + ,CD25 bright and CD25 − T cells in young and elderly humans. (A – D) PBMCs from young (n = 15) and elderly (n = 15) individuals were stained with Abs

Techniques Used: Expressing, Staining

Correlation of CD25, FOXP3 and IL-7Rα expression by CD4 + T cells in young and elderly humans. Purified peripheral blood mononuclear cells (PBMCs) from young (n = 10) and elderly (n = 10) individuals were stained with Abs to CD4 and CD25, followed
Figure Legend Snippet: Correlation of CD25, FOXP3 and IL-7Rα expression by CD4 + T cells in young and elderly humans. Purified peripheral blood mononuclear cells (PBMCs) from young (n = 10) and elderly (n = 10) individuals were stained with Abs to CD4 and CD25, followed

Techniques Used: Expressing, Purification, Staining

Comparing the frequency of CD4 + ,FOXP3 + T cells and their naïve (CD45RA + ) and memory (CD45RA − ) phenotypes between young and elderly humans. PBMCs from young (n = 15) and elderly (n = 15) individuals were stained with Abs to CD4, CD45RA
Figure Legend Snippet: Comparing the frequency of CD4 + ,FOXP3 + T cells and their naïve (CD45RA + ) and memory (CD45RA − ) phenotypes between young and elderly humans. PBMCs from young (n = 15) and elderly (n = 15) individuals were stained with Abs to CD4, CD45RA

Techniques Used: Staining

Comparing the capacity of CD4 + ,CD25 bright T cells in suppressing the proliferation and cytokine production of CD4 + ,CD25 − T cells between young and elderly humans. PBMCs from young and elderly individuals were stained with Abs to CD4 and CD25 and
Figure Legend Snippet: Comparing the capacity of CD4 + ,CD25 bright T cells in suppressing the proliferation and cytokine production of CD4 + ,CD25 − T cells between young and elderly humans. PBMCs from young and elderly individuals were stained with Abs to CD4 and CD25 and

Techniques Used: Staining

Chemokine receptor expression on CD4 + ,FOXP3 + T cells in young and elderly humans. PBMCs from young (n = 15) and elderly (n = 15) individuals were stained with Abs to CD4, CCR7, CCR4, CCR5, CXCR3 or isotype Abs, followed by permeabilization and staining
Figure Legend Snippet: Chemokine receptor expression on CD4 + ,FOXP3 + T cells in young and elderly humans. PBMCs from young (n = 15) and elderly (n = 15) individuals were stained with Abs to CD4, CCR7, CCR4, CCR5, CXCR3 or isotype Abs, followed by permeabilization and staining

Techniques Used: Expressing, Staining

14) Product Images from "SAMHD1 post-transcriptionally controls the expression of Foxp3 and Helios in Human T regulatory Cells"

Article Title: SAMHD1 post-transcriptionally controls the expression of Foxp3 and Helios in Human T regulatory Cells

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

doi: 10.4049/jimmunol.1800613

SAMHD1 specifically binds to 3’-UTR of Foxp3 and Helios, and stabilize their post-transcription by TCR stimulation as well as ODNps25 addition (a) Relative induction of Foxp3 and Helios proteins by in vitro CD3/CD28-stimulation in Tregs. Rested polyclonal Tregs, expanded as in Methods, were re-stimulated by adding anti-CD3ε (0.5 µg/ml), CD28 antibodies (0.2 µg/ml) and recombinant IL-2 (200 IU/ml) together with -irradiated PBMCs for 36 hrs. The number positioned in upper left corner in the histogram plots indicates median of MFI of Foxp3 or Helios, respectively. The plots shown is one of three independent experiments with different donors. (B) Relative expression of total mRNA of Foxp3 and Helios in 17195TCR-transduced Tregs stimulated with ODNps25, anti-CD3ε antibody, or FVIII C2 peptide. Preparation and in vitro ). The experiments of B, C, and D are one of two independent experiments with different donors. (D) Relative quantification was measured by SYBR green-qPCR. (E) Confocal microscopy of SAMHD1 (red) in re-stimulated 17195 TCR transduced (green) Tregs. Cells were re-stimulated by adding anti-CD3ε antibody (0.5 µg/ml) or FVIII C2 peptide (1 µg/ml) for 36 hrs. FVIII-specific TCR expressed cells are recognized by the expression of green fluorescent protein (GFP). Nuclei were counterstained with DAPI (blue). Scale bars, 10 µm.
Figure Legend Snippet: SAMHD1 specifically binds to 3’-UTR of Foxp3 and Helios, and stabilize their post-transcription by TCR stimulation as well as ODNps25 addition (a) Relative induction of Foxp3 and Helios proteins by in vitro CD3/CD28-stimulation in Tregs. Rested polyclonal Tregs, expanded as in Methods, were re-stimulated by adding anti-CD3ε (0.5 µg/ml), CD28 antibodies (0.2 µg/ml) and recombinant IL-2 (200 IU/ml) together with -irradiated PBMCs for 36 hrs. The number positioned in upper left corner in the histogram plots indicates median of MFI of Foxp3 or Helios, respectively. The plots shown is one of three independent experiments with different donors. (B) Relative expression of total mRNA of Foxp3 and Helios in 17195TCR-transduced Tregs stimulated with ODNps25, anti-CD3ε antibody, or FVIII C2 peptide. Preparation and in vitro ). The experiments of B, C, and D are one of two independent experiments with different donors. (D) Relative quantification was measured by SYBR green-qPCR. (E) Confocal microscopy of SAMHD1 (red) in re-stimulated 17195 TCR transduced (green) Tregs. Cells were re-stimulated by adding anti-CD3ε antibody (0.5 µg/ml) or FVIII C2 peptide (1 µg/ml) for 36 hrs. FVIII-specific TCR expressed cells are recognized by the expression of green fluorescent protein (GFP). Nuclei were counterstained with DAPI (blue). Scale bars, 10 µm.

