Structured Review

Millipore pha
LL T-cells display restricted peptide recognition. S.I. of 1 × 10 4 T-cells isolated from 8 LL donors upon stimulation with either whole hHSP60 protein or hHSP60 peptide pools (indicated by Roman numerals, all at 10 μg/mL). <t>PHA</t> and monoclonal <t>anti-CD3/anti-CD28</t> antibodies (αCD3/αCD28) were used as positive control. Antigen was omitted in the negative control (Control). Data are shown as stimulation index (S.I. ± SEM). All assays were carried out in triplicates.
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1) Product Images from "Autoreactive HSP60 epitope-specific T-cells in early human atherosclerotic lesions"

Article Title: Autoreactive HSP60 epitope-specific T-cells in early human atherosclerotic lesions

Journal: Journal of Autoimmunity

doi: 10.1016/j.jaut.2012.07.006

LL T-cells display restricted peptide recognition. S.I. of 1 × 10 4 T-cells isolated from 8 LL donors upon stimulation with either whole hHSP60 protein or hHSP60 peptide pools (indicated by Roman numerals, all at 10 μg/mL). PHA and monoclonal anti-CD3/anti-CD28 antibodies (αCD3/αCD28) were used as positive control. Antigen was omitted in the negative control (Control). Data are shown as stimulation index (S.I. ± SEM). All assays were carried out in triplicates.
Figure Legend Snippet: LL T-cells display restricted peptide recognition. S.I. of 1 × 10 4 T-cells isolated from 8 LL donors upon stimulation with either whole hHSP60 protein or hHSP60 peptide pools (indicated by Roman numerals, all at 10 μg/mL). PHA and monoclonal anti-CD3/anti-CD28 antibodies (αCD3/αCD28) were used as positive control. Antigen was omitted in the negative control (Control). Data are shown as stimulation index (S.I. ± SEM). All assays were carried out in triplicates.

Techniques Used: Isolation, Positive Control, Negative Control

EL T-cells proliferate in response to whole hHSP60 protein and recognize hHSP60-derived peptide pools. The proliferative cellular response of 1 × 10 4 intralesional T-cells isolated from 7 EL donors was evaluated upon stimulation with either whole hHSP60 or hHSP60 peptide pools (indicated by Roman numerals, all at 10 μg/mL). PHA and monoclonal anti-CD3/anti-CD28 antibodies (αCD3/αCD28) were used for positive controls. Antigen was omitted in the negative control (Control). Data are shown as stimulation index (S.I. ± SEM). All assays were carried out in triplicates.
Figure Legend Snippet: EL T-cells proliferate in response to whole hHSP60 protein and recognize hHSP60-derived peptide pools. The proliferative cellular response of 1 × 10 4 intralesional T-cells isolated from 7 EL donors was evaluated upon stimulation with either whole hHSP60 or hHSP60 peptide pools (indicated by Roman numerals, all at 10 μg/mL). PHA and monoclonal anti-CD3/anti-CD28 antibodies (αCD3/αCD28) were used for positive controls. Antigen was omitted in the negative control (Control). Data are shown as stimulation index (S.I. ± SEM). All assays were carried out in triplicates.

Techniques Used: Derivative Assay, Isolation, Negative Control

2) Product Images from "IL-2 receptor ? chain cytokines differentially regulate human CD8+CD127+ and CD8+CD127− T cell division and susceptibility to apoptosis"

Article Title: IL-2 receptor ? chain cytokines differentially regulate human CD8+CD127+ and CD8+CD127− T cell division and susceptibility to apoptosis

Journal: International Immunology

doi: 10.1093/intimm/dxn120

Cell division of subsets of CD8 + CD127 + and CD8 + CD127 − T cells. Isolated CD8 + CD127 + and CD8 + CD127 − T cells were cultured with media only (Ctl) and cells incubated with PHA (2.5 μg ml −1 ), PHA + IL-4 (1, 10 or 100 ng ml −1 ) or PHA + IL-15 (10 ng ml −1 ) or PHA + IL-2 (1, 10 or 100 units ml −1 ). Representative dot plots ( y -axis, CD8 + : x -axis, CFSE) of cell division profiles of CD8 + T cell subsets are shown: (A) CD127 + CD45RA + , (B) CD127 + CD45RA − , (C) CD127 − CD45RA + and (D) CD127 − CD45RA − . The CFSE lo cells (shown in hatched boxes) have undergone the maximum number of detectable divisions. The cytokine concentrations shown here include IL-4 (100 ng ml −1 ), IL-15 (10 ng ml −1 ) and IL-2 (100 units ml −1 ). In addition, the proportion of CFSE lo cells from experiments in six individuals is summarized in bar graphs. Statistically significant changes in the proportion of CFSE lo cells are indicated with a ‘‡’ compared with unstimulated controls (Ctl) or ‘*’ when comparing PHA + IL-4 (paired Student's t -test, P
Figure Legend Snippet: Cell division of subsets of CD8 + CD127 + and CD8 + CD127 − T cells. Isolated CD8 + CD127 + and CD8 + CD127 − T cells were cultured with media only (Ctl) and cells incubated with PHA (2.5 μg ml −1 ), PHA + IL-4 (1, 10 or 100 ng ml −1 ) or PHA + IL-15 (10 ng ml −1 ) or PHA + IL-2 (1, 10 or 100 units ml −1 ). Representative dot plots ( y -axis, CD8 + : x -axis, CFSE) of cell division profiles of CD8 + T cell subsets are shown: (A) CD127 + CD45RA + , (B) CD127 + CD45RA − , (C) CD127 − CD45RA + and (D) CD127 − CD45RA − . The CFSE lo cells (shown in hatched boxes) have undergone the maximum number of detectable divisions. The cytokine concentrations shown here include IL-4 (100 ng ml −1 ), IL-15 (10 ng ml −1 ) and IL-2 (100 units ml −1 ). In addition, the proportion of CFSE lo cells from experiments in six individuals is summarized in bar graphs. Statistically significant changes in the proportion of CFSE lo cells are indicated with a ‘‡’ compared with unstimulated controls (Ctl) or ‘*’ when comparing PHA + IL-4 (paired Student's t -test, P

Techniques Used: Isolation, Cell Culture, Incubation

Cell division of CD8 + CD127 + and CD8 + CD127 − T cells in response to γ C cytokines. Isolated CD8 + CD127 + and CD8 + CD127 − T cells were cultured with media only (Ctl) and cells incubated with PHA (2.5 μg ml −1 ), PHA + IL-4 (1, 10 or 100 ng ml −1 ) or PHA + IL-15 (10 ng ml −1 ) or PHA + IL-2 (1, 10 or 100 units ml −1 ). Representative dot plots of cell division profiles of CD8 + T cell subsets are shown: (A) CD8 + CD127 + and (B) CD8 + CD127 − . The CFSE lo cells (shown in hatched boxes), have undergone ≥3 divisions. The cytokine concentrations shown here include IL-4 (100 ng ml −1 ), IL-15 (10 ng ml −1 ) and IL-2 (100 units ml −1 ). In addition, the proportion of CFSE lo cells from experiments in six individuals is summarized in bar graphs. Statistically significant changes in the proportion of CFSE lo cells are indicated with a ‘‡’ compared with unstimulated controls (Ctl) or ‘*’ when comparing PHA + IL-4 (paired Student's t -test, P
Figure Legend Snippet: Cell division of CD8 + CD127 + and CD8 + CD127 − T cells in response to γ C cytokines. Isolated CD8 + CD127 + and CD8 + CD127 − T cells were cultured with media only (Ctl) and cells incubated with PHA (2.5 μg ml −1 ), PHA + IL-4 (1, 10 or 100 ng ml −1 ) or PHA + IL-15 (10 ng ml −1 ) or PHA + IL-2 (1, 10 or 100 units ml −1 ). Representative dot plots of cell division profiles of CD8 + T cell subsets are shown: (A) CD8 + CD127 + and (B) CD8 + CD127 − . The CFSE lo cells (shown in hatched boxes), have undergone ≥3 divisions. The cytokine concentrations shown here include IL-4 (100 ng ml −1 ), IL-15 (10 ng ml −1 ) and IL-2 (100 units ml −1 ). In addition, the proportion of CFSE lo cells from experiments in six individuals is summarized in bar graphs. Statistically significant changes in the proportion of CFSE lo cells are indicated with a ‘‡’ compared with unstimulated controls (Ctl) or ‘*’ when comparing PHA + IL-4 (paired Student's t -test, P

Techniques Used: Isolation, Cell Culture, Incubation

Cell gating strategy for the analysis of cell division by flow cytometry. Isolated CD8 + CD127 + and CD8 + CD127 − T cells were stained with CFSE and then cultured for 4 days before analysis. Activated and/or dividing lymphocytes within the isolated subsets were identified on the basis of increased forward and side scatter profiles (R1) relative to the proportionally smaller non-dividing cells. Approximately 10 000 total cells were analyzed for each sample. Representative scatter plots of (A) untreated CD8 + T cells and (B) CD8 + T cells treated with a submaximal concentration of PHA (2.5 μg ml −1 ). Cells within the R1 gate (1000–5000 cells) were then gated into CD8 + CD45RA + and CD8 + CD45RA − subsets for cell division analysis. (C) A representative histogram of gated CD8 + T cells depicts cell frequencies versus CFSE staining. Markers delineating cell divisions are included. A bracket delineates cells that have undergone ≥3 divisions, i.e. ‘CFSE lo ’ cells. This histrogram displays the CFSE profile of colchicine-treated (mitotically arrested) cells (black), cells stimulated with submaximal (white) and maximal (gray, 10 μg ml −1 ) concentrations of PHA and non-CFSE-labeled cells (dotted line).
Figure Legend Snippet: Cell gating strategy for the analysis of cell division by flow cytometry. Isolated CD8 + CD127 + and CD8 + CD127 − T cells were stained with CFSE and then cultured for 4 days before analysis. Activated and/or dividing lymphocytes within the isolated subsets were identified on the basis of increased forward and side scatter profiles (R1) relative to the proportionally smaller non-dividing cells. Approximately 10 000 total cells were analyzed for each sample. Representative scatter plots of (A) untreated CD8 + T cells and (B) CD8 + T cells treated with a submaximal concentration of PHA (2.5 μg ml −1 ). Cells within the R1 gate (1000–5000 cells) were then gated into CD8 + CD45RA + and CD8 + CD45RA − subsets for cell division analysis. (C) A representative histogram of gated CD8 + T cells depicts cell frequencies versus CFSE staining. Markers delineating cell divisions are included. A bracket delineates cells that have undergone ≥3 divisions, i.e. ‘CFSE lo ’ cells. This histrogram displays the CFSE profile of colchicine-treated (mitotically arrested) cells (black), cells stimulated with submaximal (white) and maximal (gray, 10 μg ml −1 ) concentrations of PHA and non-CFSE-labeled cells (dotted line).

Techniques Used: Flow Cytometry, Isolation, Staining, Cell Culture, Concentration Assay, Labeling

3) Product Images from "Comparison of the biological characteristics of human mesenchymal stem cells derived from exfoliated deciduous teeth, bone marrow, gingival tissue, and umbilical cord"

Article Title: Comparison of the biological characteristics of human mesenchymal stem cells derived from exfoliated deciduous teeth, bone marrow, gingival tissue, and umbilical cord

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2018.9501

Immunogenicity characterization of MSCs. (A) The proliferation capacity of peripheral blood mononuclear cells following mixed co-culture for 3 days. Cell Counting kit-8 assay revealed that the relative OD values for the four types of MSCs were significantly lower than those in the control and PHA groups. When comparing the four types of MSCs, the OD value for UCMSCs was lower than that of BMSCs and GMSCs. Results represent the mean ± standard deviation of five independent experiments. (B and C) All MSCs exhibited high expression of HLA-I prior to or following IFN-γ treatment. (D and E) All MSCs had a lower expression of HLA-DR prior to IFN-γ treatment, but following IFN-γ treatment, the expression of HLA-DR was significantly upregulated. (F and G) The expression of CD80 was very low prior to and following stimulation with IFN-γ. (H and I) The expression of CD86 was very low prior to and following stimulation with IFN-γ. Results represent the mean ± standard deviation of three independent experiments. *P
Figure Legend Snippet: Immunogenicity characterization of MSCs. (A) The proliferation capacity of peripheral blood mononuclear cells following mixed co-culture for 3 days. Cell Counting kit-8 assay revealed that the relative OD values for the four types of MSCs were significantly lower than those in the control and PHA groups. When comparing the four types of MSCs, the OD value for UCMSCs was lower than that of BMSCs and GMSCs. Results represent the mean ± standard deviation of five independent experiments. (B and C) All MSCs exhibited high expression of HLA-I prior to or following IFN-γ treatment. (D and E) All MSCs had a lower expression of HLA-DR prior to IFN-γ treatment, but following IFN-γ treatment, the expression of HLA-DR was significantly upregulated. (F and G) The expression of CD80 was very low prior to and following stimulation with IFN-γ. (H and I) The expression of CD86 was very low prior to and following stimulation with IFN-γ. Results represent the mean ± standard deviation of three independent experiments. *P

Techniques Used: Co-Culture Assay, Cell Counting, Standard Deviation, Expressing

4) Product Images from "Functional Studies of T Regulatory Lymphocytes in Human Schistosomiasis in Western Kenya"

Article Title: Functional Studies of T Regulatory Lymphocytes in Human Schistosomiasis in Western Kenya

Journal: The American Journal of Tropical Medicine and Hygiene

doi: 10.4269/ajtmh.17-0966

Peripheral blood mononuclear cells (PBMCs) and their parallel T regulatory-depleted populations were cultured in the presence of phytohemagglutinin (PHA), schistosome soluble egg antigen (SEA) or schistosome soluble worm antigenic preparation (SWAP) and their level of proliferation determined by incorporation of BrdU/labeled anti-BrdU as optical density values (O.D.) as Experimental (E; PHA, SEA or SWAP) or Control (C; media alone). Panels are as follows: A , PHA; B , SEA; C , SWAP. Panels D and E are replotted and reanalyzed from Panels B and C , respectively, showing the responses of very low (E–C O.D. values
Figure Legend Snippet: Peripheral blood mononuclear cells (PBMCs) and their parallel T regulatory-depleted populations were cultured in the presence of phytohemagglutinin (PHA), schistosome soluble egg antigen (SEA) or schistosome soluble worm antigenic preparation (SWAP) and their level of proliferation determined by incorporation of BrdU/labeled anti-BrdU as optical density values (O.D.) as Experimental (E; PHA, SEA or SWAP) or Control (C; media alone). Panels are as follows: A , PHA; B , SEA; C , SWAP. Panels D and E are replotted and reanalyzed from Panels B and C , respectively, showing the responses of very low (E–C O.D. values

Techniques Used: Cell Culture, Labeling

5) Product Images from "Isolation of Two Novel Human Anti-CTLA-4 mAbs with Intriguing Biological Properties on Tumor and NK Cells"

Article Title: Isolation of Two Novel Human Anti-CTLA-4 mAbs with Intriguing Biological Properties on Tumor and NK Cells

