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Sanquin pbmc
Identification of immunogenic epitope(s) of Rv2034 recognized by CD4 T-cell clone. To identify the immunogenic epitope(s) in Rv2034, the CD4 + T-cell clone was stimulated with all individual Rv2034 20-mer peptides with 10 aa overlap; Rv2034 recombinant protein; Rv2034 peptide pool; control ESAT-6/CFP10 fusion protein; an Ag85B/ESAT-6/Rv2034 trimeric fusion protein; and negative and positive control conditions. Autologous irradiated <t>PBMC</t> were used as APCs. Both <t>IFN-γ</t> (open bars) and T-cell proliferation (black bars) were determined. CPM bars represent median ranging the highest and lowest value (n = 3) (A). To determine the Rv2034 p81–100 specific response by flow cytometry, the CD4 + T-cell clone was stimulated with Rv2034 p81–100 (B), Rv2034 protein (C) and Rv2034 p11–30 (D) using autologous irradiated PBMC, in the presence of BFA. Intracellular CD154 and Th1 cytokine expression was determined. Data is representative of three independent experiments. Flow cytometry plots show single live CD14 − CD19 − CD3 + CD4 + T cells, the frequency of all subsets of CD3 + CD4 + T cells are indicated in the corners of each plot.
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1) Product Images from "Clonal Analysis of the T-Cell Response to In Vivo Expressed Mycobacterium tuberculosis Protein Rv2034, Using a CD154 Expression Based T-Cell Cloning Method"

Article Title: Clonal Analysis of the T-Cell Response to In Vivo Expressed Mycobacterium tuberculosis Protein Rv2034, Using a CD154 Expression Based T-Cell Cloning Method

Journal: PLoS ONE

doi: 10.1371/journal.pone.0099203

Identification of immunogenic epitope(s) of Rv2034 recognized by CD4 T-cell clone. To identify the immunogenic epitope(s) in Rv2034, the CD4 + T-cell clone was stimulated with all individual Rv2034 20-mer peptides with 10 aa overlap; Rv2034 recombinant protein; Rv2034 peptide pool; control ESAT-6/CFP10 fusion protein; an Ag85B/ESAT-6/Rv2034 trimeric fusion protein; and negative and positive control conditions. Autologous irradiated PBMC were used as APCs. Both IFN-γ (open bars) and T-cell proliferation (black bars) were determined. CPM bars represent median ranging the highest and lowest value (n = 3) (A). To determine the Rv2034 p81–100 specific response by flow cytometry, the CD4 + T-cell clone was stimulated with Rv2034 p81–100 (B), Rv2034 protein (C) and Rv2034 p11–30 (D) using autologous irradiated PBMC, in the presence of BFA. Intracellular CD154 and Th1 cytokine expression was determined. Data is representative of three independent experiments. Flow cytometry plots show single live CD14 − CD19 − CD3 + CD4 + T cells, the frequency of all subsets of CD3 + CD4 + T cells are indicated in the corners of each plot.
Figure Legend Snippet: Identification of immunogenic epitope(s) of Rv2034 recognized by CD4 T-cell clone. To identify the immunogenic epitope(s) in Rv2034, the CD4 + T-cell clone was stimulated with all individual Rv2034 20-mer peptides with 10 aa overlap; Rv2034 recombinant protein; Rv2034 peptide pool; control ESAT-6/CFP10 fusion protein; an Ag85B/ESAT-6/Rv2034 trimeric fusion protein; and negative and positive control conditions. Autologous irradiated PBMC were used as APCs. Both IFN-γ (open bars) and T-cell proliferation (black bars) were determined. CPM bars represent median ranging the highest and lowest value (n = 3) (A). To determine the Rv2034 p81–100 specific response by flow cytometry, the CD4 + T-cell clone was stimulated with Rv2034 p81–100 (B), Rv2034 protein (C) and Rv2034 p11–30 (D) using autologous irradiated PBMC, in the presence of BFA. Intracellular CD154 and Th1 cytokine expression was determined. Data is representative of three independent experiments. Flow cytometry plots show single live CD14 − CD19 − CD3 + CD4 + T cells, the frequency of all subsets of CD3 + CD4 + T cells are indicated in the corners of each plot.

Techniques Used: Recombinant, Positive Control, Irradiation, Flow Cytometry, Cytometry, Expressing

PBMC recognition of TB10.4 and IVE-TB antigen Rv2034. PBMC from a PPD + donor were stimulated with different stimuli for 6 days and IFN-γ production (pg/ml) was determined in the supernatants. Both TB10.4 protein (10 µg/ml) (A) and Rv2034 protein (10 µg/ml) (B) were analyzed as well as control mitogen PHA and Mtb derived PPD (A and B). Medium values (unstimulated PBMC) were subtracted. IFN-γ concentrations were determined from triplicate-pooled supernatant. A cut-off value was set arbitrarily at 100 pg/ml.
Figure Legend Snippet: PBMC recognition of TB10.4 and IVE-TB antigen Rv2034. PBMC from a PPD + donor were stimulated with different stimuli for 6 days and IFN-γ production (pg/ml) was determined in the supernatants. Both TB10.4 protein (10 µg/ml) (A) and Rv2034 protein (10 µg/ml) (B) were analyzed as well as control mitogen PHA and Mtb derived PPD (A and B). Medium values (unstimulated PBMC) were subtracted. IFN-γ concentrations were determined from triplicate-pooled supernatant. A cut-off value was set arbitrarily at 100 pg/ml.

Techniques Used: Derivative Assay

Restriction of Rv2034 p81–100 and p88–107 presentation by HLA-DR and -DQ molecules. To determine the HLA-DR and -DQ restriction of Rv2034 p81–100 responding CD4 + T-cell clone, T cells were incubated with Rv2034 p81–100 (A), Rv2034 protein (B) or Ag85B/ESAT-6/Rv2034 fusion protein (C), in the presence of irradiated PBMC with (mis)matched HLA-DR and/or DQ molecules. Both IFN-γ production (white bars) and proliferation (CPM) (black bars) was determined. CPM bars represent median ranging the highest and lowest value (n = 3). Matching HLA alleles are indicated in bold on the x -axis. HLA-DR and HLA-DQ molecules of APCs pulsed with either Rv2034 protein, Rv2034 p81–100, Rv2034 p88–107 (5 µg/ml) or control conditions, were blocked using monoclonal antibodies directed against HLA-DR or -DQ [21] , and T-cell proliferation was analyzed (CPM) (D). Data is representative for three independent experiments.
Figure Legend Snippet: Restriction of Rv2034 p81–100 and p88–107 presentation by HLA-DR and -DQ molecules. To determine the HLA-DR and -DQ restriction of Rv2034 p81–100 responding CD4 + T-cell clone, T cells were incubated with Rv2034 p81–100 (A), Rv2034 protein (B) or Ag85B/ESAT-6/Rv2034 fusion protein (C), in the presence of irradiated PBMC with (mis)matched HLA-DR and/or DQ molecules. Both IFN-γ production (white bars) and proliferation (CPM) (black bars) was determined. CPM bars represent median ranging the highest and lowest value (n = 3). Matching HLA alleles are indicated in bold on the x -axis. HLA-DR and HLA-DQ molecules of APCs pulsed with either Rv2034 protein, Rv2034 p81–100, Rv2034 p88–107 (5 µg/ml) or control conditions, were blocked using monoclonal antibodies directed against HLA-DR or -DQ [21] , and T-cell proliferation was analyzed (CPM) (D). Data is representative for three independent experiments.

