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Becton Dickinson human peripheral blood mononuclear cells pbmcs
CD40L expressed by recombinant ALVAC virus enhances specific antiviral CTL activity. CD4 + T cell-containing or -depleted <t>PBMCs</t> from four HIV-1-infected individuals and three HIV-1-uninfected individuals were cocultured with autologous MDDCs that were either pulsed or not with HLA-restricted epitopes of HIV-1 proteins (for HIV-1 infected individuals) or EBV proteins (for HIV-1-uninfected individuals). MDDCs were previously infected or not (medium) with parental ALVAC II or ALVAC recombinant vA3131-2 expressing human CD40L at an MOI of 10 for 48 h. On day 10, specific CTL activity was assessed by intracellular flow cytometric analysis of IFN-γproducing CD8 + T cells and 51 Cr release assay. (A) Representative intracellular IFN-γ flow cytometric data obtained from HIV-1-positive participant #1. (B) Summary data from intracellular IFN-γ flow <t>cytometry</t> of HIV-1-positive participant #2–#4 are graphically depicted. Open bars represent CD4 + T cell-containing condition and dark bar represent CD4 + T cell -depleted conditions. (C) A representative 51 Cr release assay result from HIV-1-positive participant #1 is shown. Similar results were obtained with HIV-1-positive participant #2 (data not shown). (D) Summary data from intracellular IFN-γ flow cytometric analysis of EBV-positive participant #5–#7 are graphically depicted. Open bars represent CD4 + T cell -containing condition and dark bars represent CD4 + T cell -depleted conditions. (E) A representative 51 Cr release assay result from EBV-positive participant #6 is shown. Similar results were obtained with EBV-positive participant #5 (data not shown). DC, MDDCs not pulsed with peptide; DCp, MDDCs pulsed with peptide; vA3131-2/DCp, vA3131-2-infected MDDCs pulsed with peptide; ALVAC/DCp, parental ALVAC II-infected MDDCs pulsed with peptide.
Human Peripheral Blood Mononuclear Cells Pbmcs, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Price from $9.99 to $1999.99
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1) Product Images from "CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8+ T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals"

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8+ T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