Journal: Cancers

doi: 10.3390/cancers12082204

Effects of the novel anti-CTLA-4 mAbs on IL-2 and IFNγ cytokines secretion by stimulated human or mouse PBMC or NK cells. Unfractioned human ( A ) or mouse ( B ) PBMCs or ( C ) natural killer (NK) cells were incubated with ID-1 (triangles), ID-8 (circles), or ID-5 (rhomboids) mAbs at increasing concentrations (0.5–50 nM), in the presence of SEB (50 ng/mL) or PHA (2.5 μg/mL), for 66 h at 37 °C. The levels of cytokine secretion were evaluated by ELISA assays on supernatants of the treated lymphocytes. Ipilimumab or an unrelated IgG antibody was used as positive or negative control, respectively. Error bars were calculated by considering the results obtained by at least three determinations obtained in three independent experiments. Error bars depicted means ± SD. p -values for the indicated treatments relative to untreated cells, or to the treatment with Ipilimumab when indicated with vertical bars, are: *** p ≤ 0.001; ** p
Figure Legend Snippet: Effects of the novel anti-CTLA-4 mAbs on IL-2 and IFNγ cytokines secretion by stimulated human or mouse PBMC or NK cells. Unfractioned human ( A ) or mouse ( B ) PBMCs or ( C ) natural killer (NK) cells were incubated with ID-1 (triangles), ID-8 (circles), or ID-5 (rhomboids) mAbs at increasing concentrations (0.5–50 nM), in the presence of SEB (50 ng/mL) or PHA (2.5 μg/mL), for 66 h at 37 °C. The levels of cytokine secretion were evaluated by ELISA assays on supernatants of the treated lymphocytes. Ipilimumab or an unrelated IgG antibody was used as positive or negative control, respectively. Error bars were calculated by considering the results obtained by at least three determinations obtained in three independent experiments. Error bars depicted means ± SD. p -values for the indicated treatments relative to untreated cells, or to the treatment with Ipilimumab when indicated with vertical bars, are: *** p ≤ 0.001; ** p

Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay, Negative Control

6) Product Images from "A peripheral circulating compartment of natural naive CD4+ Tregs"

Article Title: A peripheral circulating compartment of natural naive CD4+ Tregs

Journal: Journal of Clinical Investigation

doi: 10.1172/JCI23963

Growth of CD4 + T cell subsets following stimulation with PHA or anti-CD3/CD28 antibodies in the absence or presence of IL-2 at different doses. Ex vivo–sorted CD4 + T cell subsets were labeled with CFSE and stimulated with PHA or with anti-CD3/CD28 antibodies, autologous CD4 – CD8 – APCs, and IL-2 at the indicated doses. ( A ) Example of data obtained for 1 donor following stimulation with PHA. Cell division was measured at day 5. The dotted lines indicate CFSE intensity of undivided cells. Percentage cell recovery ( B ) and percentage of undivided cells ( C ) are shown as mean values from 3 independent donors.
Figure Legend Snippet: Growth of CD4 + T cell subsets following stimulation with PHA or anti-CD3/CD28 antibodies in the absence or presence of IL-2 at different doses. Ex vivo–sorted CD4 + T cell subsets were labeled with CFSE and stimulated with PHA or with anti-CD3/CD28 antibodies, autologous CD4 – CD8 – APCs, and IL-2 at the indicated doses. ( A ) Example of data obtained for 1 donor following stimulation with PHA. Cell division was measured at day 5. The dotted lines indicate CFSE intensity of undivided cells. Percentage cell recovery ( B ) and percentage of undivided cells ( C ) are shown as mean values from 3 independent donors.

Techniques Used: Ex Vivo, Labeling

Proliferation, cytokine production, and suppressor functions of NnTregs. ( A ) NnTregs and other CD4 + T cell subsets were isolated by FACS cell sorting and stimulated with plate-bound anti-CD3/CD28 antibodies in the presence of APCs. Cell proliferation was assessed on day 7 after stimulation. Results of duplicate cultures are shown for 2 independent donors. ( B ) T cell subsets were stimulated by PMA/ionomycin. The concentration of cytokines in the culture supernatants was assessed by ELISA 24 hours after stimulation. Results of duplicate cultures are shown for 2 independent donors. No detectable cytokines were produced by any of the populations in the absence of stimulation (data not shown). ( C ) Ex vivo–sorted NnTreg and Treg populations were cocultured with responders (CFSE-labeled CD4 + CD25 – T cells) in the presence of PHA at the indicated suppressor/responder cell ratio. As internal control, CFSE-responders were cocultured with unlabeled responders or with the indicated suppressor population in transwell plates. The growth of CSFE-labeled responders was assessed by flow cytometry at day 5 after stimulation. Results are shown as mean values from 3 independent donors.
Figure Legend Snippet: Proliferation, cytokine production, and suppressor functions of NnTregs. ( A ) NnTregs and other CD4 + T cell subsets were isolated by FACS cell sorting and stimulated with plate-bound anti-CD3/CD28 antibodies in the presence of APCs. Cell proliferation was assessed on day 7 after stimulation. Results of duplicate cultures are shown for 2 independent donors. ( B ) T cell subsets were stimulated by PMA/ionomycin. The concentration of cytokines in the culture supernatants was assessed by ELISA 24 hours after stimulation. Results of duplicate cultures are shown for 2 independent donors. No detectable cytokines were produced by any of the populations in the absence of stimulation (data not shown). ( C ) Ex vivo–sorted NnTreg and Treg populations were cocultured with responders (CFSE-labeled CD4 + CD25 – T cells) in the presence of PHA at the indicated suppressor/responder cell ratio. As internal control, CFSE-responders were cocultured with unlabeled responders or with the indicated suppressor population in transwell plates. The growth of CSFE-labeled responders was assessed by flow cytometry at day 5 after stimulation. Results are shown as mean values from 3 independent donors.

Techniques Used: Isolation, FACS, Concentration Assay, Enzyme-linked Immunosorbent Assay, Produced, Ex Vivo, Labeling, Flow Cytometry, Cytometry

7) Product Images from "Heterogeneity of Ara h Component-Specific CD4 T Cell Responses in Peanut-Allergic Subjects"

Article Title: Heterogeneity of Ara h Component-Specific CD4 T Cell Responses in Peanut-Allergic Subjects

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01408

“Modified” CD154 upregulation T-cell epitope mapping. CD154 + CD45RA − CD4 + T cells were sorted (A) and expended in presence of autologous-irradiated peripheral blood mononuclear cells (PBMCs), 1 µg/mL PHA, and human IL-2 (B) . (B,C) For epitope mapping, 10 5 cells were stimulated for 3 h with 5 µg/mL of synthesized Ara h 2 peptide pools with autologous PBMCs. Pool giving a positive response in the CD154 assay was retested with 40 µg/mL blocking antibodies anti-HLA-DR or anti-HLA-DQ. Peptides from pool giving a positive response were then tested individually as descried before. Then, individual peptides, giving a positive response, were loaded into the biotinylated HLA-DR protein to generate tetramers and tested in vitro . (D) Table of Ara h 2 T cell epitopes identified.
Figure Legend Snippet: “Modified” CD154 upregulation T-cell epitope mapping. CD154 + CD45RA − CD4 + T cells were sorted (A) and expended in presence of autologous-irradiated peripheral blood mononuclear cells (PBMCs), 1 µg/mL PHA, and human IL-2 (B) . (B,C) For epitope mapping, 10 5 cells were stimulated for 3 h with 5 µg/mL of synthesized Ara h 2 peptide pools with autologous PBMCs. Pool giving a positive response in the CD154 assay was retested with 40 µg/mL blocking antibodies anti-HLA-DR or anti-HLA-DQ. Peptides from pool giving a positive response were then tested individually as descried before. Then, individual peptides, giving a positive response, were loaded into the biotinylated HLA-DR protein to generate tetramers and tested in vitro . (D) Table of Ara h 2 T cell epitopes identified.

Techniques Used: Irradiation, Synthesized, Acetylene Reduction Assay, Blocking Assay, In Vitro

8) Product Images from "Expression and reactivation of HIV in a chemokine induced model of HIV latency in primary resting CD4+ T cells"

Article Title: Expression and reactivation of HIV in a chemokine induced model of HIV latency in primary resting CD4+ T cells

Journal: Retrovirology

doi: 10.1186/1742-4690-8-80

No productive HIV infection in CCL19-treated infected cells . Resting CD4+ T cells were cultured for 2 days with PHA/IL-2, CCL19 or without activation (unactivated). Cells were infected with NL4.3 or NL4.3-Δnef/EGFP for 2 hrs. The virus was washed off and the infected cells were cultured with media containing IL-2 (10 IU/mL) for up to 4 days. Productive infection was detected by flow cytometry of (A) intracellular HIV p24 (Alexa-488) following infection with NL4.3 (left panels; two representative donors). The right hand panels show the corresponding integrated copies of HIV per million cells for the same donors (as the mean of two donors (column) and as individual data (open symbols)). (B) Productive infection was detected by flow cytometry of EGFP expression following infection with NL4.3-Δnef/EGFP (one donor). The integrated copies of HIV per million cells are shown for this donor (right hand panel). SSC = side scatter.
Figure Legend Snippet: No productive HIV infection in CCL19-treated infected cells . Resting CD4+ T cells were cultured for 2 days with PHA/IL-2, CCL19 or without activation (unactivated). Cells were infected with NL4.3 or NL4.3-Δnef/EGFP for 2 hrs. The virus was washed off and the infected cells were cultured with media containing IL-2 (10 IU/mL) for up to 4 days. Productive infection was detected by flow cytometry of (A) intracellular HIV p24 (Alexa-488) following infection with NL4.3 (left panels; two representative donors). The right hand panels show the corresponding integrated copies of HIV per million cells for the same donors (as the mean of two donors (column) and as individual data (open symbols)). (B) Productive infection was detected by flow cytometry of EGFP expression following infection with NL4.3-Δnef/EGFP (one donor). The integrated copies of HIV per million cells are shown for this donor (right hand panel). SSC = side scatter.

Techniques Used: Infection, Cell Culture, Activation Assay, Flow Cytometry, Cytometry, Expressing

High levels of HIV integration with minimal virus production in CCL19-treated infected CD4+ T-cells consistent with latent infection . (A). Schematic diagram of the experimental protocol used for isolation of CD4+ T-cells and HIV infection. Resting CD4+ T-cells were cultured for 2 days with PHA/IL-2, CCL19 or without activation (unactivated). Cells were then infected with WT NL4.3, NL4.3Δenv or NL4.3-Δnef/EGFP or mock for 2 hrs and virus was washed off. The infected cells were cultured with media containing IL-2 (10 IU/mL) for 4 days. Infection of cells with WT NL4.3 (open bars) or NL4.3Δenv (grey bars) was quantified by detection of (B) integrated HIV DNA (C) RT activity (CPM/μl) in culture supernatant or (D) luciferase activity of supernatants using the TZM-bl indicator cell line. In all graphs, the mean (column) and individual data (open symbols) from two donors are shown.
Figure Legend Snippet: High levels of HIV integration with minimal virus production in CCL19-treated infected CD4+ T-cells consistent with latent infection . (A). Schematic diagram of the experimental protocol used for isolation of CD4+ T-cells and HIV infection. Resting CD4+ T-cells were cultured for 2 days with PHA/IL-2, CCL19 or without activation (unactivated). Cells were then infected with WT NL4.3, NL4.3Δenv or NL4.3-Δnef/EGFP or mock for 2 hrs and virus was washed off. The infected cells were cultured with media containing IL-2 (10 IU/mL) for 4 days. Infection of cells with WT NL4.3 (open bars) or NL4.3Δenv (grey bars) was quantified by detection of (B) integrated HIV DNA (C) RT activity (CPM/μl) in culture supernatant or (D) luciferase activity of supernatants using the TZM-bl indicator cell line. In all graphs, the mean (column) and individual data (open symbols) from two donors are shown.

Techniques Used: Infection, Isolation, Cell Culture, Activation Assay, Activity Assay, Luciferase

High level of nuclear MS RNA in CCL19-treated latently infected CD4+ T-cells . (A) Schematic diagram of the broad classes of HIV mRNA, including multiply spliced (MS) singly spliced (SS) and unspliced (US) HIV RNA (adapted from [ 56 ]). Location of primers and probes used for real-time PCR quantification of US, SS and MS RNA are shown. (B) Resting CD4+ T cells were activated with IL-2/PHA, CCL19 or unactivated and infected with NL4.3 or NL4.3Δenv. The fold increase in US RNA at day 4 post infection (relative to day 0; left panels) and the absolute copies per million cells of MS RNA (right panels) is shown for (B) total RNA following infection with WT NL4.3 (n = 5) and (C and D) nuclear (open) and cytoplasmic (grey) fractions following infection with (C) NL4.3 and (D) NL4.3Δenv. The mean (column) and individual data (open symbols) are shown. The ratio of MS RNA to integrated DNA for each donor is also shown (C and D, far right panel). The detection limit for US RNA and MS RNA was 200 copies/10 6 cells and is shown as dashed line for MS RNA. * = P
Figure Legend Snippet: High level of nuclear MS RNA in CCL19-treated latently infected CD4+ T-cells . (A) Schematic diagram of the broad classes of HIV mRNA, including multiply spliced (MS) singly spliced (SS) and unspliced (US) HIV RNA (adapted from [ 56 ]). Location of primers and probes used for real-time PCR quantification of US, SS and MS RNA are shown. (B) Resting CD4+ T cells were activated with IL-2/PHA, CCL19 or unactivated and infected with NL4.3 or NL4.3Δenv. The fold increase in US RNA at day 4 post infection (relative to day 0; left panels) and the absolute copies per million cells of MS RNA (right panels) is shown for (B) total RNA following infection with WT NL4.3 (n = 5) and (C and D) nuclear (open) and cytoplasmic (grey) fractions following infection with (C) NL4.3 and (D) NL4.3Δenv. The mean (column) and individual data (open symbols) are shown. The ratio of MS RNA to integrated DNA for each donor is also shown (C and D, far right panel). The detection limit for US RNA and MS RNA was 200 copies/10 6 cells and is shown as dashed line for MS RNA. * = P

Techniques Used: Mass Spectrometry, Infection, Real-time Polymerase Chain Reaction

Virus production from latently infected CCL19 stimulated cells . (A) Schematic diagram of the experimental protocol used for infection and restimulation of CCL19-treated resting CD4+ T-cells. Resting CD4+ T-cells were cultured for 2 days with CCL19 and infected with NL4.8 and then restimulated with different activation agents at day 4 post- infection (PI). PHA-stimulated activated PBMCs were added at a ratio of 2:1. The cultures were maintained in IL-2 alone. Supernatant was harvested at day 7 and 10 PI to detect RT activity. (B) RT activity (CPM/μl) was measured following incubation of CCL19-treated infected CD4+ T-cells with (i) PHA/PMA (maximal stimulation) or with DMSO, (ii) TNFα, IL-7, prostratin and the combination of IL-7 with prostratin (expressed as percentage of maximal stimulation), (iii) PHA/PMA or DMSO (from different donors for vorinostat (SAHA) experiments) or (iv) vorionostat (SAHA) expressed as percentage of maximal stimulation (from (iii)). The mean (column) and individual data (open symbols) from four donors are shown. (C) The latently infected cell line ACH2 was also incubated with the same stimuli as in (B) and supernatant collected at day 1, 2 and 3 post stimulation. The mean (column) and individual data (open symbols) from three separate experiments, following activation with vorinostat (SAHA) are shown.
Figure Legend Snippet: Virus production from latently infected CCL19 stimulated cells . (A) Schematic diagram of the experimental protocol used for infection and restimulation of CCL19-treated resting CD4+ T-cells. Resting CD4+ T-cells were cultured for 2 days with CCL19 and infected with NL4.8 and then restimulated with different activation agents at day 4 post- infection (PI). PHA-stimulated activated PBMCs were added at a ratio of 2:1. The cultures were maintained in IL-2 alone. Supernatant was harvested at day 7 and 10 PI to detect RT activity. (B) RT activity (CPM/μl) was measured following incubation of CCL19-treated infected CD4+ T-cells with (i) PHA/PMA (maximal stimulation) or with DMSO, (ii) TNFα, IL-7, prostratin and the combination of IL-7 with prostratin (expressed as percentage of maximal stimulation), (iii) PHA/PMA or DMSO (from different donors for vorinostat (SAHA) experiments) or (iv) vorionostat (SAHA) expressed as percentage of maximal stimulation (from (iii)). The mean (column) and individual data (open symbols) from four donors are shown. (C) The latently infected cell line ACH2 was also incubated with the same stimuli as in (B) and supernatant collected at day 1, 2 and 3 post stimulation. The mean (column) and individual data (open symbols) from three separate experiments, following activation with vorinostat (SAHA) are shown.

