Structured Review

Becton Dickinson human pbmcs
<t>EAT-2</t> over-expression enhances human DC maturation. Human <t>PBMCs</t> were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-EAT2 or rAd5-Null. At 72 hpi, cells were stained for surface expression of CD86 (A), HLA-DR (B), CD80 (C) and CD83 (D) and FACS analysis was performed. Data are representative of at least three independent experiments from three different blood donors with similar results. Samples were plated in quadruplicates. Bars represent mean ± SD. Statistical analysis was completed using a one way ANOVA with a Student–Newman–Keuls post-hoc test. P
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1) Product Images from "Manipulation of EAT-2 expression promotes induction of multiple beneficial regulatory and effector functions of the human innate immune system as a novel immunomodulatory strategy"

Article Title: Manipulation of EAT-2 expression promotes induction of multiple beneficial regulatory and effector functions of the human innate immune system as a novel immunomodulatory strategy

Journal: International Immunology

doi: 10.1093/intimm/dxt061

EAT-2 over-expression enhances human DC maturation. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-EAT2 or rAd5-Null. At 72 hpi, cells were stained for surface expression of CD86 (A), HLA-DR (B), CD80 (C) and CD83 (D) and FACS analysis was performed. Data are representative of at least three independent experiments from three different blood donors with similar results. Samples were plated in quadruplicates. Bars represent mean ± SD. Statistical analysis was completed using a one way ANOVA with a Student–Newman–Keuls post-hoc test. P
Figure Legend Snippet: EAT-2 over-expression enhances human DC maturation. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-EAT2 or rAd5-Null. At 72 hpi, cells were stained for surface expression of CD86 (A), HLA-DR (B), CD80 (C) and CD83 (D) and FACS analysis was performed. Data are representative of at least three independent experiments from three different blood donors with similar results. Samples were plated in quadruplicates. Bars represent mean ± SD. Statistical analysis was completed using a one way ANOVA with a Student–Newman–Keuls post-hoc test. P

Techniques Used: Over Expression, Isolation, Infection, Staining, Expressing, FACS

Transduction of human innate and adaptive immune cells by GFP-expressing rAd5 vector. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-GFP. FACS analysis for GFP expression at 48h following rAd5-GFP infection of human NK cells (A), CD4 + and CD8 + T cells (B), CD14 + monocytes and CD1a + cells (C) is shown. Data were collected using a BD LSRII flow cytometer and analyzed using FlowJo software.
Figure Legend Snippet: Transduction of human innate and adaptive immune cells by GFP-expressing rAd5 vector. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-GFP. FACS analysis for GFP expression at 48h following rAd5-GFP infection of human NK cells (A), CD4 + and CD8 + T cells (B), CD14 + monocytes and CD1a + cells (C) is shown. Data were collected using a BD LSRII flow cytometer and analyzed using FlowJo software.

Techniques Used: Transduction, Expressing, Plasmid Preparation, Isolation, Infection, FACS, Flow Cytometry, Cytometry, Software

Intracellular staining analysis for EAT-2 expression in human immune cells following rAd5-EAT2 infection. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-EAT2 or rAd5-EAT2(R31Q). Intracellular staining analysis for EAT-2 expression at 48h following Ad infection of human NK cells (A), CD14 + monocytes (B) and CD1a + DCs (C) is shown. Data were collected using a BD LSRII flow cytometer and analyzed using FlowJo software.
Figure Legend Snippet: Intracellular staining analysis for EAT-2 expression in human immune cells following rAd5-EAT2 infection. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-EAT2 or rAd5-EAT2(R31Q). Intracellular staining analysis for EAT-2 expression at 48h following Ad infection of human NK cells (A), CD14 + monocytes (B) and CD1a + DCs (C) is shown. Data were collected using a BD LSRII flow cytometer and analyzed using FlowJo software.

Techniques Used: Staining, Expressing, Infection, Isolation, Flow Cytometry, Cytometry, Software

EAT-2 over-expression enhances the cytolytic activity of human NK cells. Human PBMCs isolated from fresh buffy coat material were either mock infected or infected with MOI of 10000 with rAd5-EAT2, rAd5-EAT2(R31Q) or rAd5-Null for 72h and then co-cultured with CFSE-labeled K562 cells for an additional 48h at an E:T ratio of 1:10. Viability of K562 cells was assessed by either PI (A and B) or ViViD (C). Data are representative of two (A) or three (B) independent experiments from three different blood donors with similar results. Samples were plated in quadruplicates. Bars represent mean ± SD. Statistical analysis was completed using a one way ANOVA with a Student–Newman–Keuls post-hoc test. P
Figure Legend Snippet: EAT-2 over-expression enhances the cytolytic activity of human NK cells. Human PBMCs isolated from fresh buffy coat material were either mock infected or infected with MOI of 10000 with rAd5-EAT2, rAd5-EAT2(R31Q) or rAd5-Null for 72h and then co-cultured with CFSE-labeled K562 cells for an additional 48h at an E:T ratio of 1:10. Viability of K562 cells was assessed by either PI (A and B) or ViViD (C). Data are representative of two (A) or three (B) independent experiments from three different blood donors with similar results. Samples were plated in quadruplicates. Bars represent mean ± SD. Statistical analysis was completed using a one way ANOVA with a Student–Newman–Keuls post-hoc test. P

Techniques Used: Over Expression, Activity Assay, Isolation, Infection, Cell Culture, Labeling

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

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0088441

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

Techniques Used: Labeling, Flow Cytometry, Cytometry

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

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

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

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

3) Product Images from "Potential of PEGylated Toll-Like Receptor 7 Ligands for Controlling Inflammation and Functional Changes in Mouse Models of Asthma and Silicosis"

Article Title: Potential of PEGylated Toll-Like Receptor 7 Ligands for Controlling Inflammation and Functional Changes in Mouse Models of Asthma and Silicosis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2016.00095

Concentration of IL1-β (A) , IL-6 (B) , IL-12 (C) , IL-8 (D) , TNF-α (E) and IL-10 (F) in the supernatant of human PBMCs, after 24 h stimulation with TMX-202, TMX-302 or TMX-306 at 1 or 10 μM. Data are presented as mean ± SEM from n = 6.
Figure Legend Snippet: Concentration of IL1-β (A) , IL-6 (B) , IL-12 (C) , IL-8 (D) , TNF-α (E) and IL-10 (F) in the supernatant of human PBMCs, after 24 h stimulation with TMX-202, TMX-302 or TMX-306 at 1 or 10 μM. Data are presented as mean ± SEM from n = 6.

Techniques Used: Concentration Assay

4) Product Images from "TGF-?1 Down-Regulation of NKG2D/DAP10 and 2B4/SAP Expression on Human NK Cells Contributes to HBV Persistence"

Article Title: TGF-?1 Down-Regulation of NKG2D/DAP10 and 2B4/SAP Expression on Human NK Cells Contributes to HBV Persistence

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002594

Deficient NK cell function in IT patients. (A) Deficient NK cell cytotoxicity from IT patients. NK cell cytotoxicity was assessed in primary NK cells from IT, IA, and IN patients and healthy controls using a 51 Cr-release assay with K562 target cells. (B) Results show the mean ± SEM values in 19 patients and 5 controls. Values for IT patients were less than controls (P = 0.0384). (C) Positive correlation between NK cell cytotoxicity and the percentage of NK cells expressing NKG2D and 2B4. (D) Fresh PBMCs were stimulated with/without IL-12 as described in the Materials and Methods . After 16 h, IFN-γ production was determined using flow cytometry by gating on CD3 − CD56 + NK cells. A representative dot plot displaying intracellular IFNγ staining in NK cells with the subsets indicated is shown. (E) Cumulative data are shown. (F, G) Fresh PBMCs from patients before treatment (black) or after treatment (white) were stimulated with IL-12 for 16 h. IFNγ (F) and CD107a (G) production was determined using flow cytometry by gating on CD3 − CD56 + NK cells. (H) Induction of ex vivo NK cell cytotoxic activity after in vivo administration of antiviral treatment with nucleoside analogues until ALT reached normal levels. NK cytotoxicity was determined ex vivo by measuring the lysis of 51 Cr-labelled target cells before treatment (red) or after treatment (green). Cumulative data are shown.
Figure Legend Snippet: Deficient NK cell function in IT patients. (A) Deficient NK cell cytotoxicity from IT patients. NK cell cytotoxicity was assessed in primary NK cells from IT, IA, and IN patients and healthy controls using a 51 Cr-release assay with K562 target cells. (B) Results show the mean ± SEM values in 19 patients and 5 controls. Values for IT patients were less than controls (P = 0.0384). (C) Positive correlation between NK cell cytotoxicity and the percentage of NK cells expressing NKG2D and 2B4. (D) Fresh PBMCs were stimulated with/without IL-12 as described in the Materials and Methods . After 16 h, IFN-γ production was determined using flow cytometry by gating on CD3 − CD56 + NK cells. A representative dot plot displaying intracellular IFNγ staining in NK cells with the subsets indicated is shown. (E) Cumulative data are shown. (F, G) Fresh PBMCs from patients before treatment (black) or after treatment (white) were stimulated with IL-12 for 16 h. IFNγ (F) and CD107a (G) production was determined using flow cytometry by gating on CD3 − CD56 + NK cells. (H) Induction of ex vivo NK cell cytotoxic activity after in vivo administration of antiviral treatment with nucleoside analogues until ALT reached normal levels. NK cytotoxicity was determined ex vivo by measuring the lysis of 51 Cr-labelled target cells before treatment (red) or after treatment (green). Cumulative data are shown.