Techniques Used: In Vitro, Recombinant, Irradiation, Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction, Confocal Microscopy

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Flow Cytometry:

Article Title: In Vivo Safety and Persistence of Endoribonuclease Gene-Transduced CD4+ T Cells in Cynomolgus Macaques for HIV-1 Gene Therapy Model
Article Snippet: .. Flow cytometry analysis The cell surface markers of the expanded cells and peripheral blood mononuclear cells (PBMC) were analyzed using FACSCalibur (BD Bioscience) and FACSCanto (BD Bioscience), and data analysis was performed using CellQuest software (BD Bioscience), FACSDiva software (BD Bioscience) or FlowJo software (Tree Star, Inc., Ashland, OR). .. The following antibodies were used for staining: anti-CD3 (SP34-2, PerCP), anti-CD4 (L200, FITC), anti-CD25 (2A3, FITC), anti-CD28 (CD28.2, PE), anti-CD95 (DX2, FITC), anti-CXCR4 (12G5, PE) and anti-integrin-β7 (FIB504, PE), which were obtained from BD Bioscience.

Article Title: Mechanisms of Gastrointestinal CD4+ T-Cell Depletion during Acute and Early Human Immunodeficiency Virus Type 1 Infection ▿
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Article Title: The isolation and characterization of CTC subsets related to breast cancer dormancy
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In Vitro:

Article Title: The isolation and characterization of CTC subsets related to breast cancer dormancy
Article Snippet: .. Subsets of CTCs isolated from breast cancer patients grow in vitro and are capable of generating CTC tumorspheres To establish whether subsets of CTCs isolated from the same patient and possessing a combinatorial uPAR/int β1 expression could be expanded in culture, we analyzed blood from patients’ peripheral blood mononuclear cells (PBMCs) employing multi-parametric flow cytometry analysis (FACS, ARIA IID, BD Biosciences™) by selecting DAPI− / CD45− /EpCAM-negative/CD44+ /CD24− /uPAR/int β1 expression markers to capture four combinatorial subsets (uPAR+ /int β1+ , uPAR+ /int β1− , uPAR- /int β1+ , uPAR− /int β1− ) respectively ( ). ..

Isolation:

Article Title: Mechanisms of Gastrointestinal CD4+ T-Cell Depletion during Acute and Early Human Immunodeficiency Virus Type 1 Infection ▿
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Article Title: The isolation and characterization of CTC subsets related to breast cancer dormancy
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Cytometry:

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Concentration Assay:

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Expressing:

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Neutralization:

Article Title: Restricted Replication of Xenotropic Murine Leukemia Virus-Related Virus in Pigtailed Macaques
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Sequencing:

Article Title: Restricted Replication of Xenotropic Murine Leukemia Virus-Related Virus in Pigtailed Macaques
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FACS:

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Software:

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Article Title: Mechanisms of Gastrointestinal CD4+ T-Cell Depletion during Acute and Early Human Immunodeficiency Virus Type 1 Infection ▿
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Derivative Assay:

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Staining:

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Article Snippet: .. Peripheral blood mononuclear cells (PBMC) were stained with CD56-phycoerythrin (PE), CD3-Cychrome (Cyc), CD27-fluoroscein isothiocyanate (FITC) or CD70-FITC monoclonal antibodies (BD Pharmingen, San Diego, CA). .. NK cells were defined as CD56+ CD3– lymphocytes and expression of CD27 and CD70 on NK cells was analysed using three-colour (freshly isolated PBMC) or four-colour (cultured cells) analysis on the gated CD56+ CD3– cells.