Techniques Used: Infection, Cell Culture, Activation Assay, Activity Assay, Incubation

9) Product Images from "The Regulated Secretory Pathway in CD4+ T cells Contributes to Human Immunodeficiency Virus Type-1 Cell-to-Cell Spread at the Virological Synapse"

Article Title: The Regulated Secretory Pathway in CD4+ T cells Contributes to Human Immunodeficiency Virus Type-1 Cell-to-Cell Spread at the Virological Synapse

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002226

CHS CD4 + T cells are less able to support HIV-1 replication in culture. A ) CHS CD4 + T cells (black bars) and cells from normal WT donors (white bars) were infected with HIV-1 and supernatants were taken at various days post-infection and virus release was quantified by HIV-1 Gag p24 ELISA. Values in the upper panel were normalized to viable cell count and are ng p24/ml/10 6 cells and show Gag p24 from supernatants taken at 10–12 days post-infection. The lower panel shows the Gag p24 levels from the same experiments at all time points. Data are from four independent experiments performed using four different WT donors and three different CHS cell lines from two unrelated patients. Error bars show the SEM. B ) CHS cells produce infectious virus. Cell-free supernatants were harvested at 10–12 days post-infection and viral infectivity was quantified on HeLa Tzm-bl reporter cells by luciferase assay. The TCID50 was calculated and data are shown as the TCID50 per ng of p24 to normalize for differences in the amount of virus in the supernatant. Data are from three independent experiments performed using four different WT donors and three different CHS cell lines from two unrelated patients. Error bars show the SEM. C ) Western blotting analysis of HIV-1 Env incorporation into purified virions . Viral particles from HIV-1 infected cultures were purified by ultracentrifugation and concentrated 10-fold. An equal volume was loaded onto polyacrylamide gels and viral proteins were separated by SDS-PAGE. Western blotting was performed with rabbit anti-Env and rabbit anti-Gag serum and proteins were visualized by ECL. Western blots of two WT donors and two CHS patients are shown and are representative of results obtained using four WT virus samples and four CHS virus samples. One example of PHA blasts (left panels) and one example of cloned CD4 + T cells (right panels) are shown. Note that HIV-1 Env gp120 and unprocessed gp160 can be detected in all virus samples. D ) Quantification of HIV-1 infected cells. CHS cells and WT cells were fixed, stained for HIV-1 Gag and the number of Gag + infected cells was quantified by LSCM. Data are from three independent experiments with the SEM. E ) Confocal microscopy of a VS formed between an infected CHS T cell and a target T cell. HIV-1 infected CHS CD4 + T cells were mixed with an equal number of uninfected CHS CD4 + T cells (prestained with the CD4 mAb L120 (green)) and conjugates were incubated for 1 h at 37°C (upper panel). Cells were then fixed, permeabilized and stained for HIV-1 Env with 2G12 (red) and HIV-1 Gag (blue) and examined by LSCM. The target cell is indicated with an asterisk on the corresponding DIC image. A representative VS formed between two WT cells is shown for comparison (lower panel).
Figure Legend Snippet: CHS CD4 + T cells are less able to support HIV-1 replication in culture. A ) CHS CD4 + T cells (black bars) and cells from normal WT donors (white bars) were infected with HIV-1 and supernatants were taken at various days post-infection and virus release was quantified by HIV-1 Gag p24 ELISA. Values in the upper panel were normalized to viable cell count and are ng p24/ml/10 6 cells and show Gag p24 from supernatants taken at 10–12 days post-infection. The lower panel shows the Gag p24 levels from the same experiments at all time points. Data are from four independent experiments performed using four different WT donors and three different CHS cell lines from two unrelated patients. Error bars show the SEM. B ) CHS cells produce infectious virus. Cell-free supernatants were harvested at 10–12 days post-infection and viral infectivity was quantified on HeLa Tzm-bl reporter cells by luciferase assay. The TCID50 was calculated and data are shown as the TCID50 per ng of p24 to normalize for differences in the amount of virus in the supernatant. Data are from three independent experiments performed using four different WT donors and three different CHS cell lines from two unrelated patients. Error bars show the SEM. C ) Western blotting analysis of HIV-1 Env incorporation into purified virions . Viral particles from HIV-1 infected cultures were purified by ultracentrifugation and concentrated 10-fold. An equal volume was loaded onto polyacrylamide gels and viral proteins were separated by SDS-PAGE. Western blotting was performed with rabbit anti-Env and rabbit anti-Gag serum and proteins were visualized by ECL. Western blots of two WT donors and two CHS patients are shown and are representative of results obtained using four WT virus samples and four CHS virus samples. One example of PHA blasts (left panels) and one example of cloned CD4 + T cells (right panels) are shown. Note that HIV-1 Env gp120 and unprocessed gp160 can be detected in all virus samples. D ) Quantification of HIV-1 infected cells. CHS cells and WT cells were fixed, stained for HIV-1 Gag and the number of Gag + infected cells was quantified by LSCM. Data are from three independent experiments with the SEM. E ) Confocal microscopy of a VS formed between an infected CHS T cell and a target T cell. HIV-1 infected CHS CD4 + T cells were mixed with an equal number of uninfected CHS CD4 + T cells (prestained with the CD4 mAb L120 (green)) and conjugates were incubated for 1 h at 37°C (upper panel). Cells were then fixed, permeabilized and stained for HIV-1 Env with 2G12 (red) and HIV-1 Gag (blue) and examined by LSCM. The target cell is indicated with an asterisk on the corresponding DIC image. A representative VS formed between two WT cells is shown for comparison (lower panel).

Techniques Used: Infection, Enzyme-linked Immunosorbent Assay, Cell Counting, Luciferase, Western Blot, Purification, SDS Page, Clone Assay, Staining, Confocal Microscopy, Incubation

10) Product Images from "Simian immunodeficiency virus infection and immune responses in the pig-tailed macaque testis"

Article Title: Simian immunodeficiency virus infection and immune responses in the pig-tailed macaque testis

Journal: Journal of Leukocyte Biology

doi: 10.1189/jlb.4A0914-438R

Cytokine production by testicular and blood T cells after mitogen stimulation. Blood or testicular cells from uninfected or SIV-infected animals were stimulated with 2 μ g/ml PMA, 15 mg/ml PHA, and 1 mg/ml Staphylococcus Enterotoxin-B for 6 h. T cells producing TNF, IFN- γ , or both cytokines were detected by use of an intracellular cytokine staining assay. (A) Examples of gating on testicular interstitial cells expressing TNF, IFN- γ , or both cytokines after stimulation. Before these plots, cells were gated on live, single CD45 + and CD3 + cells, as well as CD4 + (upper) or CD8 + (lower). The proportion of CD4 + cells (B) and CD8 + cells (C) producing TNF, IFN- γ , or both cytokines was calculated for all animals by use of the percentage of cells that were producing either or both cytokines. Horizontal lines on the graphs denote medians. Triangles on graphs denote the SIV-controlling animal.
Figure Legend Snippet: Cytokine production by testicular and blood T cells after mitogen stimulation. Blood or testicular cells from uninfected or SIV-infected animals were stimulated with 2 μ g/ml PMA, 15 mg/ml PHA, and 1 mg/ml Staphylococcus Enterotoxin-B for 6 h. T cells producing TNF, IFN- γ , or both cytokines were detected by use of an intracellular cytokine staining assay. (A) Examples of gating on testicular interstitial cells expressing TNF, IFN- γ , or both cytokines after stimulation. Before these plots, cells were gated on live, single CD45 + and CD3 + cells, as well as CD4 + (upper) or CD8 + (lower). The proportion of CD4 + cells (B) and CD8 + cells (C) producing TNF, IFN- γ , or both cytokines was calculated for all animals by use of the percentage of cells that were producing either or both cytokines. Horizontal lines on the graphs denote medians. Triangles on graphs denote the SIV-controlling animal.

Techniques Used: Infection, Staining, Expressing

11) Product Images from "Heterogeneity of Ara h Component-Specific CD4 T Cell Responses in Peanut-Allergic Subjects"

Article Title: Heterogeneity of Ara h Component-Specific CD4 T Cell Responses in Peanut-Allergic Subjects

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01408

“Modified” CD154 upregulation T-cell epitope mapping. CD154 + CD45RA − CD4 + T cells were sorted (A) and expended in presence of autologous-irradiated peripheral blood mononuclear cells (PBMCs), 1 µg/mL PHA, and human IL-2 (B) . (B,C) For epitope mapping, 10 5 cells were stimulated for 3 h with 5 µg/mL of synthesized Ara h 2 peptide pools with autologous PBMCs. Pool giving a positive response in the CD154 assay was retested with 40 µg/mL blocking antibodies anti-HLA-DR or anti-HLA-DQ. Peptides from pool giving a positive response were then tested individually as descried before. Then, individual peptides, giving a positive response, were loaded into the biotinylated HLA-DR protein to generate tetramers and tested in vitro . (D) Table of Ara h 2 T cell epitopes identified.
Figure Legend Snippet: “Modified” CD154 upregulation T-cell epitope mapping. CD154 + CD45RA − CD4 + T cells were sorted (A) and expended in presence of autologous-irradiated peripheral blood mononuclear cells (PBMCs), 1 µg/mL PHA, and human IL-2 (B) . (B,C) For epitope mapping, 10 5 cells were stimulated for 3 h with 5 µg/mL of synthesized Ara h 2 peptide pools with autologous PBMCs. Pool giving a positive response in the CD154 assay was retested with 40 µg/mL blocking antibodies anti-HLA-DR or anti-HLA-DQ. Peptides from pool giving a positive response were then tested individually as descried before. Then, individual peptides, giving a positive response, were loaded into the biotinylated HLA-DR protein to generate tetramers and tested in vitro . (D) Table of Ara h 2 T cell epitopes identified.

Techniques Used: Irradiation, Synthesized, Acetylene Reduction Assay, Blocking Assay, In Vitro

12) Product Images from "Phenotype, specificity and avidity of antitumour CD8+ T cells in melanoma"

Article Title: Phenotype, specificity and avidity of antitumour CD8+ T cells in melanoma

Journal: Nature

doi: 10.1038/s41586-021-03704-y

Cell states of tumor-specific CD8+ TILs a-c. Antigen specificity screening of 94 TCRs sequenced from clonally expanded CD8+ T cells isolated from tumor biopsies of 7 patients with metastatic melanoma from Sade-Feldman et al . a . After TCR reconstruction and expression in T cells, reactivity was measured as CD137 upregulation on TCR-transduced (mTRBC+) CD8+ cells upon culture with autologous EBV-LCLs pulsed with peptide pools covering immunogenic viral epitopes (CEF). Unstimulated cells were analyzed as negative control. Results are reported after subtraction of background CD137 expression on T cells transduced with an irrelevant TCR. Five TCRs (black dots) recognized unpulsed EBV-LCLs, thereby documenting specificity for EBV epitopes. b . TCR antitumor reactivity, evaluated upon culture with autologous EBV-LCLs pulsed with peptide pools derived from 12 known MAAs. Background detected upon culture with DMSO-pulsed EBV-LCLs was subtracted. Additional positive and negative controls were an irrelevant peptide (Ova) and polyclonal stimulators (PHA or PMA/ionomycin), respectively. Colored dots denote MAA-reactive TCRs. c. ). d-e-f. Single-cell phenotype of TILs with antiviral or anti-MAA TCRs identified in the validation cohort from Sade-Feldman et al . d. t-SNE plot of CD8+ TILs highlighting the spatial distribution of cells harboring TCRs with identified antigen specificity. Each color denotes a distinct specificity, with crosses representing two cells with identical spatial coordinates. e. . f . RNA transcripts differentially expressed between antiviral and anti-MAA cells (log 2 FC > 1.5, adj. p value
Figure Legend Snippet: Cell states of tumor-specific CD8+ TILs a-c. Antigen specificity screening of 94 TCRs sequenced from clonally expanded CD8+ T cells isolated from tumor biopsies of 7 patients with metastatic melanoma from Sade-Feldman et al . a . After TCR reconstruction and expression in T cells, reactivity was measured as CD137 upregulation on TCR-transduced (mTRBC+) CD8+ cells upon culture with autologous EBV-LCLs pulsed with peptide pools covering immunogenic viral epitopes (CEF). Unstimulated cells were analyzed as negative control. Results are reported after subtraction of background CD137 expression on T cells transduced with an irrelevant TCR. Five TCRs (black dots) recognized unpulsed EBV-LCLs, thereby documenting specificity for EBV epitopes. b . TCR antitumor reactivity, evaluated upon culture with autologous EBV-LCLs pulsed with peptide pools derived from 12 known MAAs. Background detected upon culture with DMSO-pulsed EBV-LCLs was subtracted. Additional positive and negative controls were an irrelevant peptide (Ova) and polyclonal stimulators (PHA or PMA/ionomycin), respectively. Colored dots denote MAA-reactive TCRs. c. ). d-e-f. Single-cell phenotype of TILs with antiviral or anti-MAA TCRs identified in the validation cohort from Sade-Feldman et al . d. t-SNE plot of CD8+ TILs highlighting the spatial distribution of cells harboring TCRs with identified antigen specificity. Each color denotes a distinct specificity, with crosses representing two cells with identical spatial coordinates. e. . f . RNA transcripts differentially expressed between antiviral and anti-MAA cells (log 2 FC > 1.5, adj. p value

Techniques Used: Isolation, Expressing, Negative Control, Transduction, Derivative Assay