Techniques Used: Cell Function Assay, IA, Release Assay, Expressing, Flow Cytometry, Cytometry, Staining, Ex Vivo, Activity Assay, In Vivo, Lysis

5) Product Images from "TLR3 and Rig-Like Receptor on Myeloid Dendritic Cells and Rig-Like Receptor on Human NK Cells Are Both Mandatory for Production of IFN-? in Response to Double-Stranded RNA"

Article Title: TLR3 and Rig-Like Receptor on Myeloid Dendritic Cells and Rig-Like Receptor on Human NK Cells Are Both Mandatory for Production of IFN-? in Response to Double-Stranded RNA

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

doi: 10.4049/jimmunol.1000532

mDCs are required for NK cells activation in response to dsRNA. NK cells activation was evaluated by CD107a staining ( upper panels ) and the IFN-γ production ( lower panels ), as described (). A , A total of 2 × 10 6 /ml whole human PBMCs
Figure Legend Snippet: mDCs are required for NK cells activation in response to dsRNA. NK cells activation was evaluated by CD107a staining ( upper panels ) and the IFN-γ production ( lower panels ), as described (). A , A total of 2 × 10 6 /ml whole human PBMCs

Techniques Used: Activation Assay, Staining

Both poly(I:C) and poly(A:U) trigger NK cell activation within PBMCs, but only poly(I:C) induces high IFN-γ production by NK cells. A , A total of 2 × 10 6 /ml human PBMCs were stimulated for 20 h with poly(I:C) and poly(A:U). Culture SNs
Figure Legend Snippet: Both poly(I:C) and poly(A:U) trigger NK cell activation within PBMCs, but only poly(I:C) induces high IFN-γ production by NK cells. A , A total of 2 × 10 6 /ml human PBMCs were stimulated for 20 h with poly(I:C) and poly(A:U). Culture SNs

Techniques Used: Activation Assay

6) Product Images from "Distribution of circulating T follicular helper cell subsets is altered in immunoglobulin A vasculitis in children"

Article Title: Distribution of circulating T follicular helper cell subsets is altered in immunoglobulin A vasculitis in children

Journal: PLoS ONE

doi: 10.1371/journal.pone.0189133

Detection of circulating Tfh cell subsets by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated from patients with immunoglobulin A vasculitis (IgAV) (n = 30) and age and gender-matched healthy controls (HCs; n = 15), labeled with fluorophore-conjugated antibodies, and analyzed by flow cytometry. A gating strategy was used to identify CD3 + CD4 + T helper (Th) and CD4 + CXCR5 + CD45RA − , CD45RA − CXCR3 + CCR6 − , CD45RA − CXCR3 − CCR6 − , CD45RA − CXCR3 − CCR6 + , and CD45RA − CXCR3 + CCR6 + follicular helper T (Tfh) cell subsets.
Figure Legend Snippet: Detection of circulating Tfh cell subsets by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated from patients with immunoglobulin A vasculitis (IgAV) (n = 30) and age and gender-matched healthy controls (HCs; n = 15), labeled with fluorophore-conjugated antibodies, and analyzed by flow cytometry. A gating strategy was used to identify CD3 + CD4 + T helper (Th) and CD4 + CXCR5 + CD45RA − , CD45RA − CXCR3 + CCR6 − , CD45RA − CXCR3 − CCR6 − , CD45RA − CXCR3 − CCR6 + , and CD45RA − CXCR3 + CCR6 + follicular helper T (Tfh) cell subsets.

Techniques Used: Flow Cytometry, Cytometry, Isolation, Labeling

7) Product Images from "Phosphodiesterase-5 inhibition augments endogenous antitumor immunity by reducing myeloid-derived suppressor cell function"

Article Title: Phosphodiesterase-5 inhibition augments endogenous antitumor immunity by reducing myeloid-derived suppressor cell function

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20061104

PDE5 inhibition restores proliferation of head and neck and myeloma lymphocytes. (A) Unfractionated or CD14-depleted PBMCs from MM patients were stimulated with anti-CD3/CD28 antibody–coated beads in the presence of NorNOHA, l -NMMA, both NorNOHA and l -NMMA, sildenafil, or no inhibitor. The CD3 + T cell expansion was measured 5 d later by flow cytometry. (B) Ficoll-purified PBMCs from healthy donors ( n = 4), head and neck cancer patients (H N; n = 7), or MM patients ( n = 7) were stimulated as described in A in the presence or absence of sildenafil. CD4 + and CD8 + T cell expansion was measured by flow cytometry 5 d later. Data are reported as fold change. t test p-values are reported. Horizontal lines represent the median, the 10th and 90th percentile.
Figure Legend Snippet: PDE5 inhibition restores proliferation of head and neck and myeloma lymphocytes. (A) Unfractionated or CD14-depleted PBMCs from MM patients were stimulated with anti-CD3/CD28 antibody–coated beads in the presence of NorNOHA, l -NMMA, both NorNOHA and l -NMMA, sildenafil, or no inhibitor. The CD3 + T cell expansion was measured 5 d later by flow cytometry. (B) Ficoll-purified PBMCs from healthy donors ( n = 4), head and neck cancer patients (H N; n = 7), or MM patients ( n = 7) were stimulated as described in A in the presence or absence of sildenafil. CD4 + and CD8 + T cell expansion was measured by flow cytometry 5 d later. Data are reported as fold change. t test p-values are reported. Horizontal lines represent the median, the 10th and 90th percentile.

Techniques Used: Inhibition, Flow Cytometry, Cytometry, Purification

8) Product Images from "Involvement of CD226+ NK Cells in Immunopathogenesis of Systemic Lupus Erythematosus"

Article Title: Involvement of CD226+ NK Cells in Immunopathogenesis of Systemic Lupus Erythematosus

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

doi: 10.4049/jimmunol.1000569

Downregulated expression of CD226 on NK cells from SLE patients and MRL/lpr mice with active disease. Freshly isolated PBMCs were stained with anti-CD3, CD56, and CD226 mAbs. A, NK cells were gated ( top panel ), and expression of CD226 was analyzed ( bottom
Figure Legend Snippet: Downregulated expression of CD226 on NK cells from SLE patients and MRL/lpr mice with active disease. Freshly isolated PBMCs were stained with anti-CD3, CD56, and CD226 mAbs. A, NK cells were gated ( top panel ), and expression of CD226 was analyzed ( bottom

Techniques Used: Expressing, Mouse Assay, Isolation, Staining

The frequency of NK cells is reduced in SLE patients with active disease. Freshly isolated PBMCs were stained with anti-CD3, CD11c, CD19, CD56, Vα24, and Vβ11 mAbs and analyzed by flow cytometry. A and D , The relative percentages of NK
Figure Legend Snippet: The frequency of NK cells is reduced in SLE patients with active disease. Freshly isolated PBMCs were stained with anti-CD3, CD11c, CD19, CD56, Vα24, and Vβ11 mAbs and analyzed by flow cytometry. A and D , The relative percentages of NK

Techniques Used: Isolation, Staining, Flow Cytometry, Cytometry

9) Product Images from "Control of Established Colon Cancer Xenografts Using a Novel Humanized Single Chain Antibody-Streptococcal Superantigen Fusion Protein Targeting the 5T4 Oncofetal Antigen"

Article Title: Control of Established Colon Cancer Xenografts Using a Novel Humanized Single Chain Antibody-Streptococcal Superantigen Fusion Protein Targeting the 5T4 Oncofetal Antigen

Journal: PLoS ONE

doi: 10.1371/journal.pone.0095200

SpeC-based TTS therapy of established HT-29 colon cancer. A) Schematic illustration of the xenograft solid tumor model experimental timeline. NSG mice with established (3 week) intraperitoneal human HT-29 tumors were injected once with human PBMC intraperitoneally, followed by 8 daily intravenous injections of scFv5T4::SpeC D203A , or individual controls (2 µM/kg/injection). B) Primary tumor size was evaluated throughout the experimental timeline by external caliper measurements. Twenty-eight days post-final injection, final tumor volume was measured (panel C) and metastatic score (panel D) evaluated. All groups contained n = 4, with exception of saline alone control (n = 3). *p
Figure Legend Snippet: SpeC-based TTS therapy of established HT-29 colon cancer. A) Schematic illustration of the xenograft solid tumor model experimental timeline. NSG mice with established (3 week) intraperitoneal human HT-29 tumors were injected once with human PBMC intraperitoneally, followed by 8 daily intravenous injections of scFv5T4::SpeC D203A , or individual controls (2 µM/kg/injection). B) Primary tumor size was evaluated throughout the experimental timeline by external caliper measurements. Twenty-eight days post-final injection, final tumor volume was measured (panel C) and metastatic score (panel D) evaluated. All groups contained n = 4, with exception of saline alone control (n = 3). *p

Techniques Used: Mouse Assay, Injection

Functionality of SpeC mutants and fusion proteins for human PBMC proliferation and cytotoxicity in vitro . A) SpeC proteins were used to compare scFv5T4 alone, scFv5T4::SpeC Y15A/D203A and subsequently scFv5T4::SpeC D203A in the uptake of 3 H-thymidine as a measure of PBMC proliferation after 4 day incubation (n = 5). B–C) Dose-dependent SpeC-mediated PBMC cytotoxicity of scFv5T4::SpeC D203A was determined by comparing SpeC controls, scFv5T4 alone and scFv5T4::SpeC Y15A/D203A after 48 h incubation by using FACS analysis of WiDr (panel B), measuring percent cancer cell death with 7AAD-exclusion staining (n = 3) and 51 Cr-release to measure the specific cytotoxic potential (panel C) when incubated with increasing effector∶target ratios and 51 Cr-labeled HT-29 cancer cells. Data shown (mean ±SEM) is from four independent human donors each done in triplicate. *p
Figure Legend Snippet: Functionality of SpeC mutants and fusion proteins for human PBMC proliferation and cytotoxicity in vitro . A) SpeC proteins were used to compare scFv5T4 alone, scFv5T4::SpeC Y15A/D203A and subsequently scFv5T4::SpeC D203A in the uptake of 3 H-thymidine as a measure of PBMC proliferation after 4 day incubation (n = 5). B–C) Dose-dependent SpeC-mediated PBMC cytotoxicity of scFv5T4::SpeC D203A was determined by comparing SpeC controls, scFv5T4 alone and scFv5T4::SpeC Y15A/D203A after 48 h incubation by using FACS analysis of WiDr (panel B), measuring percent cancer cell death with 7AAD-exclusion staining (n = 3) and 51 Cr-release to measure the specific cytotoxic potential (panel C) when incubated with increasing effector∶target ratios and 51 Cr-labeled HT-29 cancer cells. Data shown (mean ±SEM) is from four independent human donors each done in triplicate. *p