Journal: Vaccine

doi: 10.1016/j.vaccine.2008.05.018

CD40L expressed by recombinant ALVAC virus enhances specific antiviral CTL activity. CD4 + T cell-containing or -depleted PBMCs from four HIV-1-infected individuals and three HIV-1-uninfected individuals were cocultured with autologous MDDCs that were either pulsed or not with HLA-restricted epitopes of HIV-1 proteins (for HIV-1 infected individuals) or EBV proteins (for HIV-1-uninfected individuals). MDDCs were previously infected or not (medium) with parental ALVAC II or ALVAC recombinant vA3131-2 expressing human CD40L at an MOI of 10 for 48 h. On day 10, specific CTL activity was assessed by intracellular flow cytometric analysis of IFN-γproducing CD8 + T cells and 51 Cr release assay. (A) Representative intracellular IFN-γ flow cytometric data obtained from HIV-1-positive participant #1. (B) Summary data from intracellular IFN-γ flow cytometry of HIV-1-positive participant #2–#4 are graphically depicted. Open bars represent CD4 + T cell-containing condition and dark bar represent CD4 + T cell -depleted conditions. (C) A representative 51 Cr release assay result from HIV-1-positive participant #1 is shown. Similar results were obtained with HIV-1-positive participant #2 (data not shown). (D) Summary data from intracellular IFN-γ flow cytometric analysis of EBV-positive participant #5–#7 are graphically depicted. Open bars represent CD4 + T cell -containing condition and dark bars represent CD4 + T cell -depleted conditions. (E) A representative 51 Cr release assay result from EBV-positive participant #6 is shown. Similar results were obtained with EBV-positive participant #5 (data not shown). DC, MDDCs not pulsed with peptide; DCp, MDDCs pulsed with peptide; vA3131-2/DCp, vA3131-2-infected MDDCs pulsed with peptide; ALVAC/DCp, parental ALVAC II-infected MDDCs pulsed with peptide.
Figure Legend Snippet: CD40L expressed by recombinant ALVAC virus enhances specific antiviral CTL activity. CD4 + T cell-containing or -depleted PBMCs from four HIV-1-infected individuals and three HIV-1-uninfected individuals were cocultured with autologous MDDCs that were either pulsed or not with HLA-restricted epitopes of HIV-1 proteins (for HIV-1 infected individuals) or EBV proteins (for HIV-1-uninfected individuals). MDDCs were previously infected or not (medium) with parental ALVAC II or ALVAC recombinant vA3131-2 expressing human CD40L at an MOI of 10 for 48 h. On day 10, specific CTL activity was assessed by intracellular flow cytometric analysis of IFN-γproducing CD8 + T cells and 51 Cr release assay. (A) Representative intracellular IFN-γ flow cytometric data obtained from HIV-1-positive participant #1. (B) Summary data from intracellular IFN-γ flow cytometry of HIV-1-positive participant #2–#4 are graphically depicted. Open bars represent CD4 + T cell-containing condition and dark bar represent CD4 + T cell -depleted conditions. (C) A representative 51 Cr release assay result from HIV-1-positive participant #1 is shown. Similar results were obtained with HIV-1-positive participant #2 (data not shown). (D) Summary data from intracellular IFN-γ flow cytometric analysis of EBV-positive participant #5–#7 are graphically depicted. Open bars represent CD4 + T cell -containing condition and dark bars represent CD4 + T cell -depleted conditions. (E) A representative 51 Cr release assay result from EBV-positive participant #6 is shown. Similar results were obtained with EBV-positive participant #5 (data not shown). DC, MDDCs not pulsed with peptide; DCp, MDDCs pulsed with peptide; vA3131-2/DCp, vA3131-2-infected MDDCs pulsed with peptide; ALVAC/DCp, parental ALVAC II-infected MDDCs pulsed with peptide.

Techniques Used: Recombinant, CTL Assay, Activity Assay, Infection, Expressing, Flow Cytometry, Release Assay, Cytometry

Expression of murine CD40L by recombinant canarypox vector. Recombinant canarypox viruses vCPmCD40L and vCPmSP-D-CD40L were generated as described in Materials and Methods. (A) RT-PCR. CEF cells were infected by vCPmCD40L, vCPmSP-D-CD40L or its parental control ALVAC II at 5 MOI for 3 days. Total RNA was isolated from infected CEF cells using Trizol Reagent and subjected to RT-PCR with specific primers amplifying the coding sequence of murine CD40L or SP-D-CD40L. The identity of the amplified product was further verified by DNA sequencing (not shown). (B) Flow cytometry. Human PBMCs were infected by vCPmCD40L, vCPmSP-D-CD40L or ALVAC II at 5 MOI for 24 hrs and stained with PE-labeled anti-mouse CD40L mAb. (C) Western blot. Hela cells were infected by vCPmCD40L, vCPmSP-D-CD40L, or ALVAC II at 10 MOI for 24–48hrs. Cell lysates or magnetic beads incubated with supernatant of infected cells were subjected to Western blot and detected with a goat anti-mouse CD40L or anti-mouse SP-D antibody. As expected, the membrane CD40L was about 35–40kD and soluble multimeric form of CD40L was about 70kD.
Figure Legend Snippet: Expression of murine CD40L by recombinant canarypox vector. Recombinant canarypox viruses vCPmCD40L and vCPmSP-D-CD40L were generated as described in Materials and Methods. (A) RT-PCR. CEF cells were infected by vCPmCD40L, vCPmSP-D-CD40L or its parental control ALVAC II at 5 MOI for 3 days. Total RNA was isolated from infected CEF cells using Trizol Reagent and subjected to RT-PCR with specific primers amplifying the coding sequence of murine CD40L or SP-D-CD40L. The identity of the amplified product was further verified by DNA sequencing (not shown). (B) Flow cytometry. Human PBMCs were infected by vCPmCD40L, vCPmSP-D-CD40L or ALVAC II at 5 MOI for 24 hrs and stained with PE-labeled anti-mouse CD40L mAb. (C) Western blot. Hela cells were infected by vCPmCD40L, vCPmSP-D-CD40L, or ALVAC II at 10 MOI for 24–48hrs. Cell lysates or magnetic beads incubated with supernatant of infected cells were subjected to Western blot and detected with a goat anti-mouse CD40L or anti-mouse SP-D antibody. As expected, the membrane CD40L was about 35–40kD and soluble multimeric form of CD40L was about 70kD.