Isolation, single-cell sequencing and screening of tumor-reactive TCRs from peripheral blood samples. a-b. PBMCs harvested at serial timepoints (TP1, TP1, TP3; see ) were cultured with autologous melanoma cell lines to enrich for antitumor TCRs. After two rounds of stimulation, the reactivity of effector CD8+ T cells was assessed by measuring: ( a ) degranulation and cytokine production; or ( b ) CD137 upregulation upon re-challenge with melanoma (blue line). The specificity of the response was supported by the low recognition of HLA-mismatched unrelated melanoma (dashed grey line). Negative controls (cultured in the absence of target cells) and positive controls (polyclonal stimulators, PHA or PMA-ionomycin) are displayed as solid grey and black lines, respectively. c. ) upon stimulation without any target (top panel) or in the presence of autologous melanoma (bottom panels). CD107a/b+ cells secreting at least one of the measured cytokines (IFNγ, TNFα and IL-2) were single-cell sorted and sequenced. Gates depict the detection and quantification of reactive (black) or sorted cells (magenta) from a representative sample (TP3 PBMCs from Pt-A). d . TCR clonotypes identified upon single-cell sorting and scTCRseq of melanoma-reactive CD8+ T cells from the 4 studied patients. Bars - cell counts of clonotype families, defined as CD8+ cells bearing identical TCRα and TCRβ chains, divided based on their detection at specific timepoints (TP1, TP2, TP3) or across multiple timepoints (shared). Presence of multiple TCRα or TCRβ chains is indicated with black or orange borders, respectively. e-h. , to identify: ( e ) tumor-specific TCRs, ( f ) non-tumor reactive TCRs, and ( g ) tumor/control reactive TCRs. Reactivity was calculated by subtracting from CD137 expression of CD8+ cells transduced with the reconstructed TCR the background of lymphocytes transduced with an irrelevant TCR. Floating boxes show min to max measurements, with mean values depicted as horizontal lines; white dots denote the basal level of activation measured on untransduced cells. Pie charts in h summarize the classification of TCR reactivity for all reconstructed TCRs. i. Cytotoxicity mediated by TCRs classified as tumor-specific (left panel), non-tumor reactive (middle panel) or tumor/control reactive (right panel). Degranulation (CD107a/b+) and concomitant production of cytokines (IFNγ, TNFα and IL-2) were measured through intracellular flow-cytometry on TCR transduced (mTRBC+) CD8+ T cells cultured alone or in the presence of autologous melanoma. Each dot represents the results of a single TCR isolated from CD8+ TILs (upon subtraction of background activation measured on CD8+ lymphocytes transduced with an irrelevant TCR). White dots denote the basal level of cytotoxicity of untransduced cells. j ). Extended Data Fig. 1
Figure Legend Snippet: Isolation, single-cell sequencing and screening of tumor-reactive TCRs from peripheral blood samples. a-b. PBMCs harvested at serial timepoints (TP1, TP1, TP3; see ) were cultured with autologous melanoma cell lines to enrich for antitumor TCRs. After two rounds of stimulation, the reactivity of effector CD8+ T cells was assessed by measuring: ( a ) degranulation and cytokine production; or ( b ) CD137 upregulation upon re-challenge with melanoma (blue line). The specificity of the response was supported by the low recognition of HLA-mismatched unrelated melanoma (dashed grey line). Negative controls (cultured in the absence of target cells) and positive controls (polyclonal stimulators, PHA or PMA-ionomycin) are displayed as solid grey and black lines, respectively. c. ) upon stimulation without any target (top panel) or in the presence of autologous melanoma (bottom panels). CD107a/b+ cells secreting at least one of the measured cytokines (IFNγ, TNFα and IL-2) were single-cell sorted and sequenced. Gates depict the detection and quantification of reactive (black) or sorted cells (magenta) from a representative sample (TP3 PBMCs from Pt-A). d . TCR clonotypes identified upon single-cell sorting and scTCRseq of melanoma-reactive CD8+ T cells from the 4 studied patients. Bars - cell counts of clonotype families, defined as CD8+ cells bearing identical TCRα and TCRβ chains, divided based on their detection at specific timepoints (TP1, TP2, TP3) or across multiple timepoints (shared). Presence of multiple TCRα or TCRβ chains is indicated with black or orange borders, respectively. e-h. , to identify: ( e ) tumor-specific TCRs, ( f ) non-tumor reactive TCRs, and ( g ) tumor/control reactive TCRs. Reactivity was calculated by subtracting from CD137 expression of CD8+ cells transduced with the reconstructed TCR the background of lymphocytes transduced with an irrelevant TCR. Floating boxes show min to max measurements, with mean values depicted as horizontal lines; white dots denote the basal level of activation measured on untransduced cells. Pie charts in h summarize the classification of TCR reactivity for all reconstructed TCRs. i. Cytotoxicity mediated by TCRs classified as tumor-specific (left panel), non-tumor reactive (middle panel) or tumor/control reactive (right panel). Degranulation (CD107a/b+) and concomitant production of cytokines (IFNγ, TNFα and IL-2) were measured through intracellular flow-cytometry on TCR transduced (mTRBC+) CD8+ T cells cultured alone or in the presence of autologous melanoma. Each dot represents the results of a single TCR isolated from CD8+ TILs (upon subtraction of background activation measured on CD8+ lymphocytes transduced with an irrelevant TCR). White dots denote the basal level of cytotoxicity of untransduced cells. j ). Extended Data Fig. 1

Techniques Used: Isolation, Sequencing, Cell Culture, FACS, Expressing, Transduction, Activation Assay, Flow Cytometry

13) Product Images from "Heterogeneity of Ara h Component-Specific CD4 T Cell Responses in Peanut-Allergic Subjects"

Article Title: Heterogeneity of Ara h Component-Specific CD4 T Cell Responses in Peanut-Allergic Subjects

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01408

“Modified” CD154 upregulation T-cell epitope mapping. CD154 + CD45RA − CD4 + T cells were sorted (A) and expended in presence of autologous-irradiated peripheral blood mononuclear cells (PBMCs), 1 µg/mL PHA, and human IL-2 (B) . (B,C) For epitope mapping, 10 5 cells were stimulated for 3 h with 5 µg/mL of synthesized Ara h 2 peptide pools with autologous PBMCs. Pool giving a positive response in the CD154 assay was retested with 40 µg/mL blocking antibodies anti-HLA-DR or anti-HLA-DQ. Peptides from pool giving a positive response were then tested individually as descried before. Then, individual peptides, giving a positive response, were loaded into the biotinylated HLA-DR protein to generate tetramers and tested in vitro . (D) Table of Ara h 2 T cell epitopes identified.
Figure Legend Snippet: “Modified” CD154 upregulation T-cell epitope mapping. CD154 + CD45RA − CD4 + T cells were sorted (A) and expended in presence of autologous-irradiated peripheral blood mononuclear cells (PBMCs), 1 µg/mL PHA, and human IL-2 (B) . (B,C) For epitope mapping, 10 5 cells were stimulated for 3 h with 5 µg/mL of synthesized Ara h 2 peptide pools with autologous PBMCs. Pool giving a positive response in the CD154 assay was retested with 40 µg/mL blocking antibodies anti-HLA-DR or anti-HLA-DQ. Peptides from pool giving a positive response were then tested individually as descried before. Then, individual peptides, giving a positive response, were loaded into the biotinylated HLA-DR protein to generate tetramers and tested in vitro . (D) Table of Ara h 2 T cell epitopes identified.

Techniques Used: Irradiation, Synthesized, Acetylene Reduction Assay, Blocking Assay, In Vitro

14) Product Images from "Heterogeneity of Ara h Component-Specific CD4 T Cell Responses in Peanut-Allergic Subjects"

Article Title: Heterogeneity of Ara h Component-Specific CD4 T Cell Responses in Peanut-Allergic Subjects

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01408

“Modified” CD154 upregulation T-cell epitope mapping. CD154 + CD45RA − CD4 + T cells were sorted (A) and expended in presence of autologous-irradiated peripheral blood mononuclear cells (PBMCs), 1 µg/mL PHA, and human IL-2 (B) . (B,C) For epitope mapping, 10 5 cells were stimulated for 3 h with 5 µg/mL of synthesized Ara h 2 peptide pools with autologous PBMCs. Pool giving a positive response in the CD154 assay was retested with 40 µg/mL blocking antibodies anti-HLA-DR or anti-HLA-DQ. Peptides from pool giving a positive response were then tested individually as descried before. Then, individual peptides, giving a positive response, were loaded into the biotinylated HLA-DR protein to generate tetramers and tested in vitro . (D) Table of Ara h 2 T cell epitopes identified.
Figure Legend Snippet: “Modified” CD154 upregulation T-cell epitope mapping. CD154 + CD45RA − CD4 + T cells were sorted (A) and expended in presence of autologous-irradiated peripheral blood mononuclear cells (PBMCs), 1 µg/mL PHA, and human IL-2 (B) . (B,C) For epitope mapping, 10 5 cells were stimulated for 3 h with 5 µg/mL of synthesized Ara h 2 peptide pools with autologous PBMCs. Pool giving a positive response in the CD154 assay was retested with 40 µg/mL blocking antibodies anti-HLA-DR or anti-HLA-DQ. Peptides from pool giving a positive response were then tested individually as descried before. Then, individual peptides, giving a positive response, were loaded into the biotinylated HLA-DR protein to generate tetramers and tested in vitro . (D) Table of Ara h 2 T cell epitopes identified.

Techniques Used: Irradiation, Synthesized, Acetylene Reduction Assay, Blocking Assay, In Vitro

15) Product Images from "Comparison of the biological characteristics of human mesenchymal stem cells derived from exfoliated deciduous teeth, bone marrow, gingival tissue, and umbilical cord"

Article Title: Comparison of the biological characteristics of human mesenchymal stem cells derived from exfoliated deciduous teeth, bone marrow, gingival tissue, and umbilical cord

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2018.9501

Immunogenicity characterization of MSCs. (A) The proliferation capacity of peripheral blood mononuclear cells following mixed co-culture for 3 days. Cell Counting kit-8 assay revealed that the relative OD values for the four types of MSCs were significantly lower than those in the control and PHA groups. When comparing the four types of MSCs, the OD value for UCMSCs was lower than that of BMSCs and GMSCs. Results represent the mean ± standard deviation of five independent experiments. (B and C) All MSCs exhibited high expression of HLA-I prior to or following IFN-γ treatment. (D and E) All MSCs had a lower expression of HLA-DR prior to IFN-γ treatment, but following IFN-γ treatment, the expression of HLA-DR was significantly upregulated. (F and G) The expression of CD80 was very low prior to and following stimulation with IFN-γ. (H and I) The expression of CD86 was very low prior to and following stimulation with IFN-γ. Results represent the mean ± standard deviation of three independent experiments. *P
Figure Legend Snippet: Immunogenicity characterization of MSCs. (A) The proliferation capacity of peripheral blood mononuclear cells following mixed co-culture for 3 days. Cell Counting kit-8 assay revealed that the relative OD values for the four types of MSCs were significantly lower than those in the control and PHA groups. When comparing the four types of MSCs, the OD value for UCMSCs was lower than that of BMSCs and GMSCs. Results represent the mean ± standard deviation of five independent experiments. (B and C) All MSCs exhibited high expression of HLA-I prior to or following IFN-γ treatment. (D and E) All MSCs had a lower expression of HLA-DR prior to IFN-γ treatment, but following IFN-γ treatment, the expression of HLA-DR was significantly upregulated. (F and G) The expression of CD80 was very low prior to and following stimulation with IFN-γ. (H and I) The expression of CD86 was very low prior to and following stimulation with IFN-γ. Results represent the mean ± standard deviation of three independent experiments. *P

Techniques Used: Co-Culture Assay, Cell Counting, Standard Deviation, Expressing

16) Product Images from "Hydroxytyrosol: a new class of microbicide displaying broad anti-HIV-1 activity"

Article Title: Hydroxytyrosol: a new class of microbicide displaying broad anti-HIV-1 activity

Journal: AIDS (London, England)

doi: 10.1097/QAD.0000000000001283

Combination effect in anti-HIV activity of 5-hydroxytyrosol and tenofovir or emtricitabine or lamivudine in MT-2 cells and peripheral blood mononuclear cells preactivated with PHA/interleukin-2 infected with NL4.3-Ren.
Figure Legend Snippet: Combination effect in anti-HIV activity of 5-hydroxytyrosol and tenofovir or emtricitabine or lamivudine in MT-2 cells and peripheral blood mononuclear cells preactivated with PHA/interleukin-2 infected with NL4.3-Ren.

Techniques Used: Activity Assay, Infection

(a) Evaluation of the anti-HIV activity of 5-hydroxytyrosol in MT-2 cells, TZM-Luc cells and human primary peripheral blood mononuclear cells preactivated with PHA/interleukin-2.
Figure Legend Snippet: (a) Evaluation of the anti-HIV activity of 5-hydroxytyrosol in MT-2 cells, TZM-Luc cells and human primary peripheral blood mononuclear cells preactivated with PHA/interleukin-2.

Techniques Used: Activity Assay

17) Product Images from "IL-15 prolongs CD154 expression on human CD4 T cells via STAT5 binding to the CD154 transcriptional promoter"

Article Title: IL-15 prolongs CD154 expression on human CD4 T cells via STAT5 binding to the CD154 transcriptional promoter

Journal: Genes and immunity

doi: 10.1038/gene.2014.3

IL-15 augments late CD154 expression on suboptimally stimulated CD4 T cells Primary human CD4 T cells were rested (red lines) or stimulated (blue lines) with phorbol ester and ionomycin (far left column), low dose PHA alone (left middle column), PHA plus IL-15 (right middle column), or IL-15 alone (far right column) for 6 (top row), 20 (middle row), or 48 (bottom row) hours prior to analysis of cell surface CD154 expression by flow cytometry. Results are representative of 4 similar experiments.
Figure Legend Snippet: IL-15 augments late CD154 expression on suboptimally stimulated CD4 T cells Primary human CD4 T cells were rested (red lines) or stimulated (blue lines) with phorbol ester and ionomycin (far left column), low dose PHA alone (left middle column), PHA plus IL-15 (right middle column), or IL-15 alone (far right column) for 6 (top row), 20 (middle row), or 48 (bottom row) hours prior to analysis of cell surface CD154 expression by flow cytometry. Results are representative of 4 similar experiments.

Techniques Used: Expressing, Flow Cytometry, Cytometry

STAT5 binds the proximal human CD154 transcriptional promoter in vivo Primary human CD4 T cells were rested or stimulated with PHA and IL-15 or with P+I for 2 hours as per the Materials and Methods. ChIP was carried out using anti-STAT5 or IgG isotype control antibodies as described in the Materials and Methods. Immunoprecipitated DNA was amplified by real-time PCR using oligonucleotide primers designed to amplify the proximal human CD154 promoter containing BS#1. Results are shown as a percentage of the input DNA. Data are means ±SEM of duplicate samples and are representative of 5 individual experiments.
Figure Legend Snippet: STAT5 binds the proximal human CD154 transcriptional promoter in vivo Primary human CD4 T cells were rested or stimulated with PHA and IL-15 or with P+I for 2 hours as per the Materials and Methods. ChIP was carried out using anti-STAT5 or IgG isotype control antibodies as described in the Materials and Methods. Immunoprecipitated DNA was amplified by real-time PCR using oligonucleotide primers designed to amplify the proximal human CD154 promoter containing BS#1. Results are shown as a percentage of the input DNA. Data are means ±SEM of duplicate samples and are representative of 5 individual experiments.