Techniques Used: In Vitro, Incubation, FACS, Staining, Labeling

10) Product Images from "Pathogenic role of circulating CD4+CXCR5+ cell subpopulations in patients with chronic spontaneous urticarial"

Article Title: Pathogenic role of circulating CD4+CXCR5+ cell subpopulations in patients with chronic spontaneous urticarial

Journal: American Journal of Translational Research

doi:

The status of circulating CD4 + CXCR5 + , Tfh, Tfr and Tfh/Tfr ratio in peripheral blood mononuclear cells (PBMCs) of patients with chronic spontaneous urticaria (CSU). A. Gating strategy to identify circulating Tfh (CD4 + CXCR5 + CD25 low CD127 high ) and Tfr (CD4 + CXCR5 + CD25 high CD127 low ) cells among the CD4 + CXCR5 + T cells in human peripheral blood. B. Percentage of circulating CD4 + CXCR5 + , Tfh and Tfr cells among PBMCs and the Tfh/Tfr in patients with CSU and healthy controls (HC). Each plot represented individual subject. Horizontal bars indicated the mean and error bars the SEM. Significance between two groups was evaluated with a two-tailed Student’s t -test and indicated by p value (* P
Figure Legend Snippet: The status of circulating CD4 + CXCR5 + , Tfh, Tfr and Tfh/Tfr ratio in peripheral blood mononuclear cells (PBMCs) of patients with chronic spontaneous urticaria (CSU). A. Gating strategy to identify circulating Tfh (CD4 + CXCR5 + CD25 low CD127 high ) and Tfr (CD4 + CXCR5 + CD25 high CD127 low ) cells among the CD4 + CXCR5 + T cells in human peripheral blood. B. Percentage of circulating CD4 + CXCR5 + , Tfh and Tfr cells among PBMCs and the Tfh/Tfr in patients with CSU and healthy controls (HC). Each plot represented individual subject. Horizontal bars indicated the mean and error bars the SEM. Significance between two groups was evaluated with a two-tailed Student’s t -test and indicated by p value (* P

Techniques Used: Two Tailed Test

11) Product Images from "Experimental infection of dromedaries with Middle East respiratory syndrome-Coronavirus is accompanied by massive ciliary loss and depletion of the cell surface receptor dipeptidyl peptidase 4"

Article Title: Experimental infection of dromedaries with Middle East respiratory syndrome-Coronavirus is accompanied by massive ciliary loss and depletion of the cell surface receptor dipeptidyl peptidase 4

Journal: Scientific Reports

doi: 10.1038/s41598-018-28109-2

Expression of dipeptidyl peptidase 4 (DPP4) in lymphoid organs of humans and dromedaries. ( A ) DPP4 (red) is frequently detectable on lymphoid cells (white arrows) in the human tonsil but is completely absent in tonsillar tissue of dromedaries. Immunofluorescence, 400x. In the lymph nodes of both human and dromedary, DPP4 (red) is detected in the paracortex and medulla (arrows), but in much smaller numbers in that of dromedaries (arrow). Immunohistochemistry, 400x. In human PBMC, DPP4 is mainly expressed by CD3 + T cells and hardly expressed by CD20 + B cells, CD56 + NK cells, and CD14 + monocytes ( B ). S1 protein of MERS-CoV is used to detect DPP4 + cells in both human and dromedary PBMC. Lymphocytes and monocytes population are differentiated based on their size and granularity. DPP4 is mainly expressed by lymphocytes in human PBMC while it is mainly expressed by monocytes in dromedary PBMC ( C ). Data in figure B and C are visualized as the mean percentage.
Figure Legend Snippet: Expression of dipeptidyl peptidase 4 (DPP4) in lymphoid organs of humans and dromedaries. ( A ) DPP4 (red) is frequently detectable on lymphoid cells (white arrows) in the human tonsil but is completely absent in tonsillar tissue of dromedaries. Immunofluorescence, 400x. In the lymph nodes of both human and dromedary, DPP4 (red) is detected in the paracortex and medulla (arrows), but in much smaller numbers in that of dromedaries (arrow). Immunohistochemistry, 400x. In human PBMC, DPP4 is mainly expressed by CD3 + T cells and hardly expressed by CD20 + B cells, CD56 + NK cells, and CD14 + monocytes ( B ). S1 protein of MERS-CoV is used to detect DPP4 + cells in both human and dromedary PBMC. Lymphocytes and monocytes population are differentiated based on their size and granularity. DPP4 is mainly expressed by lymphocytes in human PBMC while it is mainly expressed by monocytes in dromedary PBMC ( C ). Data in figure B and C are visualized as the mean percentage.

Techniques Used: Expressing, Immunofluorescence, Immunohistochemistry

12) Product Images from "Control of Established Colon Cancer Xenografts Using a Novel Humanized Single Chain Antibody-Streptococcal Superantigen Fusion Protein Targeting the 5T4 Oncofetal Antigen"

Article Title: Control of Established Colon Cancer Xenografts Using a Novel Humanized Single Chain Antibody-Streptococcal Superantigen Fusion Protein Targeting the 5T4 Oncofetal Antigen

Journal: PLoS ONE

doi: 10.1371/journal.pone.0095200

Functionality of SpeC mutants and fusion proteins for human PBMC proliferation and cytotoxicity in vitro . A) SpeC proteins were used to compare scFv5T4 alone, scFv5T4::SpeC Y15A/D203A and subsequently scFv5T4::SpeC D203A in the uptake of 3 H-thymidine as a measure of PBMC proliferation after 4 day incubation (n = 5). B–C) Dose-dependent SpeC-mediated PBMC cytotoxicity of scFv5T4::SpeC D203A was determined by comparing SpeC controls, scFv5T4 alone and scFv5T4::SpeC Y15A/D203A after 48 h incubation by using FACS analysis of WiDr (panel B), measuring percent cancer cell death with 7AAD-exclusion staining (n = 3) and 51 Cr-release to measure the specific cytotoxic potential (panel C) when incubated with increasing effector∶target ratios and 51 Cr-labeled HT-29 cancer cells. Data shown (mean ±SEM) is from four independent human donors each done in triplicate. *p
Figure Legend Snippet: Functionality of SpeC mutants and fusion proteins for human PBMC proliferation and cytotoxicity in vitro . A) SpeC proteins were used to compare scFv5T4 alone, scFv5T4::SpeC Y15A/D203A and subsequently scFv5T4::SpeC D203A in the uptake of 3 H-thymidine as a measure of PBMC proliferation after 4 day incubation (n = 5). B–C) Dose-dependent SpeC-mediated PBMC cytotoxicity of scFv5T4::SpeC D203A was determined by comparing SpeC controls, scFv5T4 alone and scFv5T4::SpeC Y15A/D203A after 48 h incubation by using FACS analysis of WiDr (panel B), measuring percent cancer cell death with 7AAD-exclusion staining (n = 3) and 51 Cr-release to measure the specific cytotoxic potential (panel C) when incubated with increasing effector∶target ratios and 51 Cr-labeled HT-29 cancer cells. Data shown (mean ±SEM) is from four independent human donors each done in triplicate. *p

Techniques Used: In Vitro, Incubation, FACS, Staining, Labeling

Overview of the SpeC-mediated T cell activation complex and mutations to reduce systemic toxicity. A) Structural overview of SpeC in complex with TCR and MHC-II. TCR Vα chain is colored orange, TCR Vβ chain is colored grey, MHCα-chains are colored red, MHCβ-chains are colored green, antigenic peptides are colored black, and the zinc atom is colored magenta. SpeC is colored blue with important interface residues Y15 and D203 highlighted in yellow. The ternary model of TCR-SpeC-(MHC) 2 was produced as described previously [9] and the ribbon diagram was generated using PyMOL ( http://www.pymol.org ). B) Proliferation of human PBMCs mediated by SpeC WT or proteins containing mutated residues Y15A (TCR-binding mutant), D203A (MHC-II-binding mutant) or Y15A/D203A was determined by the uptake of 3 H-thymidine after 72 h post-stimulation (n = 5 in triplicate; data representative of one individual). C) Dose-dependent cytotoxicity of 7-AAD + WiDr cells after 48 h incubation with human PBMCs and either SpeC WT , SpeC Y15A , SpeC D203A , or SpeC Y15A/D203A (n = 3–6 per group). (D–E) Proliferation of CFSE labeled-human PBMCs mediated by SpeC WT or proteins containing mutated residues was determined by FACS five days post-stimulation, specifically measuring total CD3 + T cell population, CD3 + CD4 + T cells and, CD3 + CD8 + T cells (n = 4; FACS data representative of one individual).
Figure Legend Snippet: Overview of the SpeC-mediated T cell activation complex and mutations to reduce systemic toxicity. A) Structural overview of SpeC in complex with TCR and MHC-II. TCR Vα chain is colored orange, TCR Vβ chain is colored grey, MHCα-chains are colored red, MHCβ-chains are colored green, antigenic peptides are colored black, and the zinc atom is colored magenta. SpeC is colored blue with important interface residues Y15 and D203 highlighted in yellow. The ternary model of TCR-SpeC-(MHC) 2 was produced as described previously [9] and the ribbon diagram was generated using PyMOL ( http://www.pymol.org ). B) Proliferation of human PBMCs mediated by SpeC WT or proteins containing mutated residues Y15A (TCR-binding mutant), D203A (MHC-II-binding mutant) or Y15A/D203A was determined by the uptake of 3 H-thymidine after 72 h post-stimulation (n = 5 in triplicate; data representative of one individual). C) Dose-dependent cytotoxicity of 7-AAD + WiDr cells after 48 h incubation with human PBMCs and either SpeC WT , SpeC Y15A , SpeC D203A , or SpeC Y15A/D203A (n = 3–6 per group). (D–E) Proliferation of CFSE labeled-human PBMCs mediated by SpeC WT or proteins containing mutated residues was determined by FACS five days post-stimulation, specifically measuring total CD3 + T cell population, CD3 + CD4 + T cells and, CD3 + CD8 + T cells (n = 4; FACS data representative of one individual).