Article Title: Mechanisms of Gastrointestinal CD4+ T-Cell Depletion during Acute and Early Human Immunodeficiency Virus Type 1 Infection ▿
Article Snippet: .. Cell surface expression of lymphocyte antigens was identified by monoclonal antibody staining of freshly isolated MMCs and peripheral blood mononuclear cells (PBMC), followed by flow cytometry using a FACSCalibur (Becton Dickinson Immunocytometry Systems [BDIS], Mountain View, CA) with analysis using CellQuest software (BDIS) as described previously ( ). .. The monoclonal antibodies used in the present study included anti-human CD3 fluorescein isothiocyanate (FITC) (clone UCHT1; BDIS), anti-human CD3-phycoerythrin (PE) (clone SK-7; BDIS), anti-human CD3-peridinin chlorophyll- a protein (PerCP; clone SK-7; BDIS), anti-human CD4-allophycocyanin (clone RPA T4; Pharmingen, San Diego, CA), anti-human CD8 PE (clone RPA T8; Pharmingen), anti-human CD38 FITC (clone HIT2; Pharmingen), anti-human CD45RO PE (clone UCHL1; Pharmingen), anti-human Ki67 FITC (clone B56; Pharmingen), anti-human CCR7 PE (clone 3D12; BD Biosciences, San Jose, CA), anti-human CD62L allophycocyanin (clone Dreg56; BD Biosciences), and the appropriate isotype controls.

Article Title: Cancer immunotherapy using a potent immunodominant CTL epitope
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Article Title: Control of Heterologous Simian Immunodeficiency Virus SIVsmE660 Infection by DNA and Protein Coimmunization Regimens Combined with Different Toll-Like-Receptor-4-Based Adjuvants in Macaques
Article Snippet: .. SIV-specific T cell responses were measured by intracellular cytokine staining using peripheral blood mononuclear cells (PBMC) (0.6 × 106 cells/sample) stimulated with Gag or Env (derived from M766 or CG7V) peptide pools (15-mer peptides overlapping by 11 aa) at a final concentration of 1 μg/ml for each peptide in the presence of monensin (Golgi-stop; BD Pharmingen, San Jose, CA). .. For negative and positive controls, PBMC were cultured in medium without peptides or stimulated with a phorbol 12-myristate 13-acetate (PMA) cell stimulation cocktail (eBioscience, Affymetrix, Inc., San Diego, CA, USA).

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    Intracellular <t>cytokine</t> staining of CP CD8 + T cells. A-D: CD8 + T cells of HLA-B*57 positive CP were either freshly isolated (left) or stimulated with either wild type (center) or escape variant (right) HLA-B*57-restricted Gag peptides for 7 days. CPs were either on suppressive HAART regiments with undetectable viral loads (circles), were on HAART regimens but recently had detectable levels of viremia (triangles), or were not on HAART and had high levels of viremia (diamond). Cells from each group underwent an overnight stimulation with individual peptides. Percentage of CD8 + T cells expressing IFN-γ when restimulated with TW10 (A) , KF11 (B) , and IW9 (C) in blue, or the escape mutant variant peptide containing T242N/G248A (A) , A163S (B) , and I147L (C) mutations in red is shown. E-H : Percentage of CD8 + T cells expressing both IFN-γ and perforin after overnight stimulation with TW10 (E) , KF11 (F) , and IW9 (G) in blue, or the escape mutant variant peptide containing T242N/G248A (E) , A163S (F) , and I147L (G) mutations in red is shown. D and H show CD8 + T cells that express IFN-γ or co-express IFN-γ and perforin when <t>PBMCs</t> were stimulated overnight with Gag 263-272 (KK10, HLA-B*27 + peptide). Black asterisks indicate statistically significant difference (P
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    Intracellular cytokine staining of CP CD8 + T cells. A-D: CD8 + T cells of HLA-B*57 positive CP were either freshly isolated (left) or stimulated with either wild type (center) or escape variant (right) HLA-B*57-restricted Gag peptides for 7 days. CPs were either on suppressive HAART regiments with undetectable viral loads (circles), were on HAART regimens but recently had detectable levels of viremia (triangles), or were not on HAART and had high levels of viremia (diamond). Cells from each group underwent an overnight stimulation with individual peptides. Percentage of CD8 + T cells expressing IFN-γ when restimulated with TW10 (A) , KF11 (B) , and IW9 (C) in blue, or the escape mutant variant peptide containing T242N/G248A (A) , A163S (B) , and I147L (C) mutations in red is shown. E-H : Percentage of CD8 + T cells expressing both IFN-γ and perforin after overnight stimulation with TW10 (E) , KF11 (F) , and IW9 (G) in blue, or the escape mutant variant peptide containing T242N/G248A (E) , A163S (F) , and I147L (G) mutations in red is shown. D and H show CD8 + T cells that express IFN-γ or co-express IFN-γ and perforin when PBMCs were stimulated overnight with Gag 263-272 (KK10, HLA-B*27 + peptide). Black asterisks indicate statistically significant difference (P