Techniques Used: Incubation, Irradiation

2) Product Images from "A Mutation in the HLA-B*2705-Restricted NP383-391 Epitope Affects the Human Influenza A Virus-Specific Cytotoxic T-Lymphocyte Response In Vitro"

Article Title: A Mutation in the HLA-B*2705-Restricted NP383-391 Epitope Affects the Human Influenza A Virus-Specific Cytotoxic T-Lymphocyte Response In Vitro

Journal: Journal of Virology

doi: 10.1128/JVI.78.10.5216-5222.2004

IFN-γ expression and lysis by CD3 + CD8 + cells after stimulation of PBMC with influenza virus A/NL/94-384G or A/NL/94-384R. (A to H) PBMC from an HLA-A * 0201- and HLA-B * 2705-positive donor, expanded after stimulation with influenza virus A/NL/94-384G (A, C, E, and G) or A/NL/94-384R (B, D, F, and H), were restimulated with BLCL pulsed with the M1 58-66 epitope (C and D), the NP 174-184 epitope (E and F), or the NP 383-391 epitope (G and H). Restimulation with untreated cells was used as a negative control (A and B). Virus-specific CTL were visualized after staining with MAbs specific for CD3, CD8, and IFN-γ. The data represent the percentages of IFN-γ + cells (IFN-γ-PE) within the CD8 + -T-cell population. FITC, fluorescein isothiocyanate. (Bottom panels) CTL specific for the M1 58-66 epitope (▴), the NP 174-184 epitope (○), and the NP 383-391 epitope (•) were also detected by a 51 Cr release assay with peptide-pulsed BLCL as target cells and PBMC cultures stimulated with A/NL/94-384G (left) or A/NL/94-384R (right). Untreated cells were included as negative controls (□). Effector cells were added at different E/T ratios as indicated, and specific lysis was calculated.
Figure Legend Snippet: IFN-γ expression and lysis by CD3 + CD8 + cells after stimulation of PBMC with influenza virus A/NL/94-384G or A/NL/94-384R. (A to H) PBMC from an HLA-A * 0201- and HLA-B * 2705-positive donor, expanded after stimulation with influenza virus A/NL/94-384G (A, C, E, and G) or A/NL/94-384R (B, D, F, and H), were restimulated with BLCL pulsed with the M1 58-66 epitope (C and D), the NP 174-184 epitope (E and F), or the NP 383-391 epitope (G and H). Restimulation with untreated cells was used as a negative control (A and B). Virus-specific CTL were visualized after staining with MAbs specific for CD3, CD8, and IFN-γ. The data represent the percentages of IFN-γ + cells (IFN-γ-PE) within the CD8 + -T-cell population. FITC, fluorescein isothiocyanate. (Bottom panels) CTL specific for the M1 58-66 epitope (▴), the NP 174-184 epitope (○), and the NP 383-391 epitope (•) were also detected by a 51 Cr release assay with peptide-pulsed BLCL as target cells and PBMC cultures stimulated with A/NL/94-384G (left) or A/NL/94-384R (right). Untreated cells were included as negative controls (□). Effector cells were added at different E/T ratios as indicated, and specific lysis was calculated.

Techniques Used: Expressing, Lysis, Negative Control, CTL Assay, Staining, Release Assay

Percentages of virus-specific CD8 + T cells in PBMC cultures stimulated in vitro. The percentages of IFN-γ + CD8 + T cells in PBMC from donor 2 were determined after stimulation in vitro with influenza virus A/NL/94-384G (left) or A/NL/94-384R (right), without or with the HLA-B * 2705-restricted NP 383-391 epitope, respectively. Expanded cells were restimulated with autologous BLCL that were infected with influenza virus A/NL/94-384G or A/NL/94-384R or that were incubated with peptide NP 383-391 (SRYWAIRTR), rNP-HK, or rNP-NL. Virus-specific CTL were visualized after staining with MAbs specific for CD3, CD8, and IFN-γ. The data represent the proportions of CD3 + CD8 + IFN-γ + cells in total PBMC cultures. These values were calculated as the product of the percentage of IFN-γ + cells in the CD3 + CD8 + fraction multiplied by the percentage of CD8 + T cells in the PBMC culture. Error bars indicate standard deviations.
Figure Legend Snippet: Percentages of virus-specific CD8 + T cells in PBMC cultures stimulated in vitro. The percentages of IFN-γ + CD8 + T cells in PBMC from donor 2 were determined after stimulation in vitro with influenza virus A/NL/94-384G (left) or A/NL/94-384R (right), without or with the HLA-B * 2705-restricted NP 383-391 epitope, respectively. Expanded cells were restimulated with autologous BLCL that were infected with influenza virus A/NL/94-384G or A/NL/94-384R or that were incubated with peptide NP 383-391 (SRYWAIRTR), rNP-HK, or rNP-NL. Virus-specific CTL were visualized after staining with MAbs specific for CD3, CD8, and IFN-γ. The data represent the proportions of CD3 + CD8 + IFN-γ + cells in total PBMC cultures. These values were calculated as the product of the percentage of IFN-γ + cells in the CD3 + CD8 + fraction multiplied by the percentage of CD8 + T cells in the PBMC culture. Error bars indicate standard deviations.

Techniques Used: In Vitro, Infection, Incubation, CTL Assay, Staining

3) Product Images from "Exploring dendritic cell based vaccines targeting survivin for the treatment of head and neck cancer patients"

Article Title: Exploring dendritic cell based vaccines targeting survivin for the treatment of head and neck cancer patients

Journal: Journal of Translational Medicine

doi: 10.1186/1479-5876-11-152

Spontaneously arising survivin specific T cells in patients. A ) Direct ex vivo tetramer staining of T cells derived from HNSCC patients. The patients (1 thru 5) were all HLA-A2 positive and the CD8 positive T cells were stained with HLA-A2 tetramers containing either the survivin 5 (light grey bars), survivin 96 (dark grey bars) or the modified survivin 96M (black bars) epitope. The percentage tetramer positive T cells in incidated on the Y-axis. B ) PBMC from HNSCC patients were stained with HLA-A2 tetramers containing the survivin 5 epitope and subsequently sorted by flow cytometry. Cells were plated out at a concentration of 0.2 cells per well and stimulated with feeder mix weekly. The left hand panel shows the forward scatter (X-axis; FSC) and side scatter (Y-axis; SSC) of the cells after four weeks of stimulation with feeder mix. A boxed live gate is indicated. The right hand panel shows tetramer staining of the cells in the live gates. Survivin 5 tetramer labeled with either APC (X-axis) or PE (Y-axis) were used to stain the T cells. The percentage of double tetramer positive cells is shown in the upper right quadrant. C ) Draining lymph nodes were obtained from a patient with locally advanced breast cancer, who had received neo-adjuvant chemotherapy before surgery. The spontaneously arising T cells were tested in an ELIspot assay against a number of different HLA-A2 binding peptides representing CTL epitopes; Survivin(95–104), CEA(571–579), Her-2/Neu(369–377) and Her-2/Neu(654–662) respectively and HPV16E7-(11–20) peptide as a negative control. Indicated are the number of spots per 100.000 T cells.
Figure Legend Snippet: Spontaneously arising survivin specific T cells in patients. A ) Direct ex vivo tetramer staining of T cells derived from HNSCC patients. The patients (1 thru 5) were all HLA-A2 positive and the CD8 positive T cells were stained with HLA-A2 tetramers containing either the survivin 5 (light grey bars), survivin 96 (dark grey bars) or the modified survivin 96M (black bars) epitope. The percentage tetramer positive T cells in incidated on the Y-axis. B ) PBMC from HNSCC patients were stained with HLA-A2 tetramers containing the survivin 5 epitope and subsequently sorted by flow cytometry. Cells were plated out at a concentration of 0.2 cells per well and stimulated with feeder mix weekly. The left hand panel shows the forward scatter (X-axis; FSC) and side scatter (Y-axis; SSC) of the cells after four weeks of stimulation with feeder mix. A boxed live gate is indicated. The right hand panel shows tetramer staining of the cells in the live gates. Survivin 5 tetramer labeled with either APC (X-axis) or PE (Y-axis) were used to stain the T cells. The percentage of double tetramer positive cells is shown in the upper right quadrant. C ) Draining lymph nodes were obtained from a patient with locally advanced breast cancer, who had received neo-adjuvant chemotherapy before surgery. The spontaneously arising T cells were tested in an ELIspot assay against a number of different HLA-A2 binding peptides representing CTL epitopes; Survivin(95–104), CEA(571–579), Her-2/Neu(369–377) and Her-2/Neu(654–662) respectively and HPV16E7-(11–20) peptide as a negative control. Indicated are the number of spots per 100.000 T cells.