Techniques Used: Activation Assay, Produced, Generated, Binding Assay, Mutagenesis, Incubation, Labeling, FACS

13) Product Images from "Comparison of the super agonist complex, ALT-803, to IL-15 as cancer immunotherapeutics in animal models"

Article Title: Comparison of the super agonist complex, ALT-803, to IL-15 as cancer immunotherapeutics in animal models

Journal: Cancer immunology research

doi: 10.1158/2326-6066.CIR-15-0093-T

ALT-803 induces cytokine release and proliferation of mouse and human immune cells in vitro . A , human PBMCs ( n = 3 from normal donors) were incubated for 4 days in wells containing media and the indicated concentrations of soluble or plastic-immobilized
Figure Legend Snippet: ALT-803 induces cytokine release and proliferation of mouse and human immune cells in vitro . A , human PBMCs ( n = 3 from normal donors) were incubated for 4 days in wells containing media and the indicated concentrations of soluble or plastic-immobilized

Techniques Used: In Vitro, Incubation

ALT-803 stimulates proliferation and activation of human NK cells and T cells in vitro . A , human PBMCs ( n = 7 normal donors) were cultured for 7 days in media alone or media containing 0.5 nM ALT-803 or IL-15. At the end of the incubation period, changes
Figure Legend Snippet: ALT-803 stimulates proliferation and activation of human NK cells and T cells in vitro . A , human PBMCs ( n = 7 normal donors) were cultured for 7 days in media alone or media containing 0.5 nM ALT-803 or IL-15. At the end of the incubation period, changes

Techniques Used: Activation Assay, In Vitro, Cell Culture, Incubation

14) Product Images from "Validation of a multicolor staining to monitor phosphoSTAT5 levels in regulatory T-cell subsets"

Article Title: Validation of a multicolor staining to monitor phosphoSTAT5 levels in regulatory T-cell subsets

Journal: Oncotarget

doi:

Impact of Anti-thymocyte globulin (ATG) and cyclosporin A (CSA) on T reg phospho STAT5 levels in vivo and in vitro A.-B. Human PBMCs, freshly isolated from 3 healthy donors were cultured in triplicate in the presence of either PBS, CSA (500 ng/ml) or ATG (100 μg/ml) for 24 hours. The impact of these compounds on CD4 + CD25 high FOXP3 + cell frequency A. as well as on phospho STAT5 levels in T regs B. was assessed by flow cytometry using the PFE technique for permeabilization. Data show median values of 9 replicates (3 biological replicates in triplicates / condition) with interquartile range (* p
Figure Legend Snippet: Impact of Anti-thymocyte globulin (ATG) and cyclosporin A (CSA) on T reg phospho STAT5 levels in vivo and in vitro A.-B. Human PBMCs, freshly isolated from 3 healthy donors were cultured in triplicate in the presence of either PBS, CSA (500 ng/ml) or ATG (100 μg/ml) for 24 hours. The impact of these compounds on CD4 + CD25 high FOXP3 + cell frequency A. as well as on phospho STAT5 levels in T regs B. was assessed by flow cytometry using the PFE technique for permeabilization. Data show median values of 9 replicates (3 biological replicates in triplicates / condition) with interquartile range (* p

Techniques Used: In Vivo, In Vitro, Isolation, Cell Culture, Flow Cytometry, Cytometry

15) Product Images from "STAT5B: a differential regulator of the life and death of CD4+ effector memory T cells"

Article Title: STAT5B: a differential regulator of the life and death of CD4+ effector memory T cells

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

doi: 10.4049/jimmunol.1701133

Patient with heterozygous Q206R mutation in STAT5B exhibits accumulation of CD4 + TEM cells in blood. (a) Pedigree of the affected family: black filled symbol, subject with mutation Q206R; grey filled symbol, subject with mutation Q206R and different clinical phenotype; open symbols, unaffected subjects; (?), unscreened subjects. (b) Schematic representation of the STAT5B protein domain structure. N-term, N-terminus domain (pink); Coiled-coil (CC) domain (green); DNA, DNA-binding domain (red); LINK, linker domain (yellow); SH2, src homology 2 domain (blue). Tyrosine phosphorylation (P, red circle) represented in the transcriptional activation (TA) domain (gray). The Q206R residue is located in the CC domain (orange triangle). (c) Three-dimensional provisional models of STAT5B as a tetramer bound to DNA molecule (gray). Three orientations of the STAT5B tetramer are shown, with Q206R positions indicated as orange circles. (d) Absolute numbers of lymphocytes in the patient’s peripheral blood as a function of age. Arrows indicate times of steroid treatment. (e) Flow cytometry plot of peripheral blood from healthy subject (normal control) and patient, stained for CD27 versus CCR7 (gated on CD4 + CD45RA - memory T cells), identifying central memory T cells (TCM: CD27 + CCR7 + ) and effector memory T cells (TEM: CD27 - CCR7 - ). (f) Absolute numbers of TCM and TEM cells per 10 5 cells of PBMCs from healthy donors (blue) and multiple blood draws from the patient (red). ns = not significant; * p=
Figure Legend Snippet: Patient with heterozygous Q206R mutation in STAT5B exhibits accumulation of CD4 + TEM cells in blood. (a) Pedigree of the affected family: black filled symbol, subject with mutation Q206R; grey filled symbol, subject with mutation Q206R and different clinical phenotype; open symbols, unaffected subjects; (?), unscreened subjects. (b) Schematic representation of the STAT5B protein domain structure. N-term, N-terminus domain (pink); Coiled-coil (CC) domain (green); DNA, DNA-binding domain (red); LINK, linker domain (yellow); SH2, src homology 2 domain (blue). Tyrosine phosphorylation (P, red circle) represented in the transcriptional activation (TA) domain (gray). The Q206R residue is located in the CC domain (orange triangle). (c) Three-dimensional provisional models of STAT5B as a tetramer bound to DNA molecule (gray). Three orientations of the STAT5B tetramer are shown, with Q206R positions indicated as orange circles. (d) Absolute numbers of lymphocytes in the patient’s peripheral blood as a function of age. Arrows indicate times of steroid treatment. (e) Flow cytometry plot of peripheral blood from healthy subject (normal control) and patient, stained for CD27 versus CCR7 (gated on CD4 + CD45RA - memory T cells), identifying central memory T cells (TCM: CD27 + CCR7 + ) and effector memory T cells (TEM: CD27 - CCR7 - ). (f) Absolute numbers of TCM and TEM cells per 10 5 cells of PBMCs from healthy donors (blue) and multiple blood draws from the patient (red). ns = not significant; * p=

Techniques Used: Mutagenesis, Transmission Electron Microscopy, Binding Assay, Activation Assay, Flow Cytometry, Cytometry, Staining

16) Product Images from "CD8+ T-Cell Responses to Trypanosoma cruzi Are Highly Focused on Strain-Variant trans-Sialidase Epitopes"

Article Title: CD8+ T-Cell Responses to Trypanosoma cruzi Are Highly Focused on Strain-Variant trans-Sialidase Epitopes

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.0020077

Detection of CD8 + T Lymphocytes Recognizing HLA-A2.1–Restricted ts Epitopes in Patients with Chronic Chagas Disease PBMCs were stained using MHC tetramers specific for the TSA2–38 or TSA2–44 epitopes. The percentage of CD8 + T cells staining positive for TSA2–38 is shown for a representative chronic chagasic patient and an uninfected control. Cells shown are lymphocytes gated on CD4 neg B220 neg CD11b neg lymphocytes. Five out of eight IFNγ ELISPOT + subjects stained positive with tetramers.
Figure Legend Snippet: Detection of CD8 + T Lymphocytes Recognizing HLA-A2.1–Restricted ts Epitopes in Patients with Chronic Chagas Disease PBMCs were stained using MHC tetramers specific for the TSA2–38 or TSA2–44 epitopes. The percentage of CD8 + T cells staining positive for TSA2–38 is shown for a representative chronic chagasic patient and an uninfected control. Cells shown are lymphocytes gated on CD4 neg B220 neg CD11b neg lymphocytes. Five out of eight IFNγ ELISPOT + subjects stained positive with tetramers.