    Journal: Retrovirology

    Article Title: CD8+ T cells from HLA-B*57 elite suppressors effectively suppress replication of HIV-1 escape mutants

    doi: 10.1186/1742-4690-10-152

    Figure Lengend Snippet: Intracellular cytokine staining of CP CD8 + T cells. A-D: CD8 + T cells of HLA-B*57 positive CP were either freshly isolated (left) or stimulated with either wild type (center) or escape variant (right) HLA-B*57-restricted Gag peptides for 7 days. CPs were either on suppressive HAART regiments with undetectable viral loads (circles), were on HAART regimens but recently had detectable levels of viremia (triangles), or were not on HAART and had high levels of viremia (diamond). Cells from each group underwent an overnight stimulation with individual peptides. Percentage of CD8 + T cells expressing IFN-γ when restimulated with TW10 (A) , KF11 (B) , and IW9 (C) in blue, or the escape mutant variant peptide containing T242N/G248A (A) , A163S (B) , and I147L (C) mutations in red is shown. E-H : Percentage of CD8 + T cells expressing both IFN-γ and perforin after overnight stimulation with TW10 (E) , KF11 (F) , and IW9 (G) in blue, or the escape mutant variant peptide containing T242N/G248A (E) , A163S (F) , and I147L (G) mutations in red is shown. D and H show CD8 + T cells that express IFN-γ or co-express IFN-γ and perforin when PBMCs were stimulated overnight with Gag 263-272 (KK10, HLA-B*27 + peptide). Black asterisks indicate statistically significant difference (P

    Article Snippet: Intracellular cytokine analysis 0.5 × 106 unstimulated or stimulated PBMCs (from above) were restimulated with peptide (2 ug/mL), anti-CD28, and anti-CD49d in the presence of GolgiStop and GolgiPlug (BD) for 12 h. After staining with CD3 PE and CD8 APC-H7 (BD), cells were fixed and permeablized with Cytoperm/Cytofix Kit (BD).

    Techniques: Staining, Isolation, Variant Assay, Expressing, Mutagenesis

    Intracellular cytokine staining of ES CD8 + T cells. A-D: CD8 + T cells of HLA-B*57 positive ES were either freshly isolated (left) or stimulated with either wild type (center) or escape variant (right) HLA-B*57-restricted Gag peptides for 7 days. Cells from each group underwent an overnight stimulation with individual peptides. Percentage of CD8 + T cells expressing IFN-γ when stimulated overnight with TW10 (A) , KF11 (B) , and IW9 (C) in blue, or the escape mutant variant peptide containing T242N/G248A (A) , A163S (B) , and I147L (C) mutations in red is shown. E-H : Percentage of CD8 + T cells expressing both IFN-γ and perforin after restimulation with TW10 (E) , KF11 (F) , and IW9 (B) in blue, or the escape mutant variant peptide containing T242N/G248A (E) , A163S (F) , and I147L (G) in red is shown. D and H show CD8 + T cells that express IFN-γ or co-express IFN-γ and perforin when PBMCs were stimulated overnight with Gag 263-272 (KK10, HLA-B*27 + peptide). Black horizontal bars indicate statistically significant difference (P

    Journal: Retrovirology

    Article Title: CD8+ T cells from HLA-B*57 elite suppressors effectively suppress replication of HIV-1 escape mutants

    doi: 10.1186/1742-4690-10-152

    Figure Lengend Snippet: Intracellular cytokine staining of ES CD8 + T cells. A-D: CD8 + T cells of HLA-B*57 positive ES were either freshly isolated (left) or stimulated with either wild type (center) or escape variant (right) HLA-B*57-restricted Gag peptides for 7 days. Cells from each group underwent an overnight stimulation with individual peptides. Percentage of CD8 + T cells expressing IFN-γ when stimulated overnight with TW10 (A) , KF11 (B) , and IW9 (C) in blue, or the escape mutant variant peptide containing T242N/G248A (A) , A163S (B) , and I147L (C) mutations in red is shown. E-H : Percentage of CD8 + T cells expressing both IFN-γ and perforin after restimulation with TW10 (E) , KF11 (F) , and IW9 (B) in blue, or the escape mutant variant peptide containing T242N/G248A (E) , A163S (F) , and I147L (G) in red is shown. D and H show CD8 + T cells that express IFN-γ or co-express IFN-γ and perforin when PBMCs were stimulated overnight with Gag 263-272 (KK10, HLA-B*27 + peptide). Black horizontal bars indicate statistically significant difference (P

    Article Snippet: Intracellular cytokine analysis 0.5 × 106 unstimulated or stimulated PBMCs (from above) were restimulated with peptide (2 ug/mL), anti-CD28, and anti-CD49d in the presence of GolgiStop and GolgiPlug (BD) for 12 h. After staining with CD3 PE and CD8 APC-H7 (BD), cells were fixed and permeablized with Cytoperm/Cytofix Kit (BD).