Techniques Used: In Vivo, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

18) Product Images from "Transcriptional induction of junctional adhesion molecule-C gene expression in activated T cells"

Article Title: Transcriptional induction of junctional adhesion molecule-C gene expression in activated T cells

Journal:

doi: 10.1189/jlb.0708422

Induction of JAM-C protein expression by antibodies against CD3 and CD28 in human T cells, which were treated for 18 h with PHA (2 μg/ml) and with mAb against CD3 and CD28 (1 μl/ml, respectively). Total protein (20 μg) was subjected
Figure Legend Snippet: Induction of JAM-C protein expression by antibodies against CD3 and CD28 in human T cells, which were treated for 18 h with PHA (2 μg/ml) and with mAb against CD3 and CD28 (1 μl/ml, respectively). Total protein (20 μg) was subjected

Techniques Used: Expressing

Induction of JAM-C mRNA expression by PHA and CD3 plus CD28 in human T cells, which were (A) treated with PHA at the indicated concentrations (μg/ml), mAb against CD3 plus CD28 (1 μg/ml), or control media for 18 h or (B) with PHA (2 μg/ml),
Figure Legend Snippet: Induction of JAM-C mRNA expression by PHA and CD3 plus CD28 in human T cells, which were (A) treated with PHA at the indicated concentrations (μg/ml), mAb against CD3 plus CD28 (1 μg/ml), or control media for 18 h or (B) with PHA (2 μg/ml),

Techniques Used: Expressing

19) Product Images from "A potent immunomodulatory role of exosomes derived from mesenchymal stromal cells in preventing cGVHD"

Article Title: A potent immunomodulatory role of exosomes derived from mesenchymal stromal cells in preventing cGVHD

Journal: Journal of Hematology & Oncology

doi: 10.1186/s13045-018-0680-7

MSCs-exo was uptaken by T cells and regulated cell differentiation. a Representative images of exosome uptake by isolated CD3 T cells. b PBMCs were isolated from healthy donors and stimulated with PHA and Fib-exo or MSCs-exo (10 μg/ml or 50 μg/ml). MSCs-exo with both doses upregulated the percentage of CD25 + Foxp3 + CD4+ Treg cells. The higher-dose MSCs-exo group with 50 μg/ml presented more Treg cells. c When cultured under Th17 culture conditions, MSCs-exo markedly suppressed the differentiation of Th17 cells. Data are expressed as the mean ± SEM. ** P
Figure Legend Snippet: MSCs-exo was uptaken by T cells and regulated cell differentiation. a Representative images of exosome uptake by isolated CD3 T cells. b PBMCs were isolated from healthy donors and stimulated with PHA and Fib-exo or MSCs-exo (10 μg/ml or 50 μg/ml). MSCs-exo with both doses upregulated the percentage of CD25 + Foxp3 + CD4+ Treg cells. The higher-dose MSCs-exo group with 50 μg/ml presented more Treg cells. c When cultured under Th17 culture conditions, MSCs-exo markedly suppressed the differentiation of Th17 cells. Data are expressed as the mean ± SEM. ** P

Techniques Used: Cell Differentiation, Isolation, Cell Culture

20) Product Images from "Cloned, CD117 Selected Human Amniotic Fluid Stem Cells Are Capable of Modulating the Immune Response"

Article Title: Cloned, CD117 Selected Human Amniotic Fluid Stem Cells Are Capable of Modulating the Immune Response

Journal: PLoS ONE

doi: 10.1371/journal.pone.0026535

AFS mediated immunosuppression does not require cell-cell contact. Amniotic fluid stem (AFS) cells from two independent sources or bone marrow derived mesenchymal stem cells (BM-MSCs) were cultured under growth conditions (AFS 1, AFS 2, BM-MSC) or activated by co-culture with total blood monocytes (AFS 1 PBMC, AFS 2 PBMC, BM-MSC PBMC) or IL-1β (AFS 1 IL1, AFS 2 IL1, BM-MSC IL1) to release soluble factors. One way mixed lymphocyte reactions (MLR) were incubated in the presence of 24 hour conditioned medium from stem cells cultured with either peripheral blood mononuclear cells (PBMCs) or IL-1β. PHA activated lymphocytes were cultured in 96 well plates coated with IFN-γ capture antibody in the presence of conditioned mediums for 24 hours. Positive control wells contained lymphocytes activated with PHA and negative control wells contained unactivated lymphocytes. Lymphocyte activation was assessed by counting the number of clones producing IFN-γ. Percent activation was calculated by comparing wells containing stem cells to positive control wells. It can be seen that all supernatants were capable of inhibiting T-cell activation by PHA by approximately 20–25%. All conditions are statistically significant when compared to the positive control wells.
Figure Legend Snippet: AFS mediated immunosuppression does not require cell-cell contact. Amniotic fluid stem (AFS) cells from two independent sources or bone marrow derived mesenchymal stem cells (BM-MSCs) were cultured under growth conditions (AFS 1, AFS 2, BM-MSC) or activated by co-culture with total blood monocytes (AFS 1 PBMC, AFS 2 PBMC, BM-MSC PBMC) or IL-1β (AFS 1 IL1, AFS 2 IL1, BM-MSC IL1) to release soluble factors. One way mixed lymphocyte reactions (MLR) were incubated in the presence of 24 hour conditioned medium from stem cells cultured with either peripheral blood mononuclear cells (PBMCs) or IL-1β. PHA activated lymphocytes were cultured in 96 well plates coated with IFN-γ capture antibody in the presence of conditioned mediums for 24 hours. Positive control wells contained lymphocytes activated with PHA and negative control wells contained unactivated lymphocytes. Lymphocyte activation was assessed by counting the number of clones producing IFN-γ. Percent activation was calculated by comparing wells containing stem cells to positive control wells. It can be seen that all supernatants were capable of inhibiting T-cell activation by PHA by approximately 20–25%. All conditions are statistically significant when compared to the positive control wells.

Techniques Used: Derivative Assay, Cell Culture, Co-Culture Assay, Incubation, Positive Control, Negative Control, Activation Assay, Clone Assay

Human AFS cells inhibit lymphocyte activation in a dose dependent manner similar to that of BM-MSCs. Immunoassays assessing lymphocyte activation were performed on two independent amniotic fluid stem cell lines (AFS1 and AFS2) or bone marrow-mesenchymal stem cell (BM-MSC) isolates. T lymphocytes were activated with phytohemaaglutinin (PHA) and cultured in 96 well plates coated with IFN-γ capture antibody in the presence of increasing amounts of stem cells from 1∶32 (4,688 stem cells cultured with 150,000 PBMCs) to 1∶2 (75,000 stem cells cultured with 150,000 PBMCs) for 24 hours. Positive control wells contained lymphocytes activated with PHA and negative control wells included unactivated lymphocytes. Lymphocyte activation was assessed by counting the number of lymphocyte clones producing IFN-γ. Activation is expressed as a percentage of the positive control wells. Both AFS lines and BM-MSC inhibited T-cell compared to the PHA activated control to an approximately equal extent, and was dependant on the number of stem cells added. Inhibition varied from about 40% at a 1∶32 ratio to 80–90% inhibition at the highest ratio of 1∶2.
Figure Legend Snippet: Human AFS cells inhibit lymphocyte activation in a dose dependent manner similar to that of BM-MSCs. Immunoassays assessing lymphocyte activation were performed on two independent amniotic fluid stem cell lines (AFS1 and AFS2) or bone marrow-mesenchymal stem cell (BM-MSC) isolates. T lymphocytes were activated with phytohemaaglutinin (PHA) and cultured in 96 well plates coated with IFN-γ capture antibody in the presence of increasing amounts of stem cells from 1∶32 (4,688 stem cells cultured with 150,000 PBMCs) to 1∶2 (75,000 stem cells cultured with 150,000 PBMCs) for 24 hours. Positive control wells contained lymphocytes activated with PHA and negative control wells included unactivated lymphocytes. Lymphocyte activation was assessed by counting the number of lymphocyte clones producing IFN-γ. Activation is expressed as a percentage of the positive control wells. Both AFS lines and BM-MSC inhibited T-cell compared to the PHA activated control to an approximately equal extent, and was dependant on the number of stem cells added. Inhibition varied from about 40% at a 1∶32 ratio to 80–90% inhibition at the highest ratio of 1∶2.

Techniques Used: Activation Assay, Cell Culture, Positive Control, Negative Control, Clone Assay, Inhibition

21) Product Images from "In situ scarless skin healing of a severe human burn wound induced by a hCTLA4Ig gene-transferred porcine skin graft"

Article Title: In situ scarless skin healing of a severe human burn wound induced by a hCTLA4Ig gene-transferred porcine skin graft

Journal: International Journal of Medical Sciences

doi: 10.7150/ijms.62438

The immune response of patient's PBMCs. A: T and B cells from patient and healthy volunteer differently responded to PHA or LPS stimulation. B: Statistical analysis of different numbers of T and B cells from the patient. **, p
Figure Legend Snippet: The immune response of patient's PBMCs. A: T and B cells from patient and healthy volunteer differently responded to PHA or LPS stimulation. B: Statistical analysis of different numbers of T and B cells from the patient. **, p

Techniques Used:

Mixed lymphocytes reaction (MLR). A: The different degrees of response of PBMCs from the patient, healthy volunteer to the pig's PBMCs and to added PHA. B: The statistical analysis of response of patient's PBMCs to PHA and pig's PBMCs. **, p
Figure Legend Snippet: Mixed lymphocytes reaction (MLR). A: The different degrees of response of PBMCs from the patient, healthy volunteer to the pig's PBMCs and to added PHA. B: The statistical analysis of response of patient's PBMCs to PHA and pig's PBMCs. **, p

Techniques Used:

22) Product Images from "Functional Measurement of Hepatitis C Virus Core-Specific CD8+ T-Cell Responses in the Livers or Peripheral Blood of Patients by Using Autologous Peripheral Blood Mononuclear Cells as Targets or Stimulators"

Article Title: Functional Measurement of Hepatitis C Virus Core-Specific CD8+ T-Cell Responses in the Livers or Peripheral Blood of Patients by Using Autologous Peripheral Blood Mononuclear Cells as Targets or Stimulators

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.39.11.3895-3901.2001

Optimizing the VSV-G-pseudotyped retroviral infection on primary PBMCs. A GFP/VSV-G retrovirus was used to determine the optimal conditions for infecting PBMCs. (A) Optimal MOI. Following isolation, PBMCs were stimulated with PHA and rIL-2 for 2 days and then infected with GFP/VSV-G retroviruses at different MOIs. Expression of GFP was analyzed on day 4 after retroviral infection with a FACScan. (B) Expression period. PBMCs were infected with GFP/VSV-G retroviruses at an MOI of 80, and GFP expression was measured at different time points after retroviral infection. (C) Infection frequency. The cells were infected with GFP/VSV-G retroviruses at an MOI of 80 for 1, 2, 3, or 4 consecutive days starting from day 0, and GFP expression was measured on day 4. The white peaks are the PBMCs infected with S2/VSV-G control retroviruses; the black peaks are the PBMCs infected with GFP/VSV-G retroviruses.
Figure Legend Snippet: Optimizing the VSV-G-pseudotyped retroviral infection on primary PBMCs. A GFP/VSV-G retrovirus was used to determine the optimal conditions for infecting PBMCs. (A) Optimal MOI. Following isolation, PBMCs were stimulated with PHA and rIL-2 for 2 days and then infected with GFP/VSV-G retroviruses at different MOIs. Expression of GFP was analyzed on day 4 after retroviral infection with a FACScan. (B) Expression period. PBMCs were infected with GFP/VSV-G retroviruses at an MOI of 80, and GFP expression was measured at different time points after retroviral infection. (C) Infection frequency. The cells were infected with GFP/VSV-G retroviruses at an MOI of 80 for 1, 2, 3, or 4 consecutive days starting from day 0, and GFP expression was measured on day 4. The white peaks are the PBMCs infected with S2/VSV-G control retroviruses; the black peaks are the PBMCs infected with GFP/VSV-G retroviruses.

Techniques Used: Infection, Isolation, Expressing

Infection of PBMCs with C191/VSV-G retroviruses. (A and B) After activation with PHA and rIL-2, the fresh PBMCs were immediately infected with C191/VSV-G retroviruses at an MOI of 80 (A) or stored in liquid nitrogen for a week and then thawed and infected (B). Expression of core antigen was detected by intracellular staining and analyzed with a FACScan. The black peaks show the PBMCs infected with C191/VSV-G retroviruses; the white peaks show the cells infected with S2/VSV-G retroviruses. (C) No changes of HLA type I molecules on PBMCs after retroviral infection. The levels of surface HLA type I molecules on PBMCs with or without retroviral infection were examined with mouse anti-human HLA-A, -B, and -C antibodies. The black peak shows the PBMCs infected with C191/VSV-G retroviruses, the gray peak shows the cells without infection, and the white peak shows the cells stained with the isotype control antibody.
Figure Legend Snippet: Infection of PBMCs with C191/VSV-G retroviruses. (A and B) After activation with PHA and rIL-2, the fresh PBMCs were immediately infected with C191/VSV-G retroviruses at an MOI of 80 (A) or stored in liquid nitrogen for a week and then thawed and infected (B). Expression of core antigen was detected by intracellular staining and analyzed with a FACScan. The black peaks show the PBMCs infected with C191/VSV-G retroviruses; the white peaks show the cells infected with S2/VSV-G retroviruses. (C) No changes of HLA type I molecules on PBMCs after retroviral infection. The levels of surface HLA type I molecules on PBMCs with or without retroviral infection were examined with mouse anti-human HLA-A, -B, and -C antibodies. The black peak shows the PBMCs infected with C191/VSV-G retroviruses, the gray peak shows the cells without infection, and the white peak shows the cells stained with the isotype control antibody.

Techniques Used: Infection, Activation Assay, Expressing, Staining

23) Product Images from "An in vitro based investigation of the cytotoxic effect of water extracts of the Chinese herbal remedy LD on cancer cells"

Article Title: An in vitro based investigation of the cytotoxic effect of water extracts of the Chinese herbal remedy LD on cancer cells

Journal: Chemistry Central Journal

doi: 10.1186/1752-153X-3-12

A-F: Histograms showing PI stained non-stimulated and PHA-stimulated PBLs exposed to LD at 1:10 for 24, 48 and 72 hours . The x axis of each histogram shows relative DNA content, with cells in G0/G1 over channel 200. The sub-G1 region represents apoptotic cells. The y axis represents cell number. 9A : non stimulated PBLs exposed to LD (1:10) for 24 hours, 9B : non stimulated PBLs exposed to LD (1:10) for 48 hours, 9C : non stimulated PBLs exposed to LD (1:10) for 72 hrs, 9D : PHA stimulated PBLS exposed to LD (1:10) for 24 hours, 9E : PHA stimulated PBLS exposed to LD (1:10) for 48 hours, 9F : PHA stimulated PBLS exposed to LD (1:10) for 72 hours.
Figure Legend Snippet: A-F: Histograms showing PI stained non-stimulated and PHA-stimulated PBLs exposed to LD at 1:10 for 24, 48 and 72 hours . The x axis of each histogram shows relative DNA content, with cells in G0/G1 over channel 200. The sub-G1 region represents apoptotic cells. The y axis represents cell number. 9A : non stimulated PBLs exposed to LD (1:10) for 24 hours, 9B : non stimulated PBLs exposed to LD (1:10) for 48 hours, 9C : non stimulated PBLs exposed to LD (1:10) for 72 hrs, 9D : PHA stimulated PBLS exposed to LD (1:10) for 24 hours, 9E : PHA stimulated PBLS exposed to LD (1:10) for 48 hours, 9F : PHA stimulated PBLS exposed to LD (1:10) for 72 hours.