Techniques Used: Expressing, Recombinant, Plasmid Preparation, Generated, Reverse Transcription Polymerase Chain Reaction, Infection, Isolation, Sequencing, Amplification, DNA Sequencing, Flow Cytometry, Cytometry, Staining, Labeling, Western Blot, Magnetic Beads, Incubation

Related Articles

Flow Cytometry:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8+ T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals
Article Snippet: .. For flow cytometry, human peripheral blood mononuclear cells (PBMCs) were infected with recombinant viruses or ALVAC II at 5 MOI for 24 h and then stained with PE-conjugated anti-mouse or anti-human CD40L (BD Biosciences, Mississauga, ON, Canada). .. For Western blot of membrane form of CD40L, Hela cells or human monocytic cell line THP-1 were infected with recombinant viruses or ALVAC II at 5–10 MOI for 24–48 h. Infected cells were then harvested and lysed with CelLytic ™-M (Sigma, Oakville, ON, Canada).

Article Title: Engraftment of human central memory-derived effector CD8+ T cells in immunodeficient mice
Article Snippet: .. Human peripheral blood mononuclear cells (PBMCs) and T cells were analyzed by flow cytometry after staining with fluorochrome-conjugated monoclonal antibodies (mAbs) to CD4, CD8, CD62L, CD45RO, CD127, CD28, CD45, CD3, perforin, granzyme A, Ki-67, interferon-γ (IFN-γ), CD122 (IL-2Rβ), CD132 (IL-2Rγ; BD Biosciences), and CCR7, and IL-15Rα (R & D Systems). .. Phycoerythrin (PE)-conjugated CMV pp65 (NLVPMVATV)–HLA-A2*0201 iTAg MHC tetramer, PE-conjugated multiallele negative tetramer, and the IOTestBeta Mark TCR V β Repertoire Kit (representing ∼ 70% of normal TCR Vβ repertoire) were obtained from Beckman Coulter.

Article Title: A Novel Monoclonal Antibody to CD40 Prolongs Islet Allograft Survival
Article Snippet: .. To confirm the specificity of 2C10 for rhesus and human CD40 in vitro, rhesus or human peripheral blood mononuclear cells (PBMC) were incubated with escalating concentrations of 2C10 or an isotype control then incubated with a fluorophore-labeled antibody to CD40 (clone 5C3, BD Bioscience, San Jose, CA) known to bind to CD40 of both species and analyzed by flow cytometry. .. To test the ability of 2C10 to block binding of CD154 in vitro, rhesus or human PBMC were incubated with escalating concentrations of 2C10 or an isotype control and incubated with soluble histidine-tagged recombinant human CD154 (R & D Systems, Minneapolis, MN).

Recombinant:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8+ T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals
Article Snippet: .. For flow cytometry, human peripheral blood mononuclear cells (PBMCs) were infected with recombinant viruses or ALVAC II at 5 MOI for 24 h and then stained with PE-conjugated anti-mouse or anti-human CD40L (BD Biosciences, Mississauga, ON, Canada). .. For Western blot of membrane form of CD40L, Hela cells or human monocytic cell line THP-1 were infected with recombinant viruses or ALVAC II at 5–10 MOI for 24–48 h. Infected cells were then harvested and lysed with CelLytic ™-M (Sigma, Oakville, ON, Canada).