    Techniques: Staining, Isolation, Variant Assay, Expressing, Mutagenesis

    Flow cytometry analysis of different subsets of CD4+ T cells. PBMCs were isolated form individual participants and stimulated with, or without, PMA/ionomycin and harvested. The cells were stained with APC-anti-CD4, fixed, and permeabilized, followed by intracellular staining with FITC-anti-IL-17, PE-Cy7-anti-IFNγ, and PE-anti-IL-22 and flow cytometry. Subsequently, the cells were gated first on CD4 + cells for analysis of the frequency of CD4 + IFNγ + and CD4 + IFNγ − cells. The CD4 + IFNγ + cells were further analyzed for CD4 + IFNγ + IL-17 + cells (column I), while the CD4 + IFNγ − cells were further analyzed for CD4 + IFNγ − IL-17 + , CD4 + IFNγ − IL-22 + , and CD4 + IFNγ − IL-17 + IL-22 + cells (column II), followed by quantitative analyses. Data are representative charts or expressed as the mean values of individual participants from sequential experiments. A. Representative charts of flow cytometry analysis; B. Quantitative analysis.

    Journal: PLoS ONE

    Article Title: A High Frequency of Circulating Th22 and Th17 Cells in Patients with New Onset Graves' Disease

    doi: 10.1371/journal.pone.0068446

    Figure Lengend Snippet: Flow cytometry analysis of different subsets of CD4+ T cells. PBMCs were isolated form individual participants and stimulated with, or without, PMA/ionomycin and harvested. The cells were stained with APC-anti-CD4, fixed, and permeabilized, followed by intracellular staining with FITC-anti-IL-17, PE-Cy7-anti-IFNγ, and PE-anti-IL-22 and flow cytometry. Subsequently, the cells were gated first on CD4 + cells for analysis of the frequency of CD4 + IFNγ + and CD4 + IFNγ − cells. The CD4 + IFNγ + cells were further analyzed for CD4 + IFNγ + IL-17 + cells (column I), while the CD4 + IFNγ − cells were further analyzed for CD4 + IFNγ − IL-17 + , CD4 + IFNγ − IL-22 + , and CD4 + IFNγ − IL-17 + IL-22 + cells (column II), followed by quantitative analyses. Data are representative charts or expressed as the mean values of individual participants from sequential experiments. A. Representative charts of flow cytometry analysis; B. Quantitative analysis.

    Article Snippet: Briefly, the stimulated PBMCs were harvested and stained with allophycocyanin (APC)-labeled anti-CD4, fixed with the Perm/Fix solution, and permeabilized, followed by staining with fluorescein isothiocyanate (FITC)-labeled anti–IL-17, PE-Cy7-labeled anti-IFNγ (Becton Dickinson, San Diego, USA), and PE-labeled anti–IL-22 (R & D Systems, Minneapolis, MN, USA).

    Techniques: Flow Cytometry, Cytometry, Isolation, Staining

    CD4 T cell subsets repartition and contribution to the pool of infected cells. A : Monocytes, activated and resting CD4 T-cell (CD4 T Ly) contributions to the pool of infected PBMCs were calculated with the infection level and frequency of each subset. Only significant p values are shown. B : The repartition of resting CD4 T-cell subsets was assessed in twelve acutely HIV-infected individuals (grey) and in ten uninfected individuals (white). The analyzed resting CD4+ subsets are: naive (TN, CD45RA+CCR7+CD27+), central-memory (TCM, CD45RA-CCR7+CD27+), transitional-memory (TTM, CD45RA−CCR7−CD27+) and effector-memory cells (TEM, CD45RA−CCR7−CD27−). Results are expressed as the percentage of resting CD4 T cells. C : Resting CD4 T-cell subset contributions to the pool of infected resting CD4 T cells were calculated with the infection level and frequency of each subset.

    Journal: PLoS ONE

    Article Title: A Single HIV-1 Cluster and a Skewed Immune Homeostasis Drive the Early Spread of HIV among Resting CD4+ Cell Subsets within One Month Post-Infection

    doi: 10.1371/journal.pone.0064219

    Figure Lengend Snippet: CD4 T cell subsets repartition and contribution to the pool of infected cells. A : Monocytes, activated and resting CD4 T-cell (CD4 T Ly) contributions to the pool of infected PBMCs were calculated with the infection level and frequency of each subset. Only significant p values are shown. B : The repartition of resting CD4 T-cell subsets was assessed in twelve acutely HIV-infected individuals (grey) and in ten uninfected individuals (white). The analyzed resting CD4+ subsets are: naive (TN, CD45RA+CCR7+CD27+), central-memory (TCM, CD45RA-CCR7+CD27+), transitional-memory (TTM, CD45RA−CCR7−CD27+) and effector-memory cells (TEM, CD45RA−CCR7−CD27−). Results are expressed as the percentage of resting CD4 T cells. C : Resting CD4 T-cell subset contributions to the pool of infected resting CD4 T cells were calculated with the infection level and frequency of each subset.