Techniques Used: Staining, Enzyme-linked Immunospot

17) Product Images from "Identification of Cinnabarinic Acid as a Novel Endogenous Aryl Hydrocarbon Receptor Ligand That Drives IL-22 Production"

Article Title: Identification of Cinnabarinic Acid as a Novel Endogenous Aryl Hydrocarbon Receptor Ligand That Drives IL-22 Production

Journal: PLoS ONE

doi: 10.1371/journal.pone.0087877

IL-22 + upregulation occurs in CD4 + 8 - T cells that are IL-17 negative but express IFNγ. (A) Cytokine production for live CD3 + CD4 + CD8 − T cells from human PBMC cultures that were stimulated with anti-CD3 and anti-CD28 antibodies and allogeneic APCs for six days with DMSO or 50 µM 3-HAA. Panel (right) depicts fold-changes in 9 donors relative to DMSO. *, p
Figure Legend Snippet: IL-22 + upregulation occurs in CD4 + 8 - T cells that are IL-17 negative but express IFNγ. (A) Cytokine production for live CD3 + CD4 + CD8 − T cells from human PBMC cultures that were stimulated with anti-CD3 and anti-CD28 antibodies and allogeneic APCs for six days with DMSO or 50 µM 3-HAA. Panel (right) depicts fold-changes in 9 donors relative to DMSO. *, p

Techniques Used:

CA is an endogenous AHR agonist generated by oxidative dimerization of 3-HAA. (A) Metabolic pathways downstream of 3-HAA catalyzed by the enzyme, 3-hydroxyanthranilate 3,4-dioxygenase (HAAO), including intermediates upstream of PA and QA. (B) Flow cytometric analysis of CD4 + T cells in human PBMCs stimulated in the presence of varying concentrations of 3-HAA (50 µM or 100 µM) with or without the HAAO inhibitor, 4-F-3-HAA (50 µM). Data are representative of three experiments. (C) An alternative pathway of 3-HAA metabolism can generate CA either by non-enzymatic (oxidation) or enzymatic (such as laccase or ceruloplasmin) processes. (D) Fluorescence [measured in relative fluorescence units (RFUs)] of the AHR-responsive reporter construct in cells incubated with varying concentrations of 3-HAA or CA. Tryptamine was a positive control. *, p
Figure Legend Snippet: CA is an endogenous AHR agonist generated by oxidative dimerization of 3-HAA. (A) Metabolic pathways downstream of 3-HAA catalyzed by the enzyme, 3-hydroxyanthranilate 3,4-dioxygenase (HAAO), including intermediates upstream of PA and QA. (B) Flow cytometric analysis of CD4 + T cells in human PBMCs stimulated in the presence of varying concentrations of 3-HAA (50 µM or 100 µM) with or without the HAAO inhibitor, 4-F-3-HAA (50 µM). Data are representative of three experiments. (C) An alternative pathway of 3-HAA metabolism can generate CA either by non-enzymatic (oxidation) or enzymatic (such as laccase or ceruloplasmin) processes. (D) Fluorescence [measured in relative fluorescence units (RFUs)] of the AHR-responsive reporter construct in cells incubated with varying concentrations of 3-HAA or CA. Tryptamine was a positive control. *, p

Techniques Used: Generated, Flow Cytometry, Fluorescence, Construct, Incubation, Positive Control

3-HKA and 3-HAA promote IL-22 expression in stimulated human CD4 + T cells. (A) Flow cytometric analysis of CD4 + T cells following stimulation of human PBMCs in the presence of 100 µM 3-HKA, 3-HAA, PA, or QA for six days. Data represent at least three independent experiments. (B) Flow cytometric analysis of CD4 + T cells from individual and aggregate donors following stimulation of PBMCs in the presence of increasing concentrations of 3-HAA (µM) for six days. Individual donor data are pooled from at least three independent experiments. Error bars indicate SD. Fold change in IL-22 expression versus vehicle control is statistically different from 1 (Wilcoxon signed rank test; *, p = 0.0312; **, p = 0.0078; N = 8 donors). (C) Flow cytometric analysis of CD4 + T cells following stimulation of human PBMCs in the presence of 3-HAA +/− the AHR antagonist, CH-223191. Data are representative of at least three independent experiments. (D) Comparison of IL-22 production in CD4 + T cells following stimulation of human PBMCs in the presence of DMSO, 3-HKA (50 µM), or 3-HAA (50 µM), with or without an AHR antagonist, N = 6. P values were calculated by Mann-Whitney. Error bars indicate SD.
Figure Legend Snippet: 3-HKA and 3-HAA promote IL-22 expression in stimulated human CD4 + T cells. (A) Flow cytometric analysis of CD4 + T cells following stimulation of human PBMCs in the presence of 100 µM 3-HKA, 3-HAA, PA, or QA for six days. Data represent at least three independent experiments. (B) Flow cytometric analysis of CD4 + T cells from individual and aggregate donors following stimulation of PBMCs in the presence of increasing concentrations of 3-HAA (µM) for six days. Individual donor data are pooled from at least three independent experiments. Error bars indicate SD. Fold change in IL-22 expression versus vehicle control is statistically different from 1 (Wilcoxon signed rank test; *, p = 0.0312; **, p = 0.0078; N = 8 donors). (C) Flow cytometric analysis of CD4 + T cells following stimulation of human PBMCs in the presence of 3-HAA +/− the AHR antagonist, CH-223191. Data are representative of at least three independent experiments. (D) Comparison of IL-22 production in CD4 + T cells following stimulation of human PBMCs in the presence of DMSO, 3-HKA (50 µM), or 3-HAA (50 µM), with or without an AHR antagonist, N = 6. P values were calculated by Mann-Whitney. Error bars indicate SD.

Techniques Used: Expressing, Flow Cytometry, MANN-WHITNEY

CA increases the differentiation of IL-22 + human CD4 + T cells in vitro . (A) Flow cytometric analysis of CD4 + T cells from human PBMCs stimulated in the presence of DMSO or increasing doses of CA (left). Fold change in IL-22 production in CD4 + T cells from human PBMCs from multiple donors stimulated in the presence of CA versus DMSO control (right panel). Data were analyzed by Wilcoxon signed rank test for significant deviation from a theoretical median of 1.000. *p
Figure Legend Snippet: CA increases the differentiation of IL-22 + human CD4 + T cells in vitro . (A) Flow cytometric analysis of CD4 + T cells from human PBMCs stimulated in the presence of DMSO or increasing doses of CA (left). Fold change in IL-22 production in CD4 + T cells from human PBMCs from multiple donors stimulated in the presence of CA versus DMSO control (right panel). Data were analyzed by Wilcoxon signed rank test for significant deviation from a theoretical median of 1.000. *p

Techniques Used: In Vitro, Flow Cytometry

18) Product Images from "Lentiviral Nef Proteins Manipulate T Cells in a Subset-Specific Manner"

Article Title: Lentiviral Nef Proteins Manipulate T Cells in a Subset-Specific Manner

Journal: Journal of Virology

doi: 10.1128/JVI.03104-14

Characterization of uninfected and HIV-infected CD4 + T cell subsets in HLACs and PBMC cultures. (A) Expression of the surface markers CD45RA, CD45RO, CCR5, and CD62L on CD4 + T cells from representative PBMC and HLACs. Numbers give percentages of cells
Figure Legend Snippet: Characterization of uninfected and HIV-infected CD4 + T cell subsets in HLACs and PBMC cultures. (A) Expression of the surface markers CD45RA, CD45RO, CCR5, and CD62L on CD4 + T cells from representative PBMC and HLACs. Numbers give percentages of cells

Techniques Used: Infection, Expressing

Nef modulates both TCR-CD3 and CD4 in a T cell subset-specific manner. (A) PBMCs were infected with X4 HIV-1 IRES-eGFP constructs expressing the parental NL4-3 or SIVmac239 Nef proteins. Virally infected (eGFP + ) cells were first examined for CD3 and CD4
Figure Legend Snippet: Nef modulates both TCR-CD3 and CD4 in a T cell subset-specific manner. (A) PBMCs were infected with X4 HIV-1 IRES-eGFP constructs expressing the parental NL4-3 or SIVmac239 Nef proteins. Virally infected (eGFP + ) cells were first examined for CD3 and CD4

Techniques Used: Infection, Construct, Expressing

TCR-CD3 downmodulation protects memory T cells against activation and apoptosis. (A and B) Flow cytometric analysis of CD69 (A) and CD25 (B) expression by PBMCs infected with X4 or R5 HIV-1 constructs expressing G1 or G2 nef alleles. U, uninfected control
Figure Legend Snippet: TCR-CD3 downmodulation protects memory T cells against activation and apoptosis. (A and B) Flow cytometric analysis of CD69 (A) and CD25 (B) expression by PBMCs infected with X4 or R5 HIV-1 constructs expressing G1 or G2 nef alleles. U, uninfected control

Techniques Used: Activation Assay, Flow Cytometry, Expressing, Infection, Construct

Nef-mediated modulation of TCR-CD3 is inefficient in Tdp cells. (A) PBMC cultures and HLACs were infected with X4 or R5 HIV-1 IRES-eGFP constructs expressing nef alleles capable of downmodulating TCR-CD3 and analyzed by flow cytometric analysis. HIV-1-infected
Figure Legend Snippet: Nef-mediated modulation of TCR-CD3 is inefficient in Tdp cells. (A) PBMC cultures and HLACs were infected with X4 or R5 HIV-1 IRES-eGFP constructs expressing nef alleles capable of downmodulating TCR-CD3 and analyzed by flow cytometric analysis. HIV-1-infected

Techniques Used: Infection, Construct, Expressing, Flow Cytometry

19) Product Images from "A Standardized Chemically Modified Curcuma longa Extract Modulates IRAK-MAPK Signaling in Inflammation and Potentiates Cytotoxicity"

Article Title: A Standardized Chemically Modified Curcuma longa Extract Modulates IRAK-MAPK Signaling in Inflammation and Potentiates Cytotoxicity

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2016.00223

Effects of CMCE on cell viability of HL-60, MCF-7, MDA-MB-231 cells, and PBMCs. The cells were treated with different concentrations of CMCE (1, 10, 30, and 100 μg/mL) or DOX (1 μg/mL) for 20 h and cell viability was determined by (A) PI permeability in HL-60, MCF-7, MDA-MB-231 cells, and PBMCs (B) SRB assay in HL-60 cells. Values represent the mean (at least n = 3) ± SEM; ∗ p
Figure Legend Snippet: Effects of CMCE on cell viability of HL-60, MCF-7, MDA-MB-231 cells, and PBMCs. The cells were treated with different concentrations of CMCE (1, 10, 30, and 100 μg/mL) or DOX (1 μg/mL) for 20 h and cell viability was determined by (A) PI permeability in HL-60, MCF-7, MDA-MB-231 cells, and PBMCs (B) SRB assay in HL-60 cells. Values represent the mean (at least n = 3) ± SEM; ∗ p

Techniques Used: Multiple Displacement Amplification, Permeability, Sulforhodamine B Assay

20) Product Images from "WASP regulates suppressor activity of human and murine CD4+CD25+FOXP3+ natural regulatory T cells"

Article Title: WASP regulates suppressor activity of human and murine CD4+CD25+FOXP3+ natural regulatory T cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20061334

Immunophenotype of nTreg cells from human peripheral blood. (a) CD4 + FOXP3 + cells in the peripheral blood of WAS patients. PBMCs from four representative HDs and seven WAS patients (WAS) were stained with anti-CD4 and anti-FOXP3 mAbs. Results shown are gated on the CD4 + T cells. Numbers indicate the percentages of FOXP3 + cells among CD4 + T cells. (b) Percentage of FOXP3 + cells (left), CD25 + FOXP3 + cells (middle), and CD25 − FOXP3 + cells (right) among CD4 + T cells of HDs ( n = 16) and WAS patients ( n = 7). Bars represent the median value for each group.
Figure Legend Snippet: Immunophenotype of nTreg cells from human peripheral blood. (a) CD4 + FOXP3 + cells in the peripheral blood of WAS patients. PBMCs from four representative HDs and seven WAS patients (WAS) were stained with anti-CD4 and anti-FOXP3 mAbs. Results shown are gated on the CD4 + T cells. Numbers indicate the percentages of FOXP3 + cells among CD4 + T cells. (b) Percentage of FOXP3 + cells (left), CD25 + FOXP3 + cells (middle), and CD25 − FOXP3 + cells (right) among CD4 + T cells of HDs ( n = 16) and WAS patients ( n = 7). Bars represent the median value for each group.