In Vitro:

Article Title: A Novel Monoclonal Antibody to CD40 Prolongs Islet Allograft Survival
Article Snippet: .. To confirm the specificity of 2C10 for rhesus and human CD40 in vitro, rhesus or human peripheral blood mononuclear cells (PBMC) were incubated with escalating concentrations of 2C10 or an isotype control then incubated with a fluorophore-labeled antibody to CD40 (clone 5C3, BD Bioscience, San Jose, CA) known to bind to CD40 of both species and analyzed by flow cytometry. .. To test the ability of 2C10 to block binding of CD154 in vitro, rhesus or human PBMC were incubated with escalating concentrations of 2C10 or an isotype control and incubated with soluble histidine-tagged recombinant human CD154 (R & D Systems, Minneapolis, MN).

Cytometry:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8+ T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals
Article Snippet: .. For flow cytometry, human peripheral blood mononuclear cells (PBMCs) were infected with recombinant viruses or ALVAC II at 5 MOI for 24 h and then stained with PE-conjugated anti-mouse or anti-human CD40L (BD Biosciences, Mississauga, ON, Canada). .. For Western blot of membrane form of CD40L, Hela cells or human monocytic cell line THP-1 were infected with recombinant viruses or ALVAC II at 5–10 MOI for 24–48 h. Infected cells were then harvested and lysed with CelLytic ™-M (Sigma, Oakville, ON, Canada).

Article Title: Engraftment of human central memory-derived effector CD8+ T cells in immunodeficient mice
Article Snippet: .. Human peripheral blood mononuclear cells (PBMCs) and T cells were analyzed by flow cytometry after staining with fluorochrome-conjugated monoclonal antibodies (mAbs) to CD4, CD8, CD62L, CD45RO, CD127, CD28, CD45, CD3, perforin, granzyme A, Ki-67, interferon-γ (IFN-γ), CD122 (IL-2Rβ), CD132 (IL-2Rγ; BD Biosciences), and CCR7, and IL-15Rα (R & D Systems). .. Phycoerythrin (PE)-conjugated CMV pp65 (NLVPMVATV)–HLA-A2*0201 iTAg MHC tetramer, PE-conjugated multiallele negative tetramer, and the IOTestBeta Mark TCR V β Repertoire Kit (representing ∼ 70% of normal TCR Vβ repertoire) were obtained from Beckman Coulter.

Article Title: A Novel Monoclonal Antibody to CD40 Prolongs Islet Allograft Survival
Article Snippet: .. To confirm the specificity of 2C10 for rhesus and human CD40 in vitro, rhesus or human peripheral blood mononuclear cells (PBMC) were incubated with escalating concentrations of 2C10 or an isotype control then incubated with a fluorophore-labeled antibody to CD40 (clone 5C3, BD Bioscience, San Jose, CA) known to bind to CD40 of both species and analyzed by flow cytometry. .. To test the ability of 2C10 to block binding of CD154 in vitro, rhesus or human PBMC were incubated with escalating concentrations of 2C10 or an isotype control and incubated with soluble histidine-tagged recombinant human CD154 (R & D Systems, Minneapolis, MN).

Infection:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8+ T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals
Article Snippet: .. For flow cytometry, human peripheral blood mononuclear cells (PBMCs) were infected with recombinant viruses or ALVAC II at 5 MOI for 24 h and then stained with PE-conjugated anti-mouse or anti-human CD40L (BD Biosciences, Mississauga, ON, Canada). .. For Western blot of membrane form of CD40L, Hela cells or human monocytic cell line THP-1 were infected with recombinant viruses or ALVAC II at 5–10 MOI for 24–48 h. Infected cells were then harvested and lysed with CelLytic ™-M (Sigma, Oakville, ON, Canada).

Purification:

Article Title: Increased Frequency of Th17 Cells in Children With Mycoplasma pneumoniae Pneumonia
Article Snippet: .. Human peripheral blood mononuclear cells (PBMCs) were collected into sodium heparin tubes (BD Biosciences, San Diego, CA) and purified by Ficoll‐paque plus (GE healthcare, Uppsala, Sweden) density gradient centrifugation. .. Cells recovered from the gradient interface were washed twice and stained for 30 min at 4°C with the following antibodies or isotype‐matched controls: CD3‐APC (eBioscience, San Diego, CA), CD4‐FITC (eBioscience).