    Article Snippet: CD4 T Cell Differentiation Analysis and Sorting PBMC cryopreserved and stored in liquid nitrogen, with more than 80% viability after thawing, were sorted as live monocytes (CD3−CD4+) or activated and resting CD3+CD4+ T cells on a 5-laser FACS ARIA (Becton Dickinson) on the CyPS platform (UPMC) after staining with the following combination: Live-Dead Fixable Aqua (Life Technologies), CD3-Pacific Blue (UCHT1), CD4-AlexaFluor700 (RPA-T4), CCR7-PE Cyanine7 (3D12), CD27-APC (L128), CD69-FITC (L78), HLA-DR-FITC (L243) and CD127-PE (IL7R-M21) from BD Pharmingen, CD45RA-ECD (2H4) and CD25-FITC (B1.49.9) from Beckman Coulter.

    Techniques: Infection, Transmission Electron Microscopy

    TSA, SAHA, and MS-275 decrease TLR9-induced activation of IRF-7 and NF-κB p65 transcription factors. PBMC were incubated with TSA (100 ng/ml) or DMSO (1:1000) for 1 hour, stimulated with HSV-1 (MOI of 1) or IAV (MOI of 2) for 3 hours. Samples were processed using BD Phosflow protocol. PBMC were gated based on SSC vs. FSC-A (Area) and pDC were identified by gating on +HLA-DRhigh/+CD123high events on a HLA-DR APC vs. CD123 PE intensity dot plot, then percent phosphorylated IRF-7+ pDC were gated based on mock sample for HSV-1 or IAV from which percentages were reported. Representative analysis for TSA, SAHA, and MS-275-mediated effect on IAV-mediated IRF-7 and NF-kB p65 phosphorylation in pDC. Red lines represent the position where the gate was placed from which percentages were derived. (A) . Pooled data with HSV-1 stimulation (top panels) and IAV (bottom panels) showing the effect of TSA (100 ng/ml) (B) , MS-275 (5 μM) (C) , and SAHA (500 nM) (D) , or DMSO (1:1000) on the phosphorylation of IRF-7 and NF-kB p65. Data represent the percentage of pIRF-7+ and pNF-κB p65+ pDC with gating based on mock sample. (N = 4-5, 4-5 independent experiments with different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; n.s. = not significant).

    Journal: bioRxiv

    Article Title: HDAC inhibitors decrease TLR7/9-mediated human plasmacytoid dendritic cell activation by interfering with IRF-7 and NF-κB signaling

    doi: 10.1101/2020.05.09.085456

    Figure Lengend Snippet: TSA, SAHA, and MS-275 decrease TLR9-induced activation of IRF-7 and NF-κB p65 transcription factors. PBMC were incubated with TSA (100 ng/ml) or DMSO (1:1000) for 1 hour, stimulated with HSV-1 (MOI of 1) or IAV (MOI of 2) for 3 hours. Samples were processed using BD Phosflow protocol. PBMC were gated based on SSC vs. FSC-A (Area) and pDC were identified by gating on +HLA-DRhigh/+CD123high events on a HLA-DR APC vs. CD123 PE intensity dot plot, then percent phosphorylated IRF-7+ pDC were gated based on mock sample for HSV-1 or IAV from which percentages were reported. Representative analysis for TSA, SAHA, and MS-275-mediated effect on IAV-mediated IRF-7 and NF-kB p65 phosphorylation in pDC. Red lines represent the position where the gate was placed from which percentages were derived. (A) . Pooled data with HSV-1 stimulation (top panels) and IAV (bottom panels) showing the effect of TSA (100 ng/ml) (B) , MS-275 (5 μM) (C) , and SAHA (500 nM) (D) , or DMSO (1:1000) on the phosphorylation of IRF-7 and NF-kB p65. Data represent the percentage of pIRF-7+ and pNF-κB p65+ pDC with gating based on mock sample. (N = 4-5, 4-5 independent experiments with different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; n.s. = not significant).

    Article Snippet: In order to answer this question, PBMC were isolated and pre-treated with TSA or SAHA for 1 hour, then stimulated with HSV-1 for 3 hours, then we utilized BD Phosflow™ assays to assess the phosphorylation status of IRF-7 and NF-κB p65 in the presence of TSA, MS-275, SAHA.