Techniques Used: Ex Vivo, Staining, Derivative Assay, Modification, Flow Cytometry, Cytometry, Concentration Assay, Labeling, Enzyme-linked Immunospot, Binding Assay, CTL Assay, Negative Control

4) Product Images from "Peptides Derived from Mycobacterium leprae ML1601c Discriminate between Leprosy Patients and Healthy Endemic Controls"

Article Title: Peptides Derived from Mycobacterium leprae ML1601c Discriminate between Leprosy Patients and Healthy Endemic Controls

Journal: Journal of Tropical Medicine

doi: 10.1155/2012/132049

IFN- γ production by PBMC induced in all test groups by ML1601c peptides p3, p11, p12, p13, and p16 and the sum of the IFN- γ values for p3, p11, p12, p13, and p16 combined. The proportions of responders in each test group are indicated below the x -axis.
Figure Legend Snippet: IFN- γ production by PBMC induced in all test groups by ML1601c peptides p3, p11, p12, p13, and p16 and the sum of the IFN- γ values for p3, p11, p12, p13, and p16 combined. The proportions of responders in each test group are indicated below the x -axis.

Techniques Used:

IFN- γ production by PBMC induced by SEB (a), PPD (b), M. leprae (c), and ML1601c recombinant protein (d) in MB ( n = 11), PB ( n = 11), HHC ( n = 19), TB ( n = 8), and EC ( n = 17) from Brazil as well as in Dutch NEC ( n = 21). For NEC, PHA was used instead of SEB. Values were corrected for background values. All background values were typically
Figure Legend Snippet: IFN- γ production by PBMC induced by SEB (a), PPD (b), M. leprae (c), and ML1601c recombinant protein (d) in MB ( n = 11), PB ( n = 11), HHC ( n = 19), TB ( n = 8), and EC ( n = 17) from Brazil as well as in Dutch NEC ( n = 21). For NEC, PHA was used instead of SEB. Values were corrected for background values. All background values were typically

Techniques Used: Recombinant

IFN- γ production by PBMC induced by ML1601c peptides (see Table 1 ) in Brazilian MB ( n = 11), PB ( n = 11), HHC ( n = 19), TB ( n = 8), and EC ( n = 17) as well as Dutch NEC ( n = 21). Values were corrected for background values. All background values were typically
Figure Legend Snippet: IFN- γ production by PBMC induced by ML1601c peptides (see Table 1 ) in Brazilian MB ( n = 11), PB ( n = 11), HHC ( n = 19), TB ( n = 8), and EC ( n = 17) as well as Dutch NEC ( n = 21). Values were corrected for background values. All background values were typically

Techniques Used:

5) Product Images from "The protein kinase C inhibitor sotrastaurin allows regulatory T cell function"

Article Title: The protein kinase C inhibitor sotrastaurin allows regulatory T cell function

Journal: Clinical and Experimental Immunology

doi: 10.1111/cei.12225

Proliferation of peripheral blood mononuclear cells (PBMC) and inhibition by sotrastaurin in vitro ( n = 38 healthy individuals). All proliferation assays revealed more than 10 000 counts per minute. A dose-dependent effect of the study drug on alloresponsiveness
Figure Legend Snippet: Proliferation of peripheral blood mononuclear cells (PBMC) and inhibition by sotrastaurin in vitro ( n = 38 healthy individuals). All proliferation assays revealed more than 10 000 counts per minute. A dose-dependent effect of the study drug on alloresponsiveness

Techniques Used: Inhibition, In Vitro

6) Product Images from "New Biomarkers with Relevance to Leprosy Diagnosis Applicable in Areas Hyperendemic for Leprosy"

Article Title: New Biomarkers with Relevance to Leprosy Diagnosis Applicable in Areas Hyperendemic for Leprosy

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

doi: 10.4049/jimmunol.1103452

IFN-γ responses to M. leprae antigens in PBMC from EC high and EC low in Brazil
Figure Legend Snippet: IFN-γ responses to M. leprae antigens in PBMC from EC high and EC low in Brazil

Techniques Used:

7) Product Images from "Exposure of Intestinal Epithelial Cells to 2′-Fucosyllactose and CpG Enhances Galectin Release and Instructs Dendritic Cells to Drive Th1 and Regulatory-Type Immune Development"

Article Title: Exposure of Intestinal Epithelial Cells to 2′-Fucosyllactose and CpG Enhances Galectin Release and Instructs Dendritic Cells to Drive Th1 and Regulatory-Type Immune Development

Journal: Biomolecules

doi: 10.3390/biom10050784

Phenotype of moDC after IEC/moDC co-culture. After exposure of IEC to 0.5% 2′-FL or GF, in the absence or presence of CpG, and co-culture with αCD3/CD28-activated PBMC, IEC were washed and co-cultured with immature moDC for 48 h ( Figure 1 C). The phenotype of moDC was studied after co-culture. Representative FACS plots are shown in ( A ). Expression of CD86 + ( B ) and CD80 + ( C ) was determined in the CD11c + HLA-DR + population. Data are represented as mean ± SEM of eight independent moDC donors (* p
Figure Legend Snippet: Phenotype of moDC after IEC/moDC co-culture. After exposure of IEC to 0.5% 2′-FL or GF, in the absence or presence of CpG, and co-culture with αCD3/CD28-activated PBMC, IEC were washed and co-cultured with immature moDC for 48 h ( Figure 1 C). The phenotype of moDC was studied after co-culture. Representative FACS plots are shown in ( A ). Expression of CD86 + ( B ) and CD80 + ( C ) was determined in the CD11c + HLA-DR + population. Data are represented as mean ± SEM of eight independent moDC donors (* p