Techniques Used: Staining

Related Articles

Transduction:

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Article Snippet: .. To identify human immune cells transduced by Ad-mediated EAT-2 or GFP gene transduction, human PBMCs (2×106 ) were infected with multiplicity of infection (MOI) of 10000 of Ad5-EAT-2 or Ad5-GFP for 48h and were then stained with the following antibodies: APC-Cy7-CD14, PE-CD1a, Alexa Fluor 700-CD8a, V450-CD4, APC-CD65 and PerCpCy5.5-CD3 (all at 4 µg ml−1 ; BD Biosciences). .. Human PBMCs were then fixed with 2% formaldehyde, permeabilized with saponin and intracellularly stained with FITC-labeled EAT-2 antibody and finally subjected to flow cytometry.

Flow Cytometry:

Article Title: T Follicular Helper Cells and Regulatory B Cells Dynamics in Systemic Lupus Erythematosus
Article Snippet: .. Flow Cytometry For detection of Tfh cells, human PBMCs were stained with Alexa Fluor 647-conjugated anti-CD4, Alexa Fluor 488-conjugated anti-CXCR5, and phycoerythrin (PE)-conjugated anti-PD-1 (all from BD Pharmingen, San Jose, CA). ..

Fluorescence:

Article Title: Productive Infection of Human Peripheral Blood Mononuclear Cells by Feline Immunodeficiency Virus: Implications for Vector Development
Article Snippet: .. Analysis of FIV p24 expression in human PBMC infected with Petaluma or V1 CSF was performed on a Becton Dickinson FacScan fluorescence activated cell sorter with a 488-nm laser for excitation. .. Duplicate cell cultures (2 × 106 ) were harvested on day 4 p.i., washed twice with phosphate-buffered saline (PBS) by centrifugation, and fixed with a solution of 2% formaldehyde in PBS by incubation for 30 min at room temperature.

Cytometry:

Article Title: T Follicular Helper Cells and Regulatory B Cells Dynamics in Systemic Lupus Erythematosus
Article Snippet: .. Flow Cytometry For detection of Tfh cells, human PBMCs were stained with Alexa Fluor 647-conjugated anti-CD4, Alexa Fluor 488-conjugated anti-CXCR5, and phycoerythrin (PE)-conjugated anti-PD-1 (all from BD Pharmingen, San Jose, CA). ..

Infection:

Article Title: Manipulation of EAT-2 expression promotes induction of multiple beneficial regulatory and effector functions of the human innate immune system as a novel immunomodulatory strategy
Article Snippet: .. To identify human immune cells transduced by Ad-mediated EAT-2 or GFP gene transduction, human PBMCs (2×106 ) were infected with multiplicity of infection (MOI) of 10000 of Ad5-EAT-2 or Ad5-GFP for 48h and were then stained with the following antibodies: APC-Cy7-CD14, PE-CD1a, Alexa Fluor 700-CD8a, V450-CD4, APC-CD65 and PerCpCy5.5-CD3 (all at 4 µg ml−1 ; BD Biosciences). .. Human PBMCs were then fixed with 2% formaldehyde, permeabilized with saponin and intracellularly stained with FITC-labeled EAT-2 antibody and finally subjected to flow cytometry.

Article Title: Productive Infection of Human Peripheral Blood Mononuclear Cells by Feline Immunodeficiency Virus: Implications for Vector Development
Article Snippet: .. Analysis of FIV p24 expression in human PBMC infected with Petaluma or V1 CSF was performed on a Becton Dickinson FacScan fluorescence activated cell sorter with a 488-nm laser for excitation. .. Duplicate cell cultures (2 × 106 ) were harvested on day 4 p.i., washed twice with phosphate-buffered saline (PBS) by centrifugation, and fixed with a solution of 2% formaldehyde in PBS by incubation for 30 min at room temperature.

Enzyme-linked Immunosorbent Assay:

Article Title: TLR3 and Rig-Like Receptor on Myeloid Dendritic Cells and Rig-Like Receptor on Human NK Cells Are Both Mandatory for Production of IFN-? in Response to Double-Stranded RNA
Article Snippet: .. SNs of 20-h–activated human PBMCs, mDCs, or NK cells ± mDC cultures were collected and production of IFN-γ, IL-12p40, IL-12p70, IL-15 (BD Biosciences), and IFN-β (BioSource International, Camarillo, CA) was determined by ELISA. .. Mouse IFN-γ (BD Biosciences) was also quantified by ELISA in SNs of 20-h–activated mouse NK cells ± conventional DC (cDC) cultures.

Concentration Assay:

Article Title: Potential of PEGylated Toll-Like Receptor 7 Ligands for Controlling Inflammation and Functional Changes in Mouse Models of Asthma and Silicosis
Article Snippet: .. Cytokines Detection in Cell Supernatant The concentration of IL-1β, IL-6, IL-8, IL-10, IL-12, and TNF in the supernatant of human PBMCs and human mo-DCs was determined using the BD™ cytometric bead array (CBA) (human inflammatory cytokine kit – 551811, BD Biosciences), according to manufacturer’s instructions. .. Flow Cytometric Analysis For surface staining of human specimens, cell suspensions were incubated with the appropriate combination of the following monoclonal antibodies: CD19-PC5 (J3-119, Beckman Coulter), CD80-Brilliant Violet 421™ (2D10, BioLegend® ), CD83-APC (HB15e, BioLegend® ), CD86-APC (IT2.2, BioLegend® ), and HLA-DR-V500 (G46-6, BD Horizon™).

Expressing:

Article Title: Productive Infection of Human Peripheral Blood Mononuclear Cells by Feline Immunodeficiency Virus: Implications for Vector Development
Article Snippet: .. Analysis of FIV p24 expression in human PBMC infected with Petaluma or V1 CSF was performed on a Becton Dickinson FacScan fluorescence activated cell sorter with a 488-nm laser for excitation. .. Duplicate cell cultures (2 × 106 ) were harvested on day 4 p.i., washed twice with phosphate-buffered saline (PBS) by centrifugation, and fixed with a solution of 2% formaldehyde in PBS by incubation for 30 min at room temperature.

Staining:

Article Title: T Follicular Helper Cells and Regulatory B Cells Dynamics in Systemic Lupus Erythematosus
Article Snippet: .. Flow Cytometry For detection of Tfh cells, human PBMCs were stained with Alexa Fluor 647-conjugated anti-CD4, Alexa Fluor 488-conjugated anti-CXCR5, and phycoerythrin (PE)-conjugated anti-PD-1 (all from BD Pharmingen, San Jose, CA). ..

Article Title: Manipulation of EAT-2 expression promotes induction of multiple beneficial regulatory and effector functions of the human innate immune system as a novel immunomodulatory strategy
Article Snippet: .. To identify human immune cells transduced by Ad-mediated EAT-2 or GFP gene transduction, human PBMCs (2×106 ) were infected with multiplicity of infection (MOI) of 10000 of Ad5-EAT-2 or Ad5-GFP for 48h and were then stained with the following antibodies: APC-Cy7-CD14, PE-CD1a, Alexa Fluor 700-CD8a, V450-CD4, APC-CD65 and PerCpCy5.5-CD3 (all at 4 µg ml−1 ; BD Biosciences). .. Human PBMCs were then fixed with 2% formaldehyde, permeabilized with saponin and intracellularly stained with FITC-labeled EAT-2 antibody and finally subjected to flow cytometry.

Article Title: TGF-?1 Down-Regulation of NKG2D/DAP10 and 2B4/SAP Expression on Human NK Cells Contributes to HBV Persistence
Article Snippet: .. Abs against the following proteins were used for staining human PBMCs: CD3 (SK7), CD56 (B159), CD16 (3G8), CD27 (M-T271), CD226 (DX11), NKp30 (p30-15), NKp44 (p44-8.1), NKp46 (9E2/NKP46), NKG2D (1D11) (BD PharMingen) and 2B4 (eBioC1.7) (eBioscience). .. For intracellular cytokine assays, PBMCs were fixed in 2% formaldehyde after surface protein staining.

Article Title: Distribution of circulating T follicular helper cell subsets is altered in immunoglobulin A vasculitis in children
Article Snippet: .. Human PBMCs (106 /tube) were stained with BV510-anti-CD3 (clone: UCHT1), allophycocyanin (APC)-H7-anti-CD4 (clone: RPA-T4), BB515-anti-CXCR5 (clone: RF8B2), phycoerythrin (PE)-Cy5-anti-CD45RA (clone: HI100), PE-Cy7-anti-CCR6 (clone: 11A9), or APC-anti-CD183 (clone: IC6/CXCR3) (Becton Dickinson, San Jose, CA, USA) at room temperature for 30 min. Data were processed using FlowJo v.5.7.2 software (Tree Star, Ashland, OR, USA). .. Cytometric bead array (CBA) analysis of plasma cytokines Plasma IFN-γ and IL-4 and -17A concentrations were determined using the CBA Human Soluble Protein Master Buffer kit (BD Biosciences) on a flow cytometer.