Techniques Used: Staining

24) Product Images from "Dendritic Cells Loaded with Pancreatic Cancer Stem Cells (CSCs) Lysates Induce Antitumor Immune Killing Effect In Vitro"

Article Title: Dendritic Cells Loaded with Pancreatic Cancer Stem Cells (CSCs) Lysates Induce Antitumor Immune Killing Effect In Vitro

Journal: PLoS ONE

doi: 10.1371/journal.pone.0114581

Panc-1 CSCs inhibited lymphocyte proliferation. Lymphocytes were stimulated to proliferate with PHA or anti-CD3 monoclonal antibodies. Panc-1 CSCs were co-cultured with lymphocyte at a ratio of 1∶10 in the presence of PHA or anti-CD3 monoclonal antibodies. Lymphocytes stimulated with PHA or anti-CD3 monoclonal antibodies solely were set as control. MTT assay indicated that lymphocyte proliferation promoted by PHA or anti-CD3 monoclonal antibodies was inhibited in the presence of Panc-1 CSCs.
Figure Legend Snippet: Panc-1 CSCs inhibited lymphocyte proliferation. Lymphocytes were stimulated to proliferate with PHA or anti-CD3 monoclonal antibodies. Panc-1 CSCs were co-cultured with lymphocyte at a ratio of 1∶10 in the presence of PHA or anti-CD3 monoclonal antibodies. Lymphocytes stimulated with PHA or anti-CD3 monoclonal antibodies solely were set as control. MTT assay indicated that lymphocyte proliferation promoted by PHA or anti-CD3 monoclonal antibodies was inhibited in the presence of Panc-1 CSCs.

Techniques Used: Cell Culture, MTT Assay

25) Product Images from "Tumor Cell Programmed Death Ligand 1-Mediated T Cell Suppression Is Overcome by Coexpression of CD80"

Article Title: Tumor Cell Programmed Death Ligand 1-Mediated T Cell Suppression Is Overcome by Coexpression of CD80

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

doi: 10.4049/jimmunol.1003682

CD80 blocks PDL1 suppressive activity and restores T cell activation. A , T cell suppression is reversed by CD80 inhibition of PDL1 expression. PBMCs were activated with PHA in the presence or absence of varying numbers of irradiated MEL202, MEL202/CD80,
Figure Legend Snippet: CD80 blocks PDL1 suppressive activity and restores T cell activation. A , T cell suppression is reversed by CD80 inhibition of PDL1 expression. PBMCs were activated with PHA in the presence or absence of varying numbers of irradiated MEL202, MEL202/CD80,

Techniques Used: Activity Assay, Activation Assay, Inhibition, Expressing, Irradiation

26) Product Images from "Reiterative infusions of MSCs improve pediatric osteogenesis imperfecta eliciting a pro‐osteogenic paracrine response: TERCELOI clinical trial, et al. Reiterative infusions of MSCs improve pediatric osteogenesis imperfecta eliciting a pro‐osteogenic paracrine response: TERCELOI clinical trial"

Article Title: Reiterative infusions of MSCs improve pediatric osteogenesis imperfecta eliciting a pro‐osteogenic paracrine response: TERCELOI clinical trial, et al. Reiterative infusions of MSCs improve pediatric osteogenesis imperfecta eliciting a pro‐osteogenic paracrine response: TERCELOI clinical trial

Journal: Clinical and Translational Medicine

doi: 10.1002/ctm2.265

Overview of the TERCELOI clinical trial. (A) Table 1, reflecting the inclusion and exclusion criteria of the clinical trial. (B) Diagram illustrating the clinical trial workflow. The five MSCs infusions administered in total period of time of 2.5 years are indicated (black arrows). Previously to each cell infusion, mixed lymphocyte reaction assay was performed, denoted by asterisks. The visits for clinical and analytical evaluation in addition to sera collection after each cell infusion are denoted by colored arrows. Before the cell therapy: red. During the cell therapy: blue, 1 week (1 w); green, 1 month (1 m); and purple, 4 months (4 m) after each cell infusion. After the cell therapy: follow‐up visits at 1 and 2 years after the fifth cell infusion: gray. (C) Representative histogram plots of generations of divided cells as determined by ModFit‐LT software before the first MSCs infusion in P01 patient. Upper plots: controls consisted of unstimulated (left) or PHA‐stimulated PBMCs (right) isolated from P01. Dark blue peak in each plot indicates undivided parental cells. PHA‐stimulated PBMCs show several cell generations, denoting cell proliferation. Lower plots: P01 PBMCs cultured in the presence of donor MSCs, showing either no proliferation (left) or suppressed proliferation under PHA stimulation (right)
Figure Legend Snippet: Overview of the TERCELOI clinical trial. (A) Table 1, reflecting the inclusion and exclusion criteria of the clinical trial. (B) Diagram illustrating the clinical trial workflow. The five MSCs infusions administered in total period of time of 2.5 years are indicated (black arrows). Previously to each cell infusion, mixed lymphocyte reaction assay was performed, denoted by asterisks. The visits for clinical and analytical evaluation in addition to sera collection after each cell infusion are denoted by colored arrows. Before the cell therapy: red. During the cell therapy: blue, 1 week (1 w); green, 1 month (1 m); and purple, 4 months (4 m) after each cell infusion. After the cell therapy: follow‐up visits at 1 and 2 years after the fifth cell infusion: gray. (C) Representative histogram plots of generations of divided cells as determined by ModFit‐LT software before the first MSCs infusion in P01 patient. Upper plots: controls consisted of unstimulated (left) or PHA‐stimulated PBMCs (right) isolated from P01. Dark blue peak in each plot indicates undivided parental cells. PHA‐stimulated PBMCs show several cell generations, denoting cell proliferation. Lower plots: P01 PBMCs cultured in the presence of donor MSCs, showing either no proliferation (left) or suppressed proliferation under PHA stimulation (right)

Techniques Used: Software, Isolation, Cell Culture

27) Product Images from "IL-6 triggers IL-21 production by human CD4+ T cells to drive STAT3-dependent plasma cell differentiation in B cells"

Article Title: IL-6 triggers IL-21 production by human CD4+ T cells to drive STAT3-dependent plasma cell differentiation in B cells

Journal: Immunology and cell biology

doi: 10.1038/icb.2012.17

Enforced BCL6 expression enhances lymphoid/follicular markers, including CXCR5 in primary human CD4 + T cells Total CD4 + T cells from peripheral blood were stimulated with CD3/CD28 beads for 4 days and transduced ( A ) with retroviral vectors encoding either LZRS-IRES-ΔNGFR (Control) or LZRS-BCL6-IRES-ΔNGFR (BCL6). ( B ) CXCR5 surface expression on resting, activated (4 days), or Control and BCL6-transduced CD4 + T cells at the indicated timepoints after transduction. Transduced cells were cultured on irradiated allogeneic PBMC feeders with PHA (2 μg/ml) and IL-2 (20 U/ml) (methods). ( C ) Percent CXCR5 + and ( D ) CXCR5 mean fluorescence intensity (MFI) of CD4 + NGFR + cells over time. ( E ) CCR7/CXCR4 expression in CD4 + NGFR + cells at Days 14 and 21 after transduction. ( F ) CD80 expression on CD4 + NGFR + cells 21 days after transduction. For panels E and F, Gates were set using isotype control stainings and total tonsil cells to define positive populations as described for CXCR4 and CD80 61 . CCR7 gates in panel E were defined as described 62 . Graphical results are means ± SD of two independent experiments each containing two donors and FACS plots are representative. Two-way ANOVA was used to calculate statistical significance ( P -values shown on graphs) in panels C and D. Quantitated data for CXCR4/CCR7 and CD80 are shown in Fig S 2.
Figure Legend Snippet: Enforced BCL6 expression enhances lymphoid/follicular markers, including CXCR5 in primary human CD4 + T cells Total CD4 + T cells from peripheral blood were stimulated with CD3/CD28 beads for 4 days and transduced ( A ) with retroviral vectors encoding either LZRS-IRES-ΔNGFR (Control) or LZRS-BCL6-IRES-ΔNGFR (BCL6). ( B ) CXCR5 surface expression on resting, activated (4 days), or Control and BCL6-transduced CD4 + T cells at the indicated timepoints after transduction. Transduced cells were cultured on irradiated allogeneic PBMC feeders with PHA (2 μg/ml) and IL-2 (20 U/ml) (methods). ( C ) Percent CXCR5 + and ( D ) CXCR5 mean fluorescence intensity (MFI) of CD4 + NGFR + cells over time. ( E ) CCR7/CXCR4 expression in CD4 + NGFR + cells at Days 14 and 21 after transduction. ( F ) CD80 expression on CD4 + NGFR + cells 21 days after transduction. For panels E and F, Gates were set using isotype control stainings and total tonsil cells to define positive populations as described for CXCR4 and CD80 61 . CCR7 gates in panel E were defined as described 62 . Graphical results are means ± SD of two independent experiments each containing two donors and FACS plots are representative. Two-way ANOVA was used to calculate statistical significance ( P -values shown on graphs) in panels C and D. Quantitated data for CXCR4/CCR7 and CD80 are shown in Fig S 2.

Techniques Used: Expressing, Transduction, Cell Culture, Irradiation, Fluorescence, FACS

28) Product Images from "Human IL2RA null mutation mediates immunodeficiency with lymphoproliferation and autoimmunity"

Article Title: Human IL2RA null mutation mediates immunodeficiency with lymphoproliferation and autoimmunity

Journal: Clinical Immunology (Orlando, Fla.)

doi: 10.1016/j.clim.2013.01.004

Defective T cell responses to polyclonal mitogens and viral Ags. A, PBMC from the patient and healthy controls were stimulated with anti-CD3 and anti-CD28 in the presence or absence of IL-2 (+ 10 U/ml, ++ 100 U/ml, and +++ 1000 U/ml) and/or IL-15 (10 ng/ml) and/or IL-15 (10 ng/ml), or PHA and proliferation was determined by 3 H-thymidine after 3 days. B, T cell responses to C. albicans , TT, were measured after 4 days and (C) different viruses (CMV, VZV, and HSV) were measured after 3 days of stimulation in the presence or absence of IL-2 and IL-15. D, PBMC ELIspot assays from the patient and healthy control were stimulated with CMV Ags alone or in the presence of IL-2 or IL-15, or with IL-2, IL-15, or TPA/Ionomyocin alone.
Figure Legend Snippet: Defective T cell responses to polyclonal mitogens and viral Ags. A, PBMC from the patient and healthy controls were stimulated with anti-CD3 and anti-CD28 in the presence or absence of IL-2 (+ 10 U/ml, ++ 100 U/ml, and +++ 1000 U/ml) and/or IL-15 (10 ng/ml) and/or IL-15 (10 ng/ml), or PHA and proliferation was determined by 3 H-thymidine after 3 days. B, T cell responses to C. albicans , TT, were measured after 4 days and (C) different viruses (CMV, VZV, and HSV) were measured after 3 days of stimulation in the presence or absence of IL-2 and IL-15. D, PBMC ELIspot assays from the patient and healthy control were stimulated with CMV Ags alone or in the presence of IL-2 or IL-15, or with IL-2, IL-15, or TPA/Ionomyocin alone.

Techniques Used: Enzyme-linked Immunospot

29) Product Images from "Negative Immunomodulatory Effects of Type 2 Porcine Reproductive and Respiratory Syndrome Virus-Induced Interleukin-1 Receptor Antagonist on Porcine Innate and Adaptive Immune Functions"

Article Title: Negative Immunomodulatory Effects of Type 2 Porcine Reproductive and Respiratory Syndrome Virus-Induced Interleukin-1 Receptor Antagonist on Porcine Innate and Adaptive Immune Functions

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.00579

PRRSV-induced IL-1Ra was not involved in suppression of IFN-γ-producing T lymphocytes in both (A) polyclonal and (B) recalled CSFV responses. The supernatants obtained from type 2 PRRSV or mock (MARC-145 cell lysate) were pretreated with anti-IL-1Ra Ab for 2 h prior to addition into the culture. PBL or PBMC were cultured with PHA, CSFV or controls for 48 h, in the presence of the pretreated supernatants. ± indicates presence/absence of indicated treatment within the culture. Data represents mean ± SD from 5 pigs. Statistical significance was analyzed using ANOVA followed by Tukey's test. * indicates significant difference at p
Figure Legend Snippet: PRRSV-induced IL-1Ra was not involved in suppression of IFN-γ-producing T lymphocytes in both (A) polyclonal and (B) recalled CSFV responses. The supernatants obtained from type 2 PRRSV or mock (MARC-145 cell lysate) were pretreated with anti-IL-1Ra Ab for 2 h prior to addition into the culture. PBL or PBMC were cultured with PHA, CSFV or controls for 48 h, in the presence of the pretreated supernatants. ± indicates presence/absence of indicated treatment within the culture. Data represents mean ± SD from 5 pigs. Statistical significance was analyzed using ANOVA followed by Tukey's test. * indicates significant difference at p

Techniques Used: Cell Culture

PRRSV-induced IL-1Ra inhibited lymphocyte proliferation. PRRSV-induced IL-1Ra inhibited PHA-induced proliferation of the (A) CD4 + , (B) CD8 + , and (C) CD4 + CD8 + subpopulations. PRRSV-induced IL-1Ra inhibited CSFV-specific proliferations of the (D) CD4 + , (E) CD8 + , and (F) CD4 + CD8 + subpopulations. The supernatants obtained from type 2 PRRSV or mock (MARC-145 cell lysate) were pretreated with anti-IL-1Ra Ab for 2 h prior to addition into the culture. PBL or PBMC were culture with PHA, CSFV or controls for 96 h, in the presence of the pretreated supernatants. ± indicates presence/absence of indicated treatment within the culture. Data represents mean ± SD from 5 pigs. Statistical significance was analyzed using ANOVA followed by Tukey's test. * indicates significant difference at p
Figure Legend Snippet: PRRSV-induced IL-1Ra inhibited lymphocyte proliferation. PRRSV-induced IL-1Ra inhibited PHA-induced proliferation of the (A) CD4 + , (B) CD8 + , and (C) CD4 + CD8 + subpopulations. PRRSV-induced IL-1Ra inhibited CSFV-specific proliferations of the (D) CD4 + , (E) CD8 + , and (F) CD4 + CD8 + subpopulations. The supernatants obtained from type 2 PRRSV or mock (MARC-145 cell lysate) were pretreated with anti-IL-1Ra Ab for 2 h prior to addition into the culture. PBL or PBMC were culture with PHA, CSFV or controls for 96 h, in the presence of the pretreated supernatants. ± indicates presence/absence of indicated treatment within the culture. Data represents mean ± SD from 5 pigs. Statistical significance was analyzed using ANOVA followed by Tukey's test. * indicates significant difference at p

Techniques Used:

30) Product Images from "Efficacy of Carraguard(R)-Based Microbicides In Vivo Despite Variable In Vitro Activity"

Article Title: Efficacy of Carraguard(R)-Based Microbicides In Vivo Despite Variable In Vitro Activity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0003162

Carraguard can inhibit or enhance cell-free infection. (A) TZM.bl cells were exposed to 0–10 µg/ml of Carraguard, before graded doses of HIV Bal or RT-SHIV were added. 24 h later, the media was replaced and cells were cultured for 4 d. The numbers of β-gal expressing SFCs per well are shown (mean±SD, triplicate cultures). (B) Titrated amounts of Carraguard were tested against of HIV MN (MN, up triangles), HIV Bal (Bal) (down triangles), or RT-SHIV (circles) in the TZM.bl cell line as in (A). The data are shown as the percent inhibition (mean±SD, triplicate cultures) of infection in the test conditions relative to the no Carraguard control. Negative % inhibition values represent enhancement, with no inhibitor effect at 0%. (C) Titrated amounts of Carraguard were added to PHA activated PBMCs before the cells were cultured with Bal or RT-SHIV for 5 d. Infection was measured by Q-PCR. Data are shown for triplicate cultures (mean±SD). Data in (A–C) are representative of 3 independent experiments with different donors in each case.
Figure Legend Snippet: Carraguard can inhibit or enhance cell-free infection. (A) TZM.bl cells were exposed to 0–10 µg/ml of Carraguard, before graded doses of HIV Bal or RT-SHIV were added. 24 h later, the media was replaced and cells were cultured for 4 d. The numbers of β-gal expressing SFCs per well are shown (mean±SD, triplicate cultures). (B) Titrated amounts of Carraguard were tested against of HIV MN (MN, up triangles), HIV Bal (Bal) (down triangles), or RT-SHIV (circles) in the TZM.bl cell line as in (A). The data are shown as the percent inhibition (mean±SD, triplicate cultures) of infection in the test conditions relative to the no Carraguard control. Negative % inhibition values represent enhancement, with no inhibitor effect at 0%. (C) Titrated amounts of Carraguard were added to PHA activated PBMCs before the cells were cultured with Bal or RT-SHIV for 5 d. Infection was measured by Q-PCR. Data are shown for triplicate cultures (mean±SD). Data in (A–C) are representative of 3 independent experiments with different donors in each case.