Techniques Used: Co-Culture Assay, Cell Culture, FACS, Expressing

IEC-derived galectin-9 secretion correlates with IEC/PBMC co-culture cytokine release and IEC-derived TGF-β1. After 24 h IEC/PBMC co-culture, IFNγ, IL-13 and IL-10 concentrations were measured in the basolateral supernatant ( Figure 1 A and Figure 3 ). Thereafter, IEC were washed with PBS, the medium was refreshed and IEC incubated for additional 24 h ( Figure 1 B). After the incubation, the basolateral supernatant was collected and IEC-derived galectin-9 and TGF-β1 were measured ( Figure 4 ). The correlation between IEC-derived galectin-9 and IFNγ ( A ), IL-13 ( B ), IL-10 ( C ) and TGF-β1 ( D ) release was tested using Spearman’s test (* p
Figure Legend Snippet: IEC-derived galectin-9 secretion correlates with IEC/PBMC co-culture cytokine release and IEC-derived TGF-β1. After 24 h IEC/PBMC co-culture, IFNγ, IL-13 and IL-10 concentrations were measured in the basolateral supernatant ( Figure 1 A and Figure 3 ). Thereafter, IEC were washed with PBS, the medium was refreshed and IEC incubated for additional 24 h ( Figure 1 B). After the incubation, the basolateral supernatant was collected and IEC-derived galectin-9 and TGF-β1 were measured ( Figure 4 ). The correlation between IEC-derived galectin-9 and IFNγ ( A ), IL-13 ( B ), IL-10 ( C ) and TGF-β1 ( D ) release was tested using Spearman’s test (* p

Techniques Used: Derivative Assay, Co-Culture Assay, Incubation

Cytokine and mediator secretion in IEC/PBMC co-culture. IEC were basolaterally exposed to αCD3/CD28-activated PBMC and apically to 0.25–1% NDO (2′-FL or GF) in combination with CpG ( Figure 1 A). After 24 h incubation, IFNγ ( A ), IL-13 ( B ), IL-10 ( C ) and galectin-9 ( D ) concentrations were measured in the basolateral supernatant. Data are represented as mean ± SEM of 7–8 independent PBMC donors (# p
Figure Legend Snippet: Cytokine and mediator secretion in IEC/PBMC co-culture. IEC were basolaterally exposed to αCD3/CD28-activated PBMC and apically to 0.25–1% NDO (2′-FL or GF) in combination with CpG ( Figure 1 A). After 24 h incubation, IFNγ ( A ), IL-13 ( B ), IL-10 ( C ) and galectin-9 ( D ) concentrations were measured in the basolateral supernatant. Data are represented as mean ± SEM of 7–8 independent PBMC donors (# p

Techniques Used: Co-Culture Assay, Incubation

Cytokine secretion in ccDC/T-cell assay after moDC co-culture with conditioned IEC. Conditioned moDC (ccDC), previously exposed to conditioned IEC, were incubated with naïve T-cells for 5–6 days in an allogeneic ccDC/T-cell assay ( Figure 1 D). Afterwards, IFNγ ( A ), IL-13 ( B ) and IL-10 ( C ) were measured. Data were normalized per donor and represented as mean ± SEM from 5–12 independent PBMC donors (* p
Figure Legend Snippet: Cytokine secretion in ccDC/T-cell assay after moDC co-culture with conditioned IEC. Conditioned moDC (ccDC), previously exposed to conditioned IEC, were incubated with naïve T-cells for 5–6 days in an allogeneic ccDC/T-cell assay ( Figure 1 D). Afterwards, IFNγ ( A ), IL-13 ( B ) and IL-10 ( C ) were measured. Data were normalized per donor and represented as mean ± SEM from 5–12 independent PBMC donors (* p

Techniques Used: Co-Culture Assay, Incubation

Co-culture model description. IEC were grown in 12-well transwell inserts until confluency and basolaterally exposed to either non-activated or αCD3/CD28-activated PBMC. Apically, IEC were conditioned with 2′-FL or GF in the presence or absence of CpG, a TLR9 agonist mimicking a bacterial trigger ( A ). After 24 h incubation, basolateral supernatant was collected to analyze the T-cell mediator release. The IEC were set apart and washed with PBS. Then, fresh medium was added and IEC were kept in incubation for an additional 24 h to study the IEC-derived mediator release ( B ). Alternatively, IEC were washed with PBS and co-cultured with immature moDC for 48 h ( C ). Then, the basolateral supernatant was collected where the mediator release was studied. Additionally, the phenotype of moDC after IEC/moDC co-culture was analyzed. Subsequently, conditioned moDC (ccDC) were exposed to naïve T-cells in an allogeneic DC/T-cell assay ( D ). After 5–6 days incubation, the cytokine release was measured in the supernatant.
Figure Legend Snippet: Co-culture model description. IEC were grown in 12-well transwell inserts until confluency and basolaterally exposed to either non-activated or αCD3/CD28-activated PBMC. Apically, IEC were conditioned with 2′-FL or GF in the presence or absence of CpG, a TLR9 agonist mimicking a bacterial trigger ( A ). After 24 h incubation, basolateral supernatant was collected to analyze the T-cell mediator release. The IEC were set apart and washed with PBS. Then, fresh medium was added and IEC were kept in incubation for an additional 24 h to study the IEC-derived mediator release ( B ). Alternatively, IEC were washed with PBS and co-cultured with immature moDC for 48 h ( C ). Then, the basolateral supernatant was collected where the mediator release was studied. Additionally, the phenotype of moDC after IEC/moDC co-culture was analyzed. Subsequently, conditioned moDC (ccDC) were exposed to naïve T-cells in an allogeneic DC/T-cell assay ( D ). After 5–6 days incubation, the cytokine release was measured in the supernatant.

Techniques Used: Co-Culture Assay, Incubation, Derivative Assay, Cell Culture

Cytokine secretion in IEC/PBMC co-culture after exposure to non-activated or αCD3/CD28-activated PBMC. IEC were basolaterally co-cultured with either αCD3/CD28-activated or non-activated PBMC for 24 h. Apically, IEC were exposed to 2′-FL or GF alone or in combination with CpG, a TLR9 agonist mimicking a bacterial trigger ( Figure 1 A). IFNγ ( A ), IL-13 ( B ) and IL-10 ( C ) concentrations were measured in the basolateral supernatant after IEC/PBMC co-culture. Data are represented as mean ± SEM of six independent PBMC donors. Two-way ANOVA and Bonferroni’s post-hoc tests were used to analyze statistical differences. Square root transformation was performed when data did not fit normal distribution (# p
Figure Legend Snippet: Cytokine secretion in IEC/PBMC co-culture after exposure to non-activated or αCD3/CD28-activated PBMC. IEC were basolaterally co-cultured with either αCD3/CD28-activated or non-activated PBMC for 24 h. Apically, IEC were exposed to 2′-FL or GF alone or in combination with CpG, a TLR9 agonist mimicking a bacterial trigger ( Figure 1 A). IFNγ ( A ), IL-13 ( B ) and IL-10 ( C ) concentrations were measured in the basolateral supernatant after IEC/PBMC co-culture. Data are represented as mean ± SEM of six independent PBMC donors. Two-way ANOVA and Bonferroni’s post-hoc tests were used to analyze statistical differences. Square root transformation was performed when data did not fit normal distribution (# p

Techniques Used: Co-Culture Assay, Cell Culture, Transformation Assay

IEC-derived galectins and TGF-β1 secretion. IEC were washed after IEC/PBMC co-culture and incubated with fresh medium for an additional 24 h ( Figure 1 B). After the incubation period, IEC-derived galectin-3 ( A ), galectin-4 ( B ), galectin-9 ( C ) and TGF-β1 ( D ) were measured in the basolateral supernatant. Data are represented as mean ± SEM of 6–8 independent PBMC donors (# p
Figure Legend Snippet: IEC-derived galectins and TGF-β1 secretion. IEC were washed after IEC/PBMC co-culture and incubated with fresh medium for an additional 24 h ( Figure 1 B). After the incubation period, IEC-derived galectin-3 ( A ), galectin-4 ( B ), galectin-9 ( C ) and TGF-β1 ( D ) were measured in the basolateral supernatant. Data are represented as mean ± SEM of 6–8 independent PBMC donors (# p

Techniques Used: Derivative Assay, Co-Culture Assay, Incubation

Related Articles

Selection:

Article Title: Exploring dendritic cell based vaccines targeting survivin for the treatment of head and neck cancer patients
Article Snippet: .. T cell activation and survivin protein detection Isolation of resting CD8β positive T cells from HLA-A2 positive, healthy donor PBMC (Sanquin) was performed by positive selection using a MACS column (Miltenyi Biotec). .. For this purpose, total PBL were incubated with anti-CD8β mAb (clone: 2ST8.5H7, Beckman Coulter, Inc.) and subsequently with microbead-conjugated anti-mouse IgG Abs (Miltenyi Biotec), followed by MACS sorting according to the manufacturer’s protocol.