Recombinase Polymerase Amplification:

Article Title: Distribution of circulating T follicular helper cell subsets is altered in immunoglobulin A vasculitis in children
Article Snippet: .. Human PBMCs (106 /tube) were stained with BV510-anti-CD3 (clone: UCHT1), allophycocyanin (APC)-H7-anti-CD4 (clone: RPA-T4), BB515-anti-CXCR5 (clone: RF8B2), phycoerythrin (PE)-Cy5-anti-CD45RA (clone: HI100), PE-Cy7-anti-CCR6 (clone: 11A9), or APC-anti-CD183 (clone: IC6/CXCR3) (Becton Dickinson, San Jose, CA, USA) at room temperature for 30 min. Data were processed using FlowJo v.5.7.2 software (Tree Star, Ashland, OR, USA). .. Cytometric bead array (CBA) analysis of plasma cytokines Plasma IFN-γ and IL-4 and -17A concentrations were determined using the CBA Human Soluble Protein Master Buffer kit (BD Biosciences) on a flow cytometer.

Crocin Bleaching Assay:

Article Title: Potential of PEGylated Toll-Like Receptor 7 Ligands for Controlling Inflammation and Functional Changes in Mouse Models of Asthma and Silicosis
Article Snippet: .. Cytokines Detection in Cell Supernatant The concentration of IL-1β, IL-6, IL-8, IL-10, IL-12, and TNF in the supernatant of human PBMCs and human mo-DCs was determined using the BD™ cytometric bead array (CBA) (human inflammatory cytokine kit – 551811, BD Biosciences), according to manufacturer’s instructions. .. Flow Cytometric Analysis For surface staining of human specimens, cell suspensions were incubated with the appropriate combination of the following monoclonal antibodies: CD19-PC5 (J3-119, Beckman Coulter), CD80-Brilliant Violet 421™ (2D10, BioLegend® ), CD83-APC (HB15e, BioLegend® ), CD86-APC (IT2.2, BioLegend® ), and HLA-DR-V500 (G46-6, BD Horizon™).

Software:

Article Title: Distribution of circulating T follicular helper cell subsets is altered in immunoglobulin A vasculitis in children
Article Snippet: .. Human PBMCs (106 /tube) were stained with BV510-anti-CD3 (clone: UCHT1), allophycocyanin (APC)-H7-anti-CD4 (clone: RPA-T4), BB515-anti-CXCR5 (clone: RF8B2), phycoerythrin (PE)-Cy5-anti-CD45RA (clone: HI100), PE-Cy7-anti-CCR6 (clone: 11A9), or APC-anti-CD183 (clone: IC6/CXCR3) (Becton Dickinson, San Jose, CA, USA) at room temperature for 30 min. Data were processed using FlowJo v.5.7.2 software (Tree Star, Ashland, OR, USA). .. Cytometric bead array (CBA) analysis of plasma cytokines Plasma IFN-γ and IL-4 and -17A concentrations were determined using the CBA Human Soluble Protein Master Buffer kit (BD Biosciences) on a flow cytometer.

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    Becton Dickinson flow cytometry analysis fcm human pbmcs
    FACS analysis of the numbers of circulating memory Tfh cells and ICOS+ memory Tfh cells in individual subjects. <t>PBMCs</t> were isolated from individual subjects and stained with different fluorescent antibodies. The cells were gated sequentially on living lymphocytes, CD3+ and CD4+, and then on CXCR5+ and CD45RA- cells. The frequency of CD3+CD4+CXCR5+CD45RA- (memory) Tfh cells was determined. Subsequently, memory Tfh cells were gated on ICOS and the frequency of memory Tfh and ICOS+ memory Tfh cells in lymphocytes was analyzed and the numbers of each type of cells in total lymphocytes per liter were calculated. (A) Flow <t>cytometry</t> analysis. (B) The numbers of memory Tfh cells in the HC, MS patients pre- and post-treatment. (C) The numbers of memory Tfh cells in the MS-CR patients pre- and post-treatment. (D) The numbers of memory Tfh cells in the MS-PR patients pre- and post-treatment. (E) The numbers of ICOS+ memory Tfh cells in the HC, MS patients pre- and post-treatment. (F) The numbers of ICOS+ memory Tfh cells in the MS-CR patients pre- and post-treatment. (G) The numbers of ICOS+ memory Tfh cells in the MS-PR patients pre- and post-treatment. There was no significant difference in the numbers of PD-1+, PD-1+ICOS+ and CD40L+ memory Tfh cells between the patients and HC as well as in MS patients pre- and post-treatment (data not shown). The horizontal lines indicate the median values for each group.
    Flow Cytometry Analysis Fcm Human Pbmcs, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson human pbmc
    Representative flow cytometric analysis of uninfected human <t>PBMC</t> (A) and <t>FIV-infected</t> feline (B) and human (C and D) PBMC. By using a monoclonal antibody specific to FIV p24 (clone 43-1B9), viral capsid protein was detected in 12% of Petaluma-infected human PBMC by flow cytometry (D) compared to 0.8 and 91.6% of cells in uninfected human PBMC (C) and FIV-infected feline PBMC (B) cultures, respectively. By using an isotype-matched control antibody, 2.4% of human PBMC were recognized (A).
    Human Pbmc, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson cryopreserved human pbmcs
    Serological and B cell responses in experimentally infected macaques. ( A ) Serum endpoint total IgG titers were measured by ELISA using CA09 HA-FL (blue) or stabilized CA09 HA stem (red) in macaques ( n = 8) infected intranasally with A/Auckland/1/2009. Note that 2 animals were sacrificed on day 23. Dotted lines denote the detection cutoff (dilution 1:100). ( B ) Frequency of IgG + memory B cells (CD19 + IgD – IgG + ) binding CA09 HA-FL (blue) or stabilized CA09 HA stem (red) was measured by flow cytometry within <t>cryopreserved</t> <t>PBMC</t> samples from infected macaques ( n = 6). Note that the 2 animals sacrificed on day 23 were excluded.
    Cryopreserved Human Pbmcs, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FACS analysis of the numbers of circulating memory Tfh cells and ICOS+ memory Tfh cells in individual subjects. PBMCs were isolated from individual subjects and stained with different fluorescent antibodies. The cells were gated sequentially on living lymphocytes, CD3+ and CD4+, and then on CXCR5+ and CD45RA- cells. The frequency of CD3+CD4+CXCR5+CD45RA- (memory) Tfh cells was determined. Subsequently, memory Tfh cells were gated on ICOS and the frequency of memory Tfh and ICOS+ memory Tfh cells in lymphocytes was analyzed and the numbers of each type of cells in total lymphocytes per liter were calculated. (A) Flow cytometry analysis. (B) The numbers of memory Tfh cells in the HC, MS patients pre- and post-treatment. (C) The numbers of memory Tfh cells in the MS-CR patients pre- and post-treatment. (D) The numbers of memory Tfh cells in the MS-PR patients pre- and post-treatment. (E) The numbers of ICOS+ memory Tfh cells in the HC, MS patients pre- and post-treatment. (F) The numbers of ICOS+ memory Tfh cells in the MS-CR patients pre- and post-treatment. (G) The numbers of ICOS+ memory Tfh cells in the MS-PR patients pre- and post-treatment. There was no significant difference in the numbers of PD-1+, PD-1+ICOS+ and CD40L+ memory Tfh cells between the patients and HC as well as in MS patients pre- and post-treatment (data not shown). The horizontal lines indicate the median values for each group.

    Journal: PLoS ONE

    Article Title: Circulating CCR7+ICOS+ Memory T Follicular Helper Cells in Patients with Multiple Sclerosis

    doi: 10.1371/journal.pone.0134523

    Figure Lengend Snippet: FACS analysis of the numbers of circulating memory Tfh cells and ICOS+ memory Tfh cells in individual subjects. PBMCs were isolated from individual subjects and stained with different fluorescent antibodies. The cells were gated sequentially on living lymphocytes, CD3+ and CD4+, and then on CXCR5+ and CD45RA- cells. The frequency of CD3+CD4+CXCR5+CD45RA- (memory) Tfh cells was determined. Subsequently, memory Tfh cells were gated on ICOS and the frequency of memory Tfh and ICOS+ memory Tfh cells in lymphocytes was analyzed and the numbers of each type of cells in total lymphocytes per liter were calculated. (A) Flow cytometry analysis. (B) The numbers of memory Tfh cells in the HC, MS patients pre- and post-treatment. (C) The numbers of memory Tfh cells in the MS-CR patients pre- and post-treatment. (D) The numbers of memory Tfh cells in the MS-PR patients pre- and post-treatment. (E) The numbers of ICOS+ memory Tfh cells in the HC, MS patients pre- and post-treatment. (F) The numbers of ICOS+ memory Tfh cells in the MS-CR patients pre- and post-treatment. (G) The numbers of ICOS+ memory Tfh cells in the MS-PR patients pre- and post-treatment. There was no significant difference in the numbers of PD-1+, PD-1+ICOS+ and CD40L+ memory Tfh cells between the patients and HC as well as in MS patients pre- and post-treatment (data not shown). The horizontal lines indicate the median values for each group.

    Article Snippet: Flow cytometry analysis (FCM) Human PBMCs at 106 /tube were stained in duplicate with APC-H7-anti-CD3, BV510-anti-CD4, FITC-anti-CD45RA, PE-Cy7-anti-CCR7, PerCP-Cy5.5-anti-CXCR5, PE-anti-ICOS, BV421-anti-PD-1, PE-CF594-anti-CD154, or isotype-matched control IgG (Becton Dickinson, San Diego, USA) at room temperature for 30 minutes, respectively.