Techniques Used: Infection, Cell Culture, Expressing, Inhibition, Polymerase Chain Reaction

31) Product Images from "Costimulation of Naive CD8+ Lymphocytes Induces CD4 Expression and Allows Human Immunodeficiency Virus Type 1 Infection"

Article Title: Costimulation of Naive CD8+ Lymphocytes Induces CD4 Expression and Allows Human Immunodeficiency Virus Type 1 Infection

Journal: Journal of Virology

doi:

(A) Stimulation of CD8-depleted (top row) and CD4-depleted (bottom row) PBL. PBL were depleted by panning and stimulated with either PHA, anti-CD3 MAb, or anti-CD3 and anti-CD28 MAbs. Cells were analyzed by flow cytometry for expression of CD4 (red 613) and CD8 (allophycocyanin) following 3 days of stimulation. CD4 + /CD8 + cells were not observed when cells were stimulated with anti-CD28 alone (data not shown). The percentage of cells in each quadrant is shown. (B) CD4 mRNA expression in costimulated and unstimulated CD4-depleted leukocytes. PBL were depleted of CD4 + cells by panning and stimulated with anti-CD3 and anti-CD28 MAbs or cultured unstimulated in parallel. Three days later, RNA was purified from the CD4-depleted and undepleted populations and subjected to RT-PCR for CD4 (top panel) and GAPDH (middle panel). Standards consisting of dilutions of undepleted stimulated PBL were amplified in parallel and are indicated by number of input cell equivalents. The primers for CD4 mRNA amplify a region containing an RNA splice site and therefore do not amplify potentially contaminating chromosomal DNA sequences. A “no RT” control was performed for GAPDH (bottom panel) to detect the presence of contaminating DNA sequences, of which there were none. The ratio of the level of CD4 mRNA signal to GAPDH signal was 22-fold higher in the costimulated CD8 + population than in the unstimulated CD8 + population.
Figure Legend Snippet: (A) Stimulation of CD8-depleted (top row) and CD4-depleted (bottom row) PBL. PBL were depleted by panning and stimulated with either PHA, anti-CD3 MAb, or anti-CD3 and anti-CD28 MAbs. Cells were analyzed by flow cytometry for expression of CD4 (red 613) and CD8 (allophycocyanin) following 3 days of stimulation. CD4 + /CD8 + cells were not observed when cells were stimulated with anti-CD28 alone (data not shown). The percentage of cells in each quadrant is shown. (B) CD4 mRNA expression in costimulated and unstimulated CD4-depleted leukocytes. PBL were depleted of CD4 + cells by panning and stimulated with anti-CD3 and anti-CD28 MAbs or cultured unstimulated in parallel. Three days later, RNA was purified from the CD4-depleted and undepleted populations and subjected to RT-PCR for CD4 (top panel) and GAPDH (middle panel). Standards consisting of dilutions of undepleted stimulated PBL were amplified in parallel and are indicated by number of input cell equivalents. The primers for CD4 mRNA amplify a region containing an RNA splice site and therefore do not amplify potentially contaminating chromosomal DNA sequences. A “no RT” control was performed for GAPDH (bottom panel) to detect the presence of contaminating DNA sequences, of which there were none. The ratio of the level of CD4 mRNA signal to GAPDH signal was 22-fold higher in the costimulated CD8 + population than in the unstimulated CD8 + population.

Techniques Used: Flow Cytometry, Cytometry, Expressing, Cell Culture, Purification, Reverse Transcription Polymerase Chain Reaction, Amplification

32) Product Images from "Upregulation of CD4 on CD8+ T cells: CD4dimCD8bright T cells constitute an activated phenotype of CD8+ T cells"

Article Title: Upregulation of CD4 on CD8+ T cells: CD4dimCD8bright T cells constitute an activated phenotype of CD8+ T cells

Journal: Immunology

doi: 10.1046/j.1365-2567.2001.01243.x

Induction of CD4 dim CD8 bright cells is not mediated by a soluble factor(s). Purified CD8 + T cells or CD4 + T cells separated by transwell were stimulated with PHA (4 µg/ml) and cultured in this transwell system for 6 days as described in Materials and methods. Cells in the top (CD8 + ) and bottom (CD4 + ) wells were stained with CD3 FITC: CD4 PE: CD8 PerCP-conjugated antibodies and expression of CD4 dim CD8 bright lymphocytes was analyzed by flow cytometry. Data shown is the mean of at least three independent experiments.
Figure Legend Snippet: Induction of CD4 dim CD8 bright cells is not mediated by a soluble factor(s). Purified CD8 + T cells or CD4 + T cells separated by transwell were stimulated with PHA (4 µg/ml) and cultured in this transwell system for 6 days as described in Materials and methods. Cells in the top (CD8 + ) and bottom (CD4 + ) wells were stained with CD3 FITC: CD4 PE: CD8 PerCP-conjugated antibodies and expression of CD4 dim CD8 bright lymphocytes was analyzed by flow cytometry. Data shown is the mean of at least three independent experiments.

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

Dose–response curve of the role of CD4 cells in the PHA-mediated induction of CD4 dim CD8 bright cells. Autologous CD4 + and CD8 + T cells were co-cultured at ratios from 1 : 1 to 1 : 64 and stimulated with PHA (4 µg/ml). The co-cultures were stained for flow cytometric analysis on day 6 using CD3 FITC: CD4 PE: CD8 PerCP-conjugated antibodies. Percentage CD4 dim CD8 bright cells were obtained by gating on CD3 + CD8 bright lymphocytes. Results shown are the mean of at least three independent experiments.
Figure Legend Snippet: Dose–response curve of the role of CD4 cells in the PHA-mediated induction of CD4 dim CD8 bright cells. Autologous CD4 + and CD8 + T cells were co-cultured at ratios from 1 : 1 to 1 : 64 and stimulated with PHA (4 µg/ml). The co-cultures were stained for flow cytometric analysis on day 6 using CD3 FITC: CD4 PE: CD8 PerCP-conjugated antibodies. Percentage CD4 dim CD8 bright cells were obtained by gating on CD3 + CD8 bright lymphocytes. Results shown are the mean of at least three independent experiments.

Techniques Used: Cell Culture, Staining, Flow Cytometry

The effect of CD4 + : CD8 + T-cell co-cultures on the PHA-mediated induction of CD4 dim CD8 bright T cells. (a) CD8 + T cells, PBMC, or a co-culture of autologous CD4 + : CD8 + T cells at a 1 : 1 ratio were stimulated with PHA (4 µg/ml) then stained for flow cytometric analysis at day 6 using CD3 FITC: CD4 PE: CD8 PerCP-conjugated antibodies. Percentage CD4 dim CD8 bright expression was obtained by gating on CD3 + CD8 bright lymphocytes. Results shown in (a) are the mean of at least three independent experiments. (b) Flow cytometric representation of unstimulated and PHA-stimulated cultures of CD8 + T cells (i and v), CD4 + T cells (ii and vi), CD4 + and CD8 + autologous T cell co-cultures (iii and vii), and PBMC (iv and viii). Top panel represents unstimulated cultures while bottom panel represents PHA-stimulated cultures. Gates a1–a3 represent CD4 bright cells expressing CD8 dim at 6, 25 and 27%, respectively. Gates b1–b2 represent CD8 bright cells expressing CD4 dim at 8 and 12%, respectively. The total percentage of CD4 + CD8 + (CD4 bright CD8 dim + CD4 dim CD8 bright + CD4 bright CD8 bright ) are indicated within each figure as 10, 24 and 19% of CD4 + T cells, CD4 + and CD8 + T-cell co-culture, and PBMC, respectively. (c) Kinetic analysis of CD4 dim CD8 bright T-cell expression post-PHA stimulation. CD4 and CD8 T cells were cultured as described previously and the percentage expression of CD4 dim CD8 bright T cells was analysed by flow cytometry for 12 days. Results shown are the mean of at least three independent experiments.
Figure Legend Snippet: The effect of CD4 + : CD8 + T-cell co-cultures on the PHA-mediated induction of CD4 dim CD8 bright T cells. (a) CD8 + T cells, PBMC, or a co-culture of autologous CD4 + : CD8 + T cells at a 1 : 1 ratio were stimulated with PHA (4 µg/ml) then stained for flow cytometric analysis at day 6 using CD3 FITC: CD4 PE: CD8 PerCP-conjugated antibodies. Percentage CD4 dim CD8 bright expression was obtained by gating on CD3 + CD8 bright lymphocytes. Results shown in (a) are the mean of at least three independent experiments. (b) Flow cytometric representation of unstimulated and PHA-stimulated cultures of CD8 + T cells (i and v), CD4 + T cells (ii and vi), CD4 + and CD8 + autologous T cell co-cultures (iii and vii), and PBMC (iv and viii). Top panel represents unstimulated cultures while bottom panel represents PHA-stimulated cultures. Gates a1–a3 represent CD4 bright cells expressing CD8 dim at 6, 25 and 27%, respectively. Gates b1–b2 represent CD8 bright cells expressing CD4 dim at 8 and 12%, respectively. The total percentage of CD4 + CD8 + (CD4 bright CD8 dim + CD4 dim CD8 bright + CD4 bright CD8 bright ) are indicated within each figure as 10, 24 and 19% of CD4 + T cells, CD4 + and CD8 + T-cell co-culture, and PBMC, respectively. (c) Kinetic analysis of CD4 dim CD8 bright T-cell expression post-PHA stimulation. CD4 and CD8 T cells were cultured as described previously and the percentage expression of CD4 dim CD8 bright T cells was analysed by flow cytometry for 12 days. Results shown are the mean of at least three independent experiments.

Techniques Used: Co-Culture Assay, Staining, Flow Cytometry, Expressing, Cell Culture, Cytometry

CD4 is essential for PHA-mediated induction of CD4 dim CD8 bright cells. PBMC, CD4-depleted PBMC, B-depleted PBMC, NK-depleted PBMC, and purified CD8 + T cells were stimulated with PHA (4 µg/ml). The samples were stained for flow cytometry at day 6 with CD3 FITC: CD4 PE: CD8 PerCP-conjugated antibodies. Percent expression of CD4 dim CD8 bright T cells was obtained by gating on CD3 + CD8 bright lymphocytes. Results shown are the mean of at least two independent experiments performed in duplicates.
Figure Legend Snippet: CD4 is essential for PHA-mediated induction of CD4 dim CD8 bright cells. PBMC, CD4-depleted PBMC, B-depleted PBMC, NK-depleted PBMC, and purified CD8 + T cells were stimulated with PHA (4 µg/ml). The samples were stained for flow cytometry at day 6 with CD3 FITC: CD4 PE: CD8 PerCP-conjugated antibodies. Percent expression of CD4 dim CD8 bright T cells was obtained by gating on CD3 + CD8 bright lymphocytes. Results shown are the mean of at least two independent experiments performed in duplicates.

Techniques Used: Purification, Staining, Flow Cytometry, Cytometry, Expressing

Effect of PHA on the induction of CD4 dim CD8 bright and CD4 bright CD8 dim expression in PBMC. (a) Isolated PBMC and CD8 + T cells were stimulated with PHA (4 µg/ml) for 6 days. Stimulated CD8 + T cells and PBMC were stained with CD3 FITC: CD4 PE: CD8 PerCP-conjugated antibodies and analysed by flow cytometry. Top panel represents unstimulated CD8 + (i) and PBMC (ii). Bottom panel represents PHA-stimulated CD8 + T cells (iii) and PHA-stimulated PBMC (iv). The percent of CD4 bright CD8 dim and CD4 dim CD8 bright are shown as histogram of gates a and b, respectively. Results shown in (a) are representative of one experiment. (b) The percentage of CD8 bright T cells expressing CD4 dim on their surface in PHA-stimulated PBMC cultures was obtained by gating on CD3 + CD8 bright lymphocytes. Similarly, the percentage of CD4 bright T cells expressing CD8 dim on their surface was obtained by gating on CD3 + CD4 bright lymphocytes. CD4 + CD8 + cells are indicative of CD4 bright CD8 dim+ CD4 bright CD8 dim+ CD4 bright CD8 bright cells. Results shown in B are the mean of at least three independent experiments.
Figure Legend Snippet: Effect of PHA on the induction of CD4 dim CD8 bright and CD4 bright CD8 dim expression in PBMC. (a) Isolated PBMC and CD8 + T cells were stimulated with PHA (4 µg/ml) for 6 days. Stimulated CD8 + T cells and PBMC were stained with CD3 FITC: CD4 PE: CD8 PerCP-conjugated antibodies and analysed by flow cytometry. Top panel represents unstimulated CD8 + (i) and PBMC (ii). Bottom panel represents PHA-stimulated CD8 + T cells (iii) and PHA-stimulated PBMC (iv). The percent of CD4 bright CD8 dim and CD4 dim CD8 bright are shown as histogram of gates a and b, respectively. Results shown in (a) are representative of one experiment. (b) The percentage of CD8 bright T cells expressing CD4 dim on their surface in PHA-stimulated PBMC cultures was obtained by gating on CD3 + CD8 bright lymphocytes. Similarly, the percentage of CD4 bright T cells expressing CD8 dim on their surface was obtained by gating on CD3 + CD4 bright lymphocytes. CD4 + CD8 + cells are indicative of CD4 bright CD8 dim+ CD4 bright CD8 dim+ CD4 bright CD8 bright cells. Results shown in B are the mean of at least three independent experiments.