In Vitro:

Article Title: Clonal Analysis of the T-Cell Response to In Vivo Expressed Mycobacterium tuberculosis Protein Rv2034, Using a CD154 Expression Based T-Cell Cloning Method
Article Snippet: .. Recombinant proteins were tested to exclude protein non-specific T-cell stimulation and cellular toxicity in IFN-γ release assays using PBMC of in vitro PPD-negative, healthy Dutch donors recruited at the Blood Bank Sanquin, Leiden, The Netherlands , . ..

Article Title: Peptides Derived from Mycobacterium leprae ML1601c Discriminate between Leprosy Patients and Healthy Endemic Controls
Article Snippet: .. Recombinant ML1601c protein was tested to exclude protein nonspecific T-cell stimulation and cellular toxicity in IFN-γ release assays using PBMC of in vitro PPD-negative, healthy Dutch donors recruited at the Sanquin Blood Bank, Leiden, The Netherlands. .. M. leprae Whole-Cell Sonicate Irradiated armadillo-derived M. leprae whole cells were probe-sonicated with a Sanyo sonicator to > 95% breakage.

Cell Stimulation:

Article Title: Clonal Analysis of the T-Cell Response to In Vivo Expressed Mycobacterium tuberculosis Protein Rv2034, Using a CD154 Expression Based T-Cell Cloning Method
Article Snippet: .. Recombinant proteins were tested to exclude protein non-specific T-cell stimulation and cellular toxicity in IFN-γ release assays using PBMC of in vitro PPD-negative, healthy Dutch donors recruited at the Blood Bank Sanquin, Leiden, The Netherlands , . ..

Article Title: Peptides Derived from Mycobacterium leprae ML1601c Discriminate between Leprosy Patients and Healthy Endemic Controls
Article Snippet: .. Recombinant ML1601c protein was tested to exclude protein nonspecific T-cell stimulation and cellular toxicity in IFN-γ release assays using PBMC of in vitro PPD-negative, healthy Dutch donors recruited at the Sanquin Blood Bank, Leiden, The Netherlands. .. M. leprae Whole-Cell Sonicate Irradiated armadillo-derived M. leprae whole cells were probe-sonicated with a Sanyo sonicator to > 95% breakage.

Isolation:

Article Title: Exploring dendritic cell based vaccines targeting survivin for the treatment of head and neck cancer patients
Article Snippet: .. T cell activation and survivin protein detection Isolation of resting CD8β positive T cells from HLA-A2 positive, healthy donor PBMC (Sanquin) was performed by positive selection using a MACS column (Miltenyi Biotec). .. For this purpose, total PBL were incubated with anti-CD8β mAb (clone: 2ST8.5H7, Beckman Coulter, Inc.) and subsequently with microbead-conjugated anti-mouse IgG Abs (Miltenyi Biotec), followed by MACS sorting according to the manufacturer’s protocol.

Article Title: A Mutation in the HLA-B*2705-Restricted NP383-391 Epitope Affects the Human Influenza A Virus-Specific Cytotoxic T-Lymphocyte Response In Vitro
Article Snippet: .. PBMC from healthy HLA-B* 2705-positive blood donors were isolated from heparinized blood (Sanquin Bloodbank, Rotterdam, The Netherlands) by density gradient centrifugation with lymphocyte separation medium (ICN Biomedicals/Cappel, Aurora, Ohio) and cryopreserved at −135°C. .. Genetic subtyping was performed in the Laboratory for Histocompatibility and Immunogenetics at the Sanquin Bloodbank by using a commercial typing system (GenoVision, Vienna, Austria).

Article Title: Protein versus DNA as a marker for peripheral blood mononuclear cell counting
Article Snippet: .. For the validation of the DNA-based cell counting assay, PBMCs were isolated from human leucocyte buffy coat (Sanquin, Amsterdam, The Netherlands) originating from 500 mL whole blood, as previously described [ ]. .. This method also uses a Ficoll density gradient, but is adapted for the isolation of larger numbers of PBMCs.

Cell Counting:

Article Title: Protein versus DNA as a marker for peripheral blood mononuclear cell counting
Article Snippet: .. For the validation of the DNA-based cell counting assay, PBMCs were isolated from human leucocyte buffy coat (Sanquin, Amsterdam, The Netherlands) originating from 500 mL whole blood, as previously described [ ]. .. This method also uses a Ficoll density gradient, but is adapted for the isolation of larger numbers of PBMCs.

Activation Assay:

Article Title: Exploring dendritic cell based vaccines targeting survivin for the treatment of head and neck cancer patients
Article Snippet: .. T cell activation and survivin protein detection Isolation of resting CD8β positive T cells from HLA-A2 positive, healthy donor PBMC (Sanquin) was performed by positive selection using a MACS column (Miltenyi Biotec). .. For this purpose, total PBL were incubated with anti-CD8β mAb (clone: 2ST8.5H7, Beckman Coulter, Inc.) and subsequently with microbead-conjugated anti-mouse IgG Abs (Miltenyi Biotec), followed by MACS sorting according to the manufacturer’s protocol.

Concentration Assay:

Article Title: Bifidobacterium breve Attenuates Murine Dextran Sodium Sulfate-Induced Colitis and Increases Regulatory T Cell Responses
Article Snippet: .. A total of 105 PBMCs were incubated either with anti-CD3 (Sanquin, Amsterdam, the Netherlands) alone (at a final concentration of 1∶10.000) or in combination with L. rhamnosus or B. breve. .. Both L. rhamnosus (NutRes 1 formerly known as NumRes 1) and B. breve (NutRes 204 formerly known as NumRes 204) were provided by Danone Research BV (Wageningen, the Netherlands) as live bacteria in a 20% glycerol stock.

Incubation:

Article Title: Bifidobacterium breve Attenuates Murine Dextran Sodium Sulfate-Induced Colitis and Increases Regulatory T Cell Responses
Article Snippet: .. A total of 105 PBMCs were incubated either with anti-CD3 (Sanquin, Amsterdam, the Netherlands) alone (at a final concentration of 1∶10.000) or in combination with L. rhamnosus or B. breve. .. Both L. rhamnosus (NutRes 1 formerly known as NumRes 1) and B. breve (NutRes 204 formerly known as NumRes 204) were provided by Danone Research BV (Wageningen, the Netherlands) as live bacteria in a 20% glycerol stock.