    Techniques: Activation Assay, Incubation, Derivative Assay

    TSA and MS-275 inhibit HSV-1 and IAV-induced total IFN-α production in human pDC. PBMC were isolated and pre-treated with TSA (100 ng/ml) or DMSO vehicle control (1:1000) (A) and MS-275 (5 μM) or DMSO vehicle control (1:530) (B) for 1 hour and then stimulated with HSV-1 (MOI of 1) and IAV (MOI of 2) for 18 hours. Supernatants were collected and tested for IFN-α production by an ELISA assay. (N=4, 4 independent experiments from different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; *p

    Journal: bioRxiv

    Article Title: HDAC inhibitors decrease TLR7/9-mediated human plasmacytoid dendritic cell activation by interfering with IRF-7 and NF-κB signaling

    doi: 10.1101/2020.05.09.085456

    Figure Lengend Snippet: TSA and MS-275 inhibit HSV-1 and IAV-induced total IFN-α production in human pDC. PBMC were isolated and pre-treated with TSA (100 ng/ml) or DMSO vehicle control (1:1000) (A) and MS-275 (5 μM) or DMSO vehicle control (1:530) (B) for 1 hour and then stimulated with HSV-1 (MOI of 1) and IAV (MOI of 2) for 18 hours. Supernatants were collected and tested for IFN-α production by an ELISA assay. (N=4, 4 independent experiments from different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; *p

    Article Snippet: In order to answer this question, PBMC were isolated and pre-treated with TSA or SAHA for 1 hour, then stimulated with HSV-1 for 3 hours, then we utilized BD Phosflow™ assays to assess the phosphorylation status of IRF-7 and NF-κB p65 in the presence of TSA, MS-275, SAHA.

    Techniques: Isolation, Enzyme-linked Immunosorbent Assay

    TSA and MS-275 inhibit the upregulation of pDC maturation markers upon TLR7/9 stimulation, and increase the shedding of the activation marker CD62L. PBMC were incubated with TSA (100 ng/ml), MS-275 (5 μM), or DMSO (1:530) for 1 hour and then stimulated with HSV-1 (MOI of 1), IAV (MOI of 2), or CpG-B (5 μg/ml) for 8 hours. Representative data derived from events first gated on PBMC, then pDC were identified by a BDCA-2 + /CD123 high gate. From this gate, histograms were derived and gates applied based on the unstimulated control in DMSO (Mock) (A) . Surface expression of CD62L (B, top) , CD40 (B, bottom) , CD86 (C, top), CD82 (C, bottom) were measured by flow cytometry and reported as percentage. (N = 4, 4 independent experiments with different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA and Bonferroni’s post test; *p

    Journal: bioRxiv

    Article Title: HDAC inhibitors decrease TLR7/9-mediated human plasmacytoid dendritic cell activation by interfering with IRF-7 and NF-κB signaling

    doi: 10.1101/2020.05.09.085456

    Figure Lengend Snippet: TSA and MS-275 inhibit the upregulation of pDC maturation markers upon TLR7/9 stimulation, and increase the shedding of the activation marker CD62L. PBMC were incubated with TSA (100 ng/ml), MS-275 (5 μM), or DMSO (1:530) for 1 hour and then stimulated with HSV-1 (MOI of 1), IAV (MOI of 2), or CpG-B (5 μg/ml) for 8 hours. Representative data derived from events first gated on PBMC, then pDC were identified by a BDCA-2 + /CD123 high gate. From this gate, histograms were derived and gates applied based on the unstimulated control in DMSO (Mock) (A) . Surface expression of CD62L (B, top) , CD40 (B, bottom) , CD86 (C, top), CD82 (C, bottom) were measured by flow cytometry and reported as percentage. (N = 4, 4 independent experiments with different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA and Bonferroni’s post test; *p

    Article Snippet: In order to answer this question, PBMC were isolated and pre-treated with TSA or SAHA for 1 hour, then stimulated with HSV-1 for 3 hours, then we utilized BD Phosflow™ assays to assess the phosphorylation status of IRF-7 and NF-κB p65 in the presence of TSA, MS-275, SAHA.

    Techniques: Activation Assay, Marker, Incubation, Derivative Assay, Expressing, Flow Cytometry

    TSA and MS-275, but not SAHA, inhibits IAV and HSV-1-mediated IFN-α protein upregulation without decreasing IRF-7 upregulation. PBMC were pre-treated with TSA (100 ng/ml), SAHA (500 nM) and DMSO vehicle control (1:1000) or MS-275 (5 μM) and DMSO (1:530), for 1 hour and then stimulated with HSV-1 (MOI of 1) or IAV (MOI of 2) for 6 hours. IRF-7 (black bars) and intracellular IFN-α (gray bars) protein expression was measured concurrently by intracellular flow cytometry. Representative data showing the effect, on IAV-induced IFN (A, top) and IRF-7 (A, bottom) upregulation in pDC, by TSA, MS-275, and SAHA (A). Cumulative experiments showing the effect on HSV-1 (top) and IAV (bottom)-induced upregulation of IFN-α and IRF-7, by TSA (100 ng/ml) (B) and MS-275 (5 μM) (C) and SAHA (500 nM) (D). (N = 5; 5 independent experiments with different donors for IAV. N = 3; 3 independent experiments with different donors for HSV-1. Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; (* = p