Incubation:

Article Title: A Novel Monoclonal Antibody to CD40 Prolongs Islet Allograft Survival
Article Snippet: .. To confirm the specificity of 2C10 for rhesus and human CD40 in vitro, rhesus or human peripheral blood mononuclear cells (PBMC) were incubated with escalating concentrations of 2C10 or an isotype control then incubated with a fluorophore-labeled antibody to CD40 (clone 5C3, BD Bioscience, San Jose, CA) known to bind to CD40 of both species and analyzed by flow cytometry. .. To test the ability of 2C10 to block binding of CD154 in vitro, rhesus or human PBMC were incubated with escalating concentrations of 2C10 or an isotype control and incubated with soluble histidine-tagged recombinant human CD154 (R & D Systems, Minneapolis, MN).

Activity Assay:

Article Title: Characterization of Novel Staphylococcal Enterotoxin-Like Toxin Type P
Article Snippet: .. To determine the mitogenic activity of SElP, we stimulated human peripheral blood mononuclear cells (PBMC) for 72 h in 96-well flat-bottom microplates (Becton Dickinson, Franklin Lakes, NJ) with various concentrations of SElP or TSST-1 with or without of 100 U of polymyxin B sulfate (Bio West, Nuaile, France)/ml, a lipopolysaccharide (LPS) inhibitor. ..

Staining:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8+ T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals
Article Snippet: .. For flow cytometry, human peripheral blood mononuclear cells (PBMCs) were infected with recombinant viruses or ALVAC II at 5 MOI for 24 h and then stained with PE-conjugated anti-mouse or anti-human CD40L (BD Biosciences, Mississauga, ON, Canada). .. For Western blot of membrane form of CD40L, Hela cells or human monocytic cell line THP-1 were infected with recombinant viruses or ALVAC II at 5–10 MOI for 24–48 h. Infected cells were then harvested and lysed with CelLytic ™-M (Sigma, Oakville, ON, Canada).

Article Title: Engraftment of human central memory-derived effector CD8+ T cells in immunodeficient mice
Article Snippet: .. Human peripheral blood mononuclear cells (PBMCs) and T cells were analyzed by flow cytometry after staining with fluorochrome-conjugated monoclonal antibodies (mAbs) to CD4, CD8, CD62L, CD45RO, CD127, CD28, CD45, CD3, perforin, granzyme A, Ki-67, interferon-γ (IFN-γ), CD122 (IL-2Rβ), CD132 (IL-2Rγ; BD Biosciences), and CCR7, and IL-15Rα (R & D Systems). .. Phycoerythrin (PE)-conjugated CMV pp65 (NLVPMVATV)–HLA-A2*0201 iTAg MHC tetramer, PE-conjugated multiallele negative tetramer, and the IOTestBeta Mark TCR V β Repertoire Kit (representing ∼ 70% of normal TCR Vβ repertoire) were obtained from Beckman Coulter.

Article Title: Inhibition of Epidermal Growth Factor Receptor Tyrosine Kinase Ameliorates Collagen-Induced Arthritis
Article Snippet: .. Human peripheral blood mononuclear cells (PBMCs) and RA synovial fibroblasts were stained with antibodies including CD3-PE, CD20-PerCP-Cy5.5 and CD11c-PE (BD) and CD14-PeCy7 (ebioscience). ..

Article Title: Sialyl Lewis x (CD15s) identifies highly differentiated and most suppressive FOXP3high regulatory T cells in humans
Article Snippet: .. Human peripheral blood mononuclear cells (PBMCs) and human thymocytes were prepared by Ficoll gradient centrifugation and stained with anti-hCD3, anti-hCD8, anti–hCD4-PerCP-Cy5.5 or –APC, anti–hCD25-PE, anti–hCD45RA-PE-Cy7, anti–ICOS-, anti–HLA-DR-PE (from BD Biosciences), anti-CD31 (-APC from eBioscience), anti-hCD127 (-Pacific blue). .. Intracellular detection of FOXP3 with anti-hFOXP3 (PE or Alexa Fluor 647, clone 259D/A7, BD Biosciences) and of Ki-67 antigen with Ki-67 antibody (FITC or PE from BD Biosciences) was performed on fixed and permeabilized cells using Intracellular Fixation and Permeabilization Buffer Set (eBioscience).