Gradient Centrifugation:

Article Title: A Mutation in the HLA-B*2705-Restricted NP383-391 Epitope Affects the Human Influenza A Virus-Specific Cytotoxic T-Lymphocyte Response In Vitro
Article Snippet: .. PBMC from healthy HLA-B* 2705-positive blood donors were isolated from heparinized blood (Sanquin Bloodbank, Rotterdam, The Netherlands) by density gradient centrifugation with lymphocyte separation medium (ICN Biomedicals/Cappel, Aurora, Ohio) and cryopreserved at −135°C. .. Genetic subtyping was performed in the Laboratory for Histocompatibility and Immunogenetics at the Sanquin Bloodbank by using a commercial typing system (GenoVision, Vienna, Austria).

Staining:

Article Title: The CD3-Zeta Chimeric Antigen Receptor Overcomes TCR Hypo-Responsiveness of Human Terminal Late-Stage T Cells
Article Snippet: .. Intracellular IFN-gamma was detected in PBMC's from CMV patients stimulated for 6 hrs in presence with PE-conjugated MHC tetrameric complexes HLA-A*0201/pp65495–503 (Sanquin) together with CMV peptide NLVPMVATV (“CMV pp65 Peptide Mix”) or the TCR cross-linking reagent “Cytostim” (both from Miltenyi Biotec) or HIV peptide SLYNTVATL (JPT, Berlin, Germany), respectively, and stained for CD8, CD7 and CD45RO. .. During the final 5 hrs, brefeldin A (5 µg/ml) (BD Biosciences) was added to avoid release of cytokines from the Golgi apparatus, respectively, and cells were subsequently stained for IFN-gamma using the clone 25723.11 mAb (BD Biosciences).

Recombinant:

Article Title: Clonal Analysis of the T-Cell Response to In Vivo Expressed Mycobacterium tuberculosis Protein Rv2034, Using a CD154 Expression Based T-Cell Cloning Method
Article Snippet: .. Recombinant proteins were tested to exclude protein non-specific T-cell stimulation and cellular toxicity in IFN-γ release assays using PBMC of in vitro PPD-negative, healthy Dutch donors recruited at the Blood Bank Sanquin, Leiden, The Netherlands , . ..

Article Title: Peptides Derived from Mycobacterium leprae ML1601c Discriminate between Leprosy Patients and Healthy Endemic Controls
Article Snippet: .. Recombinant ML1601c protein was tested to exclude protein nonspecific T-cell stimulation and cellular toxicity in IFN-γ release assays using PBMC of in vitro PPD-negative, healthy Dutch donors recruited at the Sanquin Blood Bank, Leiden, The Netherlands. .. M. leprae Whole-Cell Sonicate Irradiated armadillo-derived M. leprae whole cells were probe-sonicated with a Sanyo sonicator to > 95% breakage.

Magnetic Cell Separation:

Article Title: Exploring dendritic cell based vaccines targeting survivin for the treatment of head and neck cancer patients
Article Snippet: .. T cell activation and survivin protein detection Isolation of resting CD8β positive T cells from HLA-A2 positive, healthy donor PBMC (Sanquin) was performed by positive selection using a MACS column (Miltenyi Biotec). .. For this purpose, total PBL were incubated with anti-CD8β mAb (clone: 2ST8.5H7, Beckman Coulter, Inc.) and subsequently with microbead-conjugated anti-mouse IgG Abs (Miltenyi Biotec), followed by MACS sorting according to the manufacturer’s protocol.

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  • 93
    Sanquin pbmcs
    L. rhamnosus and B. breve alter T cell differentiation in human <t>PBMCs.</t> PBMCs were stimulated with <t>anti-CD3</t> alone (white bars), with a combination of anti-CD3 and L. rhamnosus (grey bar) or a combination of anti-CD3 and B. breve (black bar) for 48 hours or 7 days. A–D) The percentages of Th2 (GATA3+Tbet-), Th17 (RORγ+FOXP3-), Treg (RORγ-FOXP3+) or Th1 (GATA-Tbet+) cells within the activated T cells (CD4+CD69+) in the PBMCs were determined after 48 hours of incubation. Percentages within activated CD4+CD69+ T cell population are shown. E–H) The percentages of cytokines (IL10, IL17, IL4 or IFNγ) producing CD4+ T cells in the PBMCs were determined after 7 days of incubation. Percentages within CD4+ T cell population are shown. The Results are expressed as mean + SEM, n = 3, * p
    Pbmcs, supplied by Sanquin, used in various techniques. Bioz Stars score: 93/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbmcs/product/Sanquin
    Average 93 stars, based on 46 article reviews
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    85
    Sanquin peptide specific t cells pbmcs
    <t>PBMC</t> of a healthy donor enriched for virus-specific T cells. Peripheral blood of an HLA-A2 + healthy donor was enriched for EBV- and CMV-specific CD8 + T cells. Before culture there were low amounts of EBV- and CMV-specific CD8 + T cells present, which showed an increase after enrichment and 16 days of culture. Cells were stained with tetramers coupled to PE and gated on CD8 + tetramer + . Analysis was performed on the Cyan cytometer.
    Peptide Specific T Cells Pbmcs, supplied by Sanquin, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peptide specific t cells pbmcs/product/Sanquin
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    Price from $9.99 to $1999.99
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    93
    Sanquin blood mononuclear cells pbmc
    Spontaneously arising survivin specific T cells in patients. A ) Direct ex vivo tetramer staining of T cells derived from HNSCC patients. The patients (1 thru 5) were all <t>HLA-A2</t> positive and the CD8 positive T cells were stained with HLA-A2 tetramers containing either the survivin 5 (light grey bars), survivin 96 (dark grey bars) or the modified survivin 96M (black bars) epitope. The percentage tetramer positive T cells in incidated on the Y-axis. B ) <t>PBMC</t> from HNSCC patients were stained with HLA-A2 tetramers containing the survivin 5 epitope and subsequently sorted by flow cytometry. Cells were plated out at a concentration of 0.2 cells per well and stimulated with feeder mix weekly. The left hand panel shows the forward scatter (X-axis; FSC) and side scatter (Y-axis; SSC) of the cells after four weeks of stimulation with feeder mix. A boxed live gate is indicated. The right hand panel shows tetramer staining of the cells in the live gates. Survivin 5 tetramer labeled with either APC (X-axis) or PE (Y-axis) were used to stain the T cells. The percentage of double tetramer positive cells is shown in the upper right quadrant. C ) Draining lymph nodes were obtained from a patient with locally advanced breast cancer, who had received neo-adjuvant chemotherapy before surgery. The spontaneously arising T cells were tested in an ELIspot assay against a number of different HLA-A2 binding peptides representing CTL epitopes; Survivin(95–104), CEA(571–579), Her-2/Neu(369–377) and Her-2/Neu(654–662) respectively and HPV16E7-(11–20) peptide as a negative control. Indicated are the number of spots per 100.000 T cells.
    Blood Mononuclear Cells Pbmc, supplied by Sanquin, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/blood mononuclear cells pbmc/product/Sanquin
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    L. rhamnosus and B. breve alter T cell differentiation in human PBMCs. PBMCs were stimulated with anti-CD3 alone (white bars), with a combination of anti-CD3 and L. rhamnosus (grey bar) or a combination of anti-CD3 and B. breve (black bar) for 48 hours or 7 days. A–D) The percentages of Th2 (GATA3+Tbet-), Th17 (RORγ+FOXP3-), Treg (RORγ-FOXP3+) or Th1 (GATA-Tbet+) cells within the activated T cells (CD4+CD69+) in the PBMCs were determined after 48 hours of incubation. Percentages within activated CD4+CD69+ T cell population are shown. E–H) The percentages of cytokines (IL10, IL17, IL4 or IFNγ) producing CD4+ T cells in the PBMCs were determined after 7 days of incubation. Percentages within CD4+ T cell population are shown. The Results are expressed as mean + SEM, n = 3, * p