    Journal: bioRxiv

    Article Title: HDAC inhibitors decrease TLR7/9-mediated human plasmacytoid dendritic cell activation by interfering with IRF-7 and NF-κB signaling

    doi: 10.1101/2020.05.09.085456

    Figure Lengend Snippet: TSA and MS-275, but not SAHA, inhibits IAV and HSV-1-mediated IFN-α protein upregulation without decreasing IRF-7 upregulation. PBMC were pre-treated with TSA (100 ng/ml), SAHA (500 nM) and DMSO vehicle control (1:1000) or MS-275 (5 μM) and DMSO (1:530), for 1 hour and then stimulated with HSV-1 (MOI of 1) or IAV (MOI of 2) for 6 hours. IRF-7 (black bars) and intracellular IFN-α (gray bars) protein expression was measured concurrently by intracellular flow cytometry. Representative data showing the effect, on IAV-induced IFN (A, top) and IRF-7 (A, bottom) upregulation in pDC, by TSA, MS-275, and SAHA (A). Cumulative experiments showing the effect on HSV-1 (top) and IAV (bottom)-induced upregulation of IFN-α and IRF-7, by TSA (100 ng/ml) (B) and MS-275 (5 μM) (C) and SAHA (500 nM) (D). (N = 5; 5 independent experiments with different donors for IAV. N = 3; 3 independent experiments with different donors for HSV-1. Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; (* = p

    Article Snippet: In order to answer this question, PBMC were isolated and pre-treated with TSA or SAHA for 1 hour, then stimulated with HSV-1 for 3 hours, then we utilized BD Phosflow™ assays to assess the phosphorylation status of IRF-7 and NF-κB p65 in the presence of TSA, MS-275, SAHA.

    Techniques: Expressing, Flow Cytometry

    TSA and MS-275 inhibit HSV-1 and IAV-induced IFN-α and TNF-α production in human pDC in a dose-dependent manner. PBMC were incubated with TSA and MS-275 at decreasing concentrations for 1 hour and then stimulated with HSV-1 or IAV for 6 hours. Cells were then processed for intracellular flow cytometry to detect IFN-α and TNF-α production in pDC. Representative histograms showing the differential inhibition of intracellular HSV-1 and IAV-induced IFN-α and TNF-α production in pDC by TSA (100 ng/ml = 330 nM) or DMSO vehicle control (1:1000) (A), and MS-275 (5 μM) or DMSO vehicle control (1:530) (C). Pooled data of dose curve experiments showing a dose-dependent inhibitory effect on HSV-1- (MOI of 1; squares) or IAV (MOI of 2; triangles) -induced IFN-α and TNF-α by TSA (0-100 ng/ml) (C) and MS-275 (0-5 μM) (D) when compared to their respective vehicle controls. (TSA, N=3; MS-275, N=4; 3-4 independent experiments from different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; *p

    Journal: bioRxiv

    Article Title: HDAC inhibitors decrease TLR7/9-mediated human plasmacytoid dendritic cell activation by interfering with IRF-7 and NF-κB signaling

    doi: 10.1101/2020.05.09.085456

    Figure Lengend Snippet: TSA and MS-275 inhibit HSV-1 and IAV-induced IFN-α and TNF-α production in human pDC in a dose-dependent manner. PBMC were incubated with TSA and MS-275 at decreasing concentrations for 1 hour and then stimulated with HSV-1 or IAV for 6 hours. Cells were then processed for intracellular flow cytometry to detect IFN-α and TNF-α production in pDC. Representative histograms showing the differential inhibition of intracellular HSV-1 and IAV-induced IFN-α and TNF-α production in pDC by TSA (100 ng/ml = 330 nM) or DMSO vehicle control (1:1000) (A), and MS-275 (5 μM) or DMSO vehicle control (1:530) (C). Pooled data of dose curve experiments showing a dose-dependent inhibitory effect on HSV-1- (MOI of 1; squares) or IAV (MOI of 2; triangles) -induced IFN-α and TNF-α by TSA (0-100 ng/ml) (C) and MS-275 (0-5 μM) (D) when compared to their respective vehicle controls. (TSA, N=3; MS-275, N=4; 3-4 independent experiments from different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; *p

    Article Snippet: In order to answer this question, PBMC were isolated and pre-treated with TSA or SAHA for 1 hour, then stimulated with HSV-1 for 3 hours, then we utilized BD Phosflow™ assays to assess the phosphorylation status of IRF-7 and NF-κB p65 in the presence of TSA, MS-275, SAHA.

    Techniques: Incubation, Flow Cytometry, Inhibition