Centrifugation:

Article Title: Genome editing of CXCR4 by CRISPR/cas9 confers cells resistant to HIV-1 infection
Article Snippet: .. The human peripheral blood mononuclear cells (PBMCs) were separated from the whole blood by centrifugation with Ficoll-Paque Premium (BD). .. The primary human CD4+ T cells were further purified and enriched by the CD4+ T cell isolation Kit (Miltenyi Biotech) according to the manufacturer’s instructions and then maintained in complete RPMI medium supplemented with 10% FBS.

Gradient Centrifugation:

Article Title: Increased Frequency of Th17 Cells in Children With Mycoplasma pneumoniae Pneumonia
Article Snippet: .. Human peripheral blood mononuclear cells (PBMCs) were collected into sodium heparin tubes (BD Biosciences, San Diego, CA) and purified by Ficoll‐paque plus (GE healthcare, Uppsala, Sweden) density gradient centrifugation. .. Cells recovered from the gradient interface were washed twice and stained for 30 min at 4°C with the following antibodies or isotype‐matched controls: CD3‐APC (eBioscience, San Diego, CA), CD4‐FITC (eBioscience).

Article Title: Sialyl Lewis x (CD15s) identifies highly differentiated and most suppressive FOXP3high regulatory T cells in humans
Article Snippet: .. Human peripheral blood mononuclear cells (PBMCs) and human thymocytes were prepared by Ficoll gradient centrifugation and stained with anti-hCD3, anti-hCD8, anti–hCD4-PerCP-Cy5.5 or –APC, anti–hCD25-PE, anti–hCD45RA-PE-Cy7, anti–ICOS-, anti–HLA-DR-PE (from BD Biosciences), anti-CD31 (-APC from eBioscience), anti-hCD127 (-Pacific blue). .. Intracellular detection of FOXP3 with anti-hFOXP3 (PE or Alexa Fluor 647, clone 259D/A7, BD Biosciences) and of Ki-67 antigen with Ki-67 antibody (FITC or PE from BD Biosciences) was performed on fixed and permeabilized cells using Intracellular Fixation and Permeabilization Buffer Set (eBioscience).

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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 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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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    Becton Dickinson pbmcs
    Binding of CAT-8015 or CAT-3888 to human or monkey <t>PBMCs.</t> Human (A) or monkey (B) purified PBMCs were incubated with <t>biotinylated</t> CAT-8015 at concentrations shown and then with streptavidin-FITC. Following washing in PBS, the samples were analyzed on
    Pbmcs, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 95/100, based on 2031 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

    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

    Binding of CAT-8015 or CAT-3888 to human or monkey PBMCs. Human (A) or monkey (B) purified PBMCs were incubated with biotinylated CAT-8015 at concentrations shown and then with streptavidin-FITC. Following washing in PBS, the samples were analyzed on

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: CAT-8015: A Second-generation Pseudomonas Exotoxin A-Based Immunotherapy Targeting CD22 -Expressing Hematological Malignancies

    doi: 10.1158/1078-0432.CCR-08-1456

    Figure Lengend Snippet: Binding of CAT-8015 or CAT-3888 to human or monkey PBMCs. Human (A) or monkey (B) purified PBMCs were incubated with biotinylated CAT-8015 at concentrations shown and then with streptavidin-FITC. Following washing in PBS, the samples were analyzed on

    Article Snippet: The PBMCs were stained with biotinylated CAT-8015 and the cells were then washed and treated with streptavidin-FITC (10 ug/mL) followed by washing in PBS and analysis on a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA) following standard procedures.

    Techniques: Binding Assay, Purification, Incubation