    Journal: PLoS ONE

    Article Title: Bifidobacterium breve Attenuates Murine Dextran Sodium Sulfate-Induced Colitis and Increases Regulatory T Cell Responses

    doi: 10.1371/journal.pone.0095441

    Figure Lengend Snippet: L. rhamnosus and B. breve alter T cell differentiation in human PBMCs. PBMCs were stimulated with anti-CD3 alone (white bars), with a combination of anti-CD3 and L. rhamnosus (grey bar) or a combination of anti-CD3 and B. breve (black bar) for 48 hours or 7 days. A–D) The percentages of Th2 (GATA3+Tbet-), Th17 (RORγ+FOXP3-), Treg (RORγ-FOXP3+) or Th1 (GATA-Tbet+) cells within the activated T cells (CD4+CD69+) in the PBMCs were determined after 48 hours of incubation. Percentages within activated CD4+CD69+ T cell population are shown. E–H) The percentages of cytokines (IL10, IL17, IL4 or IFNγ) producing CD4+ T cells in the PBMCs were determined after 7 days of incubation. Percentages within CD4+ T cell population are shown. The Results are expressed as mean + SEM, n = 3, * p

    Article Snippet: A total of 105 PBMCs were incubated either with anti-CD3 (Sanquin, Amsterdam, the Netherlands) alone (at a final concentration of 1∶10.000) or in combination with L. rhamnosus or B. breve.

    Techniques: Cell Differentiation, Incubation

    Flow cytometric analysis of ectoenzyme expression on ILC across different tissues. (A) Gating strategy for flow cytometric analysis of human ILC from healthy peripheral blood, with ILC defined as: Lin − (CD1a, CD3, CD4, CD8, CD14, CD16, CD19, CD34, CD94, CD123, BDCA2, FcεRI, TCRαβ, TCRγδ), CD45 + CD127 + CD161 + cells. From this population, CRTH2 + cells define ILC2; CRTH2 − CD117 − NKp44 − cells define ILC1, and CD117 + cells define ILC3. (B) CD39-CD73 and CD38-CD73 expression by ILC3 freshly isolated from fetal intestine and mLNs, cord blood, adult peripheral blood, healthy bone marrow, and tonsils. Contour plots are representative of 2 to 3 independent experiments. (C) Contour plot showing CD39 − CD73 − (DN) ILC3 and CD39 + CD73 + (ecto + ) ILC3 gating in tonsils, and the bar graph shows the frequency of CD39 and CD73 on total tonsillar ILC3 (n = 25); each symbol represents an individual donor, error bars show the standard error of the mean (SEM), and the horizontal lines represent the mean.

    Journal: Blood Advances

    Article Title: Human ectoenzyme-expressing ILC3: immunosuppressive innate cells that are depleted in graft-versus-host disease

    doi: 10.1182/bloodadvances.2019000176

    Figure Lengend Snippet: Flow cytometric analysis of ectoenzyme expression on ILC across different tissues. (A) Gating strategy for flow cytometric analysis of human ILC from healthy peripheral blood, with ILC defined as: Lin − (CD1a, CD3, CD4, CD8, CD14, CD16, CD19, CD34, CD94, CD123, BDCA2, FcεRI, TCRαβ, TCRγδ), CD45 + CD127 + CD161 + cells. From this population, CRTH2 + cells define ILC2; CRTH2 − CD117 − NKp44 − cells define ILC1, and CD117 + cells define ILC3. (B) CD39-CD73 and CD38-CD73 expression by ILC3 freshly isolated from fetal intestine and mLNs, cord blood, adult peripheral blood, healthy bone marrow, and tonsils. Contour plots are representative of 2 to 3 independent experiments. (C) Contour plot showing CD39 − CD73 − (DN) ILC3 and CD39 + CD73 + (ecto + ) ILC3 gating in tonsils, and the bar graph shows the frequency of CD39 and CD73 on total tonsillar ILC3 (n = 25); each symbol represents an individual donor, error bars show the standard error of the mean (SEM), and the horizontal lines represent the mean.

    Article Snippet: Healthy peripheral blood mononuclear cells were isolated from buffy coats from blood donations (Sanquin, Amsterdam, The Netherlands).

    Techniques: Flow Cytometry, Expressing, Isolation

    PBMC of a healthy donor enriched for virus-specific T cells. Peripheral blood of an HLA-A2 + healthy donor was enriched for EBV- and CMV-specific CD8 + T cells. Before culture there were low amounts of EBV- and CMV-specific CD8 + T cells present, which showed an increase after enrichment and 16 days of culture. Cells were stained with tetramers coupled to PE and gated on CD8 + tetramer + . Analysis was performed on the Cyan cytometer.

    Journal: PLoS ONE

    Article Title: Concurrent Detection of Circulating Minor Histocompatibility Antigen-Specific CD8+ T Cells in SCT Recipients by Combinatorial Encoding MHC Multimers

    doi: 10.1371/journal.pone.0021266

    Figure Lengend Snippet: PBMC of a healthy donor enriched for virus-specific T cells. Peripheral blood of an HLA-A2 + healthy donor was enriched for EBV- and CMV-specific CD8 + T cells. Before culture there were low amounts of EBV- and CMV-specific CD8 + T cells present, which showed an increase after enrichment and 16 days of culture. Cells were stained with tetramers coupled to PE and gated on CD8 + tetramer + . Analysis was performed on the Cyan cytometer.

    Article Snippet: Enrichment of peptide-specific T cells PBMCs were obtained from CMV+ and EBV+ buffy coats (Sanquin blood bank, Nijmegen, the Netherlands) or from patients after SCT or DLI.

    Techniques: Staining, Cytometry

    Detection of multiple peptide-specific CD8 + T cells after enrichment of patient PBMC. Fifteen different dot plots showing multimer-enriched PBMC of representative patient UPN665 stained with the combinatorial encoded MiHA assay using the multimers of pool A. Epitope-specific T cells were detected for RMFPNAPYL (APC and Qdot705) and KLQPYFQTL (Qdot605 and Qdot655). In addition to double positive cells, single positive cells for either APC, Qdot605, Qdot655 or Qdot705 are visible in their respective channels. Cells were stained with multimers, CD8 AlexaFluor 700, CD4-, CD14-, CD16- and CD19-FITC and Sytox Blue. Subsequently, cell populations were analyzed by flow cytometry. Cells were gated on CD8 + FITC − Sytox Blue − lymphocytes and corrected for single, triple, quadruple or quintuple multimer positive cells. The percentage of tetramer + CD8 + T cells is depicted in the dot plots.

    Journal: PLoS ONE

    Article Title: Concurrent Detection of Circulating Minor Histocompatibility Antigen-Specific CD8+ T Cells in SCT Recipients by Combinatorial Encoding MHC Multimers

    doi: 10.1371/journal.pone.0021266

    Figure Lengend Snippet: Detection of multiple peptide-specific CD8 + T cells after enrichment of patient PBMC. Fifteen different dot plots showing multimer-enriched PBMC of representative patient UPN665 stained with the combinatorial encoded MiHA assay using the multimers of pool A. Epitope-specific T cells were detected for RMFPNAPYL (APC and Qdot705) and KLQPYFQTL (Qdot605 and Qdot655). In addition to double positive cells, single positive cells for either APC, Qdot605, Qdot655 or Qdot705 are visible in their respective channels. Cells were stained with multimers, CD8 AlexaFluor 700, CD4-, CD14-, CD16- and CD19-FITC and Sytox Blue. Subsequently, cell populations were analyzed by flow cytometry. Cells were gated on CD8 + FITC − Sytox Blue − lymphocytes and corrected for single, triple, quadruple or quintuple multimer positive cells. The percentage of tetramer + CD8 + T cells is depicted in the dot plots.