    Techniques: FACS, Isolation, Staining, Flow Cytometry, Cytometry, Mass Spectrometry

    Representative flow cytometric analysis of uninfected human PBMC (A) and FIV-infected feline (B) and human (C and D) PBMC. By using a monoclonal antibody specific to FIV p24 (clone 43-1B9), viral capsid protein was detected in 12% of Petaluma-infected human PBMC by flow cytometry (D) compared to 0.8 and 91.6% of cells in uninfected human PBMC (C) and FIV-infected feline PBMC (B) cultures, respectively. By using an isotype-matched control antibody, 2.4% of human PBMC were recognized (A).

    Journal: Journal of Virology

    Article Title: Productive Infection of Human Peripheral Blood Mononuclear Cells by Feline Immunodeficiency Virus: Implications for Vector Development

    doi:

    Figure Lengend Snippet: Representative flow cytometric analysis of uninfected human PBMC (A) and FIV-infected feline (B) and human (C and D) PBMC. By using a monoclonal antibody specific to FIV p24 (clone 43-1B9), viral capsid protein was detected in 12% of Petaluma-infected human PBMC by flow cytometry (D) compared to 0.8 and 91.6% of cells in uninfected human PBMC (C) and FIV-infected feline PBMC (B) cultures, respectively. By using an isotype-matched control antibody, 2.4% of human PBMC were recognized (A).

    Article Snippet: Analysis of FIV p24 expression in human PBMC infected with Petaluma or V1 CSF was performed on a Becton Dickinson FacScan fluorescence activated cell sorter with a 488-nm laser for excitation.

    Techniques: Flow Cytometry, Infection, Cytometry

    RTase activity and viral titer in culture supernatants from human and feline PBMC infected with either Petaluma or V 1 CSF strains of FIV. (A) RTase activity in human PBMC infected with either strain increased over the time course, reaching a maximum value of approximately 5 × 10 4 cpm/ml at day 16 p.i. for both viruses. Significant differences between uninfected cells (CONTROL) and PBMC infected by either virus were observed by using a two-tailed Student’s t test ( P

    Journal: Journal of Virology

    Article Title: Productive Infection of Human Peripheral Blood Mononuclear Cells by Feline Immunodeficiency Virus: Implications for Vector Development

    doi:

    Figure Lengend Snippet: RTase activity and viral titer in culture supernatants from human and feline PBMC infected with either Petaluma or V 1 CSF strains of FIV. (A) RTase activity in human PBMC infected with either strain increased over the time course, reaching a maximum value of approximately 5 × 10 4 cpm/ml at day 16 p.i. for both viruses. Significant differences between uninfected cells (CONTROL) and PBMC infected by either virus were observed by using a two-tailed Student’s t test ( P

    Article Snippet: Analysis of FIV p24 expression in human PBMC infected with Petaluma or V1 CSF was performed on a Becton Dickinson FacScan fluorescence activated cell sorter with a 488-nm laser for excitation.

    Techniques: Activity Assay, Infection, Two Tailed Test

    (A) FIV viral DNA detection after incubation with antibodies recognizing the CCR3, CXCR4, or CCR5 chemokine receptors. Uninfected PBMC and PBMC infected with either viral strain but without pretreatment with antibody served as negative and positive controls, respectively. (B) FIV DNA levels were measured relative to the amount of template DNA, as determined by amplification of the HLA-DQα gene. (C) The greatest decrease in the viral DNA levels compared to positive control cultures was observed after treatment with antibodies to the CCR3 chemokine receptor for both strains (59.8 ± 2.4% for Petaluma, 77.1 ± 1.1% for V 1 CSF). Antibodies to the CCR5 receptor significantly inhibited infection with V 1 CSF, decreasing detectable FIV DNA levels by 71.2 ± 0.7% of the control value, but not with Petaluma. For both viruses, infection was decreased in the presence of antibodies recognizing the CXCR4 receptor (29.3 ± 5.7% for Petaluma, 63.3 ± 1.6% for V 1 CSF). Viral DNA levels were not affected by preincubation with nonspecific ARPA or BSA. ∗∗, P

    Journal: Journal of Virology

    Article Title: Productive Infection of Human Peripheral Blood Mononuclear Cells by Feline Immunodeficiency Virus: Implications for Vector Development

    doi:

    Figure Lengend Snippet: (A) FIV viral DNA detection after incubation with antibodies recognizing the CCR3, CXCR4, or CCR5 chemokine receptors. Uninfected PBMC and PBMC infected with either viral strain but without pretreatment with antibody served as negative and positive controls, respectively. (B) FIV DNA levels were measured relative to the amount of template DNA, as determined by amplification of the HLA-DQα gene. (C) The greatest decrease in the viral DNA levels compared to positive control cultures was observed after treatment with antibodies to the CCR3 chemokine receptor for both strains (59.8 ± 2.4% for Petaluma, 77.1 ± 1.1% for V 1 CSF). Antibodies to the CCR5 receptor significantly inhibited infection with V 1 CSF, decreasing detectable FIV DNA levels by 71.2 ± 0.7% of the control value, but not with Petaluma. For both viruses, infection was decreased in the presence of antibodies recognizing the CXCR4 receptor (29.3 ± 5.7% for Petaluma, 63.3 ± 1.6% for V 1 CSF). Viral DNA levels were not affected by preincubation with nonspecific ARPA or BSA. ∗∗, P

    Article Snippet: Analysis of FIV p24 expression in human PBMC infected with Petaluma or V1 CSF was performed on a Becton Dickinson FacScan fluorescence activated cell sorter with a 488-nm laser for excitation.

    Techniques: Incubation, Infection, Amplification, Positive Control

    FIV-induced cytotoxicity in human PBMC cultures infected with either Petaluma or V 1 CSF strains. Results are expressed as the percentage of cell death and are assessed relative to uninfected (CONTROL) cultures. Petaluma-infected cultures exhibited increased cytotoxicity at days 7, 10, and 14 p.i., with the greatest difference occurring at day 10 when cell death was 38.9 ± 2.4% ( P

    Journal: Journal of Virology

    Article Title: Productive Infection of Human Peripheral Blood Mononuclear Cells by Feline Immunodeficiency Virus: Implications for Vector Development

    doi:

    Figure Lengend Snippet: FIV-induced cytotoxicity in human PBMC cultures infected with either Petaluma or V 1 CSF strains. Results are expressed as the percentage of cell death and are assessed relative to uninfected (CONTROL) cultures. Petaluma-infected cultures exhibited increased cytotoxicity at days 7, 10, and 14 p.i., with the greatest difference occurring at day 10 when cell death was 38.9 ± 2.4% ( P

    Article Snippet: Analysis of FIV p24 expression in human PBMC infected with Petaluma or V1 CSF was performed on a Becton Dickinson FacScan fluorescence activated cell sorter with a 488-nm laser for excitation.

    Techniques: Infection

    Concentration of IL1-β (A) , IL-6 (B) , IL-12 (C) , IL-8 (D) , TNF-α (E) and IL-10 (F) in the supernatant of human PBMCs, after 24 h stimulation with TMX-202, TMX-302 or TMX-306 at 1 or 10 μM. Data are presented as mean ± SEM from n = 6.

    Journal: Frontiers in Immunology

    Article Title: Potential of PEGylated Toll-Like Receptor 7 Ligands for Controlling Inflammation and Functional Changes in Mouse Models of Asthma and Silicosis

    doi: 10.3389/fimmu.2016.00095

    Figure Lengend Snippet: Concentration of IL1-β (A) , IL-6 (B) , IL-12 (C) , IL-8 (D) , TNF-α (E) and IL-10 (F) in the supernatant of human PBMCs, after 24 h stimulation with TMX-202, TMX-302 or TMX-306 at 1 or 10 μM. Data are presented as mean ± SEM from n = 6.

    Article Snippet: Cytokines Detection in Cell Supernatant The concentration of IL-1β, IL-6, IL-8, IL-10, IL-12, and TNF in the supernatant of human PBMCs and human mo-DCs was determined using the BD™ cytometric bead array (CBA) (human inflammatory cytokine kit – 551811, BD Biosciences), according to manufacturer’s instructions.

    Techniques: Concentration Assay

    Serological and B cell responses in experimentally infected macaques. ( A ) Serum endpoint total IgG titers were measured by ELISA using CA09 HA-FL (blue) or stabilized CA09 HA stem (red) in macaques ( n = 8) infected intranasally with A/Auckland/1/2009. Note that 2 animals were sacrificed on day 23. Dotted lines denote the detection cutoff (dilution 1:100). ( B ) Frequency of IgG + memory B cells (CD19 + IgD – IgG + ) binding CA09 HA-FL (blue) or stabilized CA09 HA stem (red) was measured by flow cytometry within cryopreserved PBMC samples from infected macaques ( n = 6). Note that the 2 animals sacrificed on day 23 were excluded.

    Journal: The Journal of Clinical Investigation

    Article Title: Subdominance and poor intrinsic immunogenicity limit humoral immunity targeting influenza HA stem

    doi: 10.1172/JCI123366

    Figure Lengend Snippet: Serological and B cell responses in experimentally infected macaques. ( A ) Serum endpoint total IgG titers were measured by ELISA using CA09 HA-FL (blue) or stabilized CA09 HA stem (red) in macaques ( n = 8) infected intranasally with A/Auckland/1/2009. Note that 2 animals were sacrificed on day 23. Dotted lines denote the detection cutoff (dilution 1:100). ( B ) Frequency of IgG + memory B cells (CD19 + IgD – IgG + ) binding CA09 HA-FL (blue) or stabilized CA09 HA stem (red) was measured by flow cytometry within cryopreserved PBMC samples from infected macaques ( n = 6). Note that the 2 animals sacrificed on day 23 were excluded.

    Article Snippet: HA-specific B cells were identified within cryopreserved human PBMCs by costaining with HA probes conjugated to SA-PE, SA-APC, SA-BV421, or SA-Ax488 (all from BD).

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, Binding Assay, Flow Cytometry, Cytometry