    Article Snippet: Enrichment of peptide-specific T cells PBMCs were obtained from CMV+ and EBV+ buffy coats (Sanquin blood bank, Nijmegen, the Netherlands) or from patients after SCT or DLI.

    Techniques: Staining, Flow Cytometry, Cytometry

    Detection of peptide-specific T cells by the combinatorial encoding MiHA-multimer assay. ( A ) Ten different flow cytometric dot plots showing thawed PBMC of representative patient UPN348, stained with the combinatorial encoded MiHA-multimer assay using the peptide set as described in Table 1 . The combination of multimers coupled to PE and to Qdot605 shows a positive response, corresponding to multimer-SMCY.A2 + T cells. Cells were stained with MiHA multimers, CD8 AlexaFluor 700, CD4-, CD14-, CD16- and CD19-FITC and Sytox Blue. Subsequently, cell populations were analyzed by flow cytometry. Cells were gated on CD8 + FITC − Sytox Blue − lymphocytes and corrected for single, triple or quadruple multimer-positive cells. The percentage of multimer + CD8 + T cells is depicted. ( B ) Ten different dot plots showing PBMC of representative patient UPN348 after culturing with SMCY.A2-peptide pulsed donor EBV-LCL. Only the response observed in non-cultured PBMC show an increase of multimer-SMCY.A2 + T cells. Staining and analysis was performed as described under (A).

    Journal: PLoS ONE

    Article Title: Concurrent Detection of Circulating Minor Histocompatibility Antigen-Specific CD8+ T Cells in SCT Recipients by Combinatorial Encoding MHC Multimers

    doi: 10.1371/journal.pone.0021266

    Figure Lengend Snippet: Detection of peptide-specific T cells by the combinatorial encoding MiHA-multimer assay. ( A ) Ten different flow cytometric dot plots showing thawed PBMC of representative patient UPN348, stained with the combinatorial encoded MiHA-multimer assay using the peptide set as described in Table 1 . The combination of multimers coupled to PE and to Qdot605 shows a positive response, corresponding to multimer-SMCY.A2 + T cells. Cells were stained with MiHA multimers, CD8 AlexaFluor 700, CD4-, CD14-, CD16- and CD19-FITC and Sytox Blue. Subsequently, cell populations were analyzed by flow cytometry. Cells were gated on CD8 + FITC − Sytox Blue − lymphocytes and corrected for single, triple or quadruple multimer-positive cells. The percentage of multimer + CD8 + T cells is depicted. ( B ) Ten different dot plots showing PBMC of representative patient UPN348 after culturing with SMCY.A2-peptide pulsed donor EBV-LCL. Only the response observed in non-cultured PBMC show an increase of multimer-SMCY.A2 + T cells. Staining and analysis was performed as described under (A).

    Article Snippet: Enrichment of peptide-specific T cells PBMCs were obtained from CMV+ and EBV+ buffy coats (Sanquin blood bank, Nijmegen, the Netherlands) or from patients after SCT or DLI.

    Techniques: Flow Cytometry, Staining, Cytometry, Cell Culture

    Spontaneously arising survivin specific T cells in patients. A ) Direct ex vivo tetramer staining of T cells derived from HNSCC patients. The patients (1 thru 5) were all HLA-A2 positive and the CD8 positive T cells were stained with HLA-A2 tetramers containing either the survivin 5 (light grey bars), survivin 96 (dark grey bars) or the modified survivin 96M (black bars) epitope. The percentage tetramer positive T cells in incidated on the Y-axis. B ) PBMC from HNSCC patients were stained with HLA-A2 tetramers containing the survivin 5 epitope and subsequently sorted by flow cytometry. Cells were plated out at a concentration of 0.2 cells per well and stimulated with feeder mix weekly. The left hand panel shows the forward scatter (X-axis; FSC) and side scatter (Y-axis; SSC) of the cells after four weeks of stimulation with feeder mix. A boxed live gate is indicated. The right hand panel shows tetramer staining of the cells in the live gates. Survivin 5 tetramer labeled with either APC (X-axis) or PE (Y-axis) were used to stain the T cells. The percentage of double tetramer positive cells is shown in the upper right quadrant. C ) Draining lymph nodes were obtained from a patient with locally advanced breast cancer, who had received neo-adjuvant chemotherapy before surgery. The spontaneously arising T cells were tested in an ELIspot assay against a number of different HLA-A2 binding peptides representing CTL epitopes; Survivin(95–104), CEA(571–579), Her-2/Neu(369–377) and Her-2/Neu(654–662) respectively and HPV16E7-(11–20) peptide as a negative control. Indicated are the number of spots per 100.000 T cells.

    Journal: Journal of Translational Medicine

    Article Title: Exploring dendritic cell based vaccines targeting survivin for the treatment of head and neck cancer patients

    doi: 10.1186/1479-5876-11-152

    Figure Lengend Snippet: Spontaneously arising survivin specific T cells in patients. A ) Direct ex vivo tetramer staining of T cells derived from HNSCC patients. The patients (1 thru 5) were all HLA-A2 positive and the CD8 positive T cells were stained with HLA-A2 tetramers containing either the survivin 5 (light grey bars), survivin 96 (dark grey bars) or the modified survivin 96M (black bars) epitope. The percentage tetramer positive T cells in incidated on the Y-axis. B ) PBMC from HNSCC patients were stained with HLA-A2 tetramers containing the survivin 5 epitope and subsequently sorted by flow cytometry. Cells were plated out at a concentration of 0.2 cells per well and stimulated with feeder mix weekly. The left hand panel shows the forward scatter (X-axis; FSC) and side scatter (Y-axis; SSC) of the cells after four weeks of stimulation with feeder mix. A boxed live gate is indicated. The right hand panel shows tetramer staining of the cells in the live gates. Survivin 5 tetramer labeled with either APC (X-axis) or PE (Y-axis) were used to stain the T cells. The percentage of double tetramer positive cells is shown in the upper right quadrant. C ) Draining lymph nodes were obtained from a patient with locally advanced breast cancer, who had received neo-adjuvant chemotherapy before surgery. The spontaneously arising T cells were tested in an ELIspot assay against a number of different HLA-A2 binding peptides representing CTL epitopes; Survivin(95–104), CEA(571–579), Her-2/Neu(369–377) and Her-2/Neu(654–662) respectively and HPV16E7-(11–20) peptide as a negative control. Indicated are the number of spots per 100.000 T cells.

    Article Snippet: Dendritic cell generation and phenotype analysis Healthy donor derived peripheral blood mononuclear cells (PBMC) were isolated from HLA-A2 positive buffycoats (Sanquin, Amsterdam, The Netherlands) by density gradient centrifugation using Lymphoprep (Nycomed, Oslo, Norway).

    Techniques: Ex Vivo, Staining, Derivative Assay, Modification, Flow Cytometry, Cytometry, Concentration Assay, Labeling, Enzyme-linked Immunospot, Binding Assay, CTL Assay, Negative Control