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

33) Product Images from "Upregulation of T-bet expression in peripheral blood mononuclear cells during Vogt-Koyanagi-Harada disease"

Article Title: Upregulation of T-bet expression in peripheral blood mononuclear cells during Vogt-Koyanagi-Harada disease

Journal: The British Journal of Ophthalmology

doi: 10.1136/bjo.2005.074062

RT-PCR products of T-bet mRNA extracted from PHA stimulated PBMC from three patients with active (lanes 1, 2, 3) and inactive (lanes 4, 5, 6) uveitis and three controls (lanes 7, 8, 9). One representative
Figure Legend Snippet: RT-PCR products of T-bet mRNA extracted from PHA stimulated PBMC from three patients with active (lanes 1, 2, 3) and inactive (lanes 4, 5, 6) uveitis and three controls (lanes 7, 8, 9). One representative

Techniques Used: Reverse Transcription Polymerase Chain Reaction

T-bet protein expression in PHA-stimulated PBMC from patients and controls. A 62 kDa protein was detectable in all tested samples from patients with active (lanes 1, 2) or inactive uveitis (lanes 3, 4) and controls
Figure Legend Snippet: T-bet protein expression in PHA-stimulated PBMC from patients and controls. A 62 kDa protein was detectable in all tested samples from patients with active (lanes 1, 2) or inactive uveitis (lanes 3, 4) and controls

Techniques Used: Expressing

34) Product Images from "Loss of ARPC1B impairs cytotoxic T lymphocyte maintenance and cytolytic activity"

Article Title: Loss of ARPC1B impairs cytotoxic T lymphocyte maintenance and cytolytic activity

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI129388

Electron and light microscopy reveal atypical synapse formation by ARPC1B-deficient hCTLs. ( A ) Electron micrograph of HD and ARPC1B patient hCTLs conjugated to P815 target cells (false colored in blue) for 25 minutes. ( B ) hCTLs from HD and ARPC1B-deficient patient were conjugated to blue P815 target cells, fixed, and labeled with anti-CD8 (green) and anti-ARPC1B (red) antibodies and phalloidin (white). 3D reconstructions of Z -stack are shown. Scale bars: 3 μm. Data are representative of 3 independent experiments ( A and B ). ( C ) Actin dynamics in HD and ARPC1B-deficient patient hCTLs expressing mApple-Lifeact (red) during interaction with blue P815 target cells. Images are single confocal slice from Video 3 at the indicated time points (min:s) from the first contact. Data are representative of 5 independent experiments. Scale bars: 5 μm.
Figure Legend Snippet: Electron and light microscopy reveal atypical synapse formation by ARPC1B-deficient hCTLs. ( A ) Electron micrograph of HD and ARPC1B patient hCTLs conjugated to P815 target cells (false colored in blue) for 25 minutes. ( B ) hCTLs from HD and ARPC1B-deficient patient were conjugated to blue P815 target cells, fixed, and labeled with anti-CD8 (green) and anti-ARPC1B (red) antibodies and phalloidin (white). 3D reconstructions of Z -stack are shown. Scale bars: 3 μm. Data are representative of 3 independent experiments ( A and B ). ( C ) Actin dynamics in HD and ARPC1B-deficient patient hCTLs expressing mApple-Lifeact (red) during interaction with blue P815 target cells. Images are single confocal slice from Video 3 at the indicated time points (min:s) from the first contact. Data are representative of 5 independent experiments. Scale bars: 5 μm.

Techniques Used: Light Microscopy, Labeling, Expressing

Differential actin structure in hCTLs from ARPC1B-deficient patient. ( A ) Immunoblot analysis of calnexin, ARPC1B, and ARPC1A in hCTL lysates from 4 different HD (lanes 1 to 4) and ARPC1B patient samples from day 20 (lanes 4 to 6) and day 12 (lanes 7 and 8) after expansion. HD 4 (lanes 4 and 7) was used for the rest of the study. MW markers in kD. Lanes were run on the same gel, but were noncontiguous. ( B ) Single plane of a Z -stack across hCTLs from HD or ARPC1B patient plated on ICAM-1– (0.5 μg/mL) or anti-CD3–coated (10 μg/mL; right panel) slide, fixed, stained with phalloidin and CD8 (not shown), and imaged using Structural Illumination Microscopy. Scale bars: 3 μm. ( C ) Quantitation of the mean intensity of phalloidin staining (F-actin) in HD or ARPC1B-deficient patient hCTLs generated from images as exemplified in B (right panel) (see Methods for description). HD n = 10 cells ARPC1B-patient n = 13 cells. *** P = 0.0053, unpaired t test. Data representative of 3 independent experiments.
Figure Legend Snippet: Differential actin structure in hCTLs from ARPC1B-deficient patient. ( A ) Immunoblot analysis of calnexin, ARPC1B, and ARPC1A in hCTL lysates from 4 different HD (lanes 1 to 4) and ARPC1B patient samples from day 20 (lanes 4 to 6) and day 12 (lanes 7 and 8) after expansion. HD 4 (lanes 4 and 7) was used for the rest of the study. MW markers in kD. Lanes were run on the same gel, but were noncontiguous. ( B ) Single plane of a Z -stack across hCTLs from HD or ARPC1B patient plated on ICAM-1– (0.5 μg/mL) or anti-CD3–coated (10 μg/mL; right panel) slide, fixed, stained with phalloidin and CD8 (not shown), and imaged using Structural Illumination Microscopy. Scale bars: 3 μm. ( C ) Quantitation of the mean intensity of phalloidin staining (F-actin) in HD or ARPC1B-deficient patient hCTLs generated from images as exemplified in B (right panel) (see Methods for description). HD n = 10 cells ARPC1B-patient n = 13 cells. *** P = 0.0053, unpaired t test. Data representative of 3 independent experiments.

Techniques Used: Staining, Microscopy, Quantitation Assay, Generated

35) Product Images from "Inhibition of HIV-1 Infection by Human ?-Defensin-5, a Natural Antimicrobial Peptide Expressed in the Genital and Intestinal Mucosae"

Article Title: Inhibition of HIV-1 Infection by Human ?-Defensin-5, a Natural Antimicrobial Peptide Expressed in the Genital and Intestinal Mucosae

Journal: PLoS ONE

doi: 10.1371/journal.pone.0045208

Effect of HD5 on cell-surface HIV-1 coreceptor expression: induction of CXCR4 downmodulation. PM1 cells and primary peripheral blood lymphocytes activated with PHA and IL-2 were preincubated for 2 hours with or without HNP1 or HD5 (each at 14.7 µM) in RPMI-ITS without serum; the cells were then stained with 12G5 or 44717.11, two_conformation-dependent mAbs that recognize antigenically distinct populations of CXCR4 [39] and analyzed by flow cytometry. The level of expression was determined by differences between the level of staining with specific mAbs and with the isotype control. The results are expressed as percent reduction of mean fluorescent intensity (MFI) compared to untreated controls. Data are representative of 4 to 6 independent experiments performed with similar results. Error bars show standard deviations from the mean. *, p = 0.032 and **, p = 0.0084. Wilcoxon Mann-Whitney rank-sum test, 2-tailed.
Figure Legend Snippet: Effect of HD5 on cell-surface HIV-1 coreceptor expression: induction of CXCR4 downmodulation. PM1 cells and primary peripheral blood lymphocytes activated with PHA and IL-2 were preincubated for 2 hours with or without HNP1 or HD5 (each at 14.7 µM) in RPMI-ITS without serum; the cells were then stained with 12G5 or 44717.11, two_conformation-dependent mAbs that recognize antigenically distinct populations of CXCR4 [39] and analyzed by flow cytometry. The level of expression was determined by differences between the level of staining with specific mAbs and with the isotype control. The results are expressed as percent reduction of mean fluorescent intensity (MFI) compared to untreated controls. Data are representative of 4 to 6 independent experiments performed with similar results. Error bars show standard deviations from the mean. *, p = 0.032 and **, p = 0.0084. Wilcoxon Mann-Whitney rank-sum test, 2-tailed.

Techniques Used: Expressing, Staining, Flow Cytometry, MANN-WHITNEY

36) Product Images from "Immunological characteristics of human umbilical cord mesenchymal stem cells and the therapeutic effects of their transplantion on hyperglycemia in diabetic rats"

Article Title: Immunological characteristics of human umbilical cord mesenchymal stem cells and the therapeutic effects of their transplantion on hyperglycemia in diabetic rats

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2013.1572

Immunological characteristics of human umbilical cord Wharton’s jelly-derived mesenchymal stem cells (HUMSCs). Cells were cultured for 3 passages, harvested and labeled with mouse anti-human monoclonal antibodies against CD40, CD40L, CD80 and CD86. (A) Flow cytometry analysis revealed low expression for all antibodies (n=3). (B) qRT-PCR analysis revealed that the cells expressed human leukocyte antigen (HLA)-I but not HLA-DR. (C) Lymphocyte proliferation assay. The CCK-8 kit was used to assess the immunomodulatory effect of HUMSCs on human peripheral blood lymphocytes (PBMCs) after phytohemagglutinin (PHA) stimulation. PBMCs + PHA (positive control), 1.90±0.25; HUMSCs (1×10 5 ) + PBMCs + PHA, 1.37±0.024 (P
Figure Legend Snippet: Immunological characteristics of human umbilical cord Wharton’s jelly-derived mesenchymal stem cells (HUMSCs). Cells were cultured for 3 passages, harvested and labeled with mouse anti-human monoclonal antibodies against CD40, CD40L, CD80 and CD86. (A) Flow cytometry analysis revealed low expression for all antibodies (n=3). (B) qRT-PCR analysis revealed that the cells expressed human leukocyte antigen (HLA)-I but not HLA-DR. (C) Lymphocyte proliferation assay. The CCK-8 kit was used to assess the immunomodulatory effect of HUMSCs on human peripheral blood lymphocytes (PBMCs) after phytohemagglutinin (PHA) stimulation. PBMCs + PHA (positive control), 1.90±0.25; HUMSCs (1×10 5 ) + PBMCs + PHA, 1.37±0.024 (P

Techniques Used: Derivative Assay, Cell Culture, Labeling, Flow Cytometry, Cytometry, Expressing, Quantitative RT-PCR, Lymphocyte Proliferation Assay, CCK-8 Assay, Positive Control

37) Product Images from "Dominant-negative IKZF1 mutations cause a T, B, and myeloid cell combined immunodeficiency"

Article Title: Dominant-negative IKZF1 mutations cause a T, B, and myeloid cell combined immunodeficiency

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI98164

Functional T cell defects associated with heterozygous IKZF1 N159S/T mutations. ( A – C ) PBMCs from patients A1, C1, and G1 and healthy donor controls (Ctrl) were stimulated with indicated agonists for 6–7 days. ( A ) Cells stimulated (stim.) with plate-coated anti-CD3 at the indicated concentrations or with anti-CD3/CD28–coated beads. ( B ) Cells stimulated with plate-coated anti-CD3 at 0.05 μg/ml with or without IL-2. unstim., unstimulated. ( C ) Cells stimulated with plate-coated anti-CD3 at the indicated concentrations with or without IL-2. Proliferation was determined by dilution of CellTrace Violet dye analyzed by flow cytometry. Histograms showing cell divisions by dilution of the CellTrace Violet dye ( A and B ) and CD25 expression ( C ). Data are representative of 3 experiments. ( D ) Histograms from PHA-derived T cell blasts from patients A1, G1, and a healthy donor control (Ctrl) stimulated with (blue) or without (red) anti-CD3/CD28–coated beads (upper panels) or with PMA/ionomycin (iono; lower panels) for 24 hours. IL-2 (lower panels) and IFN-γ (upper panels) were measured with intracellular flow cytometry assays. Black dashed line indicates isotype control. Data are representative of 2 experiments. ( E ) PBMCs from patients C1, D1, and G1 and healthy donor controls (Ctrls) were stimulated with anti-CD3/CD28–coated beads for 24 hours. Cell-free supernatants were harvested, and cytokines (IL-2, TNF-α, IL-6, and IFN-γ) were measured with the Luminex 200 System. Data indicate the mean of replicate sample for C1 ( n = 2), D1 ( n = 1), and G1 ( n = 1) compared with 6–9 different healthy donor controls (mean ± SD). The n values represent the number of replicates. ( F ) PBMCs from patients C1, D1, G1, and 2 paired healthy donor controls (Ctrls) were stimulated with PMA/ionomycin for 6 hours. IL-2, IFN-γ, and IL-4 (for Th1/2 evaluation) and IL-17A (for Th17 evaluation) were measured with intracellular flow cytometry assays. Data represent the percentage of the cells positive for the indicated cytokine among CD4 + T cells. Data are issued from 2 experiments performed in patient C1 and 1 experiment from D1 and G1.
Figure Legend Snippet: Functional T cell defects associated with heterozygous IKZF1 N159S/T mutations. ( A – C ) PBMCs from patients A1, C1, and G1 and healthy donor controls (Ctrl) were stimulated with indicated agonists for 6–7 days. ( A ) Cells stimulated (stim.) with plate-coated anti-CD3 at the indicated concentrations or with anti-CD3/CD28–coated beads. ( B ) Cells stimulated with plate-coated anti-CD3 at 0.05 μg/ml with or without IL-2. unstim., unstimulated. ( C ) Cells stimulated with plate-coated anti-CD3 at the indicated concentrations with or without IL-2. Proliferation was determined by dilution of CellTrace Violet dye analyzed by flow cytometry. Histograms showing cell divisions by dilution of the CellTrace Violet dye ( A and B ) and CD25 expression ( C ). Data are representative of 3 experiments. ( D ) Histograms from PHA-derived T cell blasts from patients A1, G1, and a healthy donor control (Ctrl) stimulated with (blue) or without (red) anti-CD3/CD28–coated beads (upper panels) or with PMA/ionomycin (iono; lower panels) for 24 hours. IL-2 (lower panels) and IFN-γ (upper panels) were measured with intracellular flow cytometry assays. Black dashed line indicates isotype control. Data are representative of 2 experiments. ( E ) PBMCs from patients C1, D1, and G1 and healthy donor controls (Ctrls) were stimulated with anti-CD3/CD28–coated beads for 24 hours. Cell-free supernatants were harvested, and cytokines (IL-2, TNF-α, IL-6, and IFN-γ) were measured with the Luminex 200 System. Data indicate the mean of replicate sample for C1 ( n = 2), D1 ( n = 1), and G1 ( n = 1) compared with 6–9 different healthy donor controls (mean ± SD). The n values represent the number of replicates. ( F ) PBMCs from patients C1, D1, G1, and 2 paired healthy donor controls (Ctrls) were stimulated with PMA/ionomycin for 6 hours. IL-2, IFN-γ, and IL-4 (for Th1/2 evaluation) and IL-17A (for Th17 evaluation) were measured with intracellular flow cytometry assays. Data represent the percentage of the cells positive for the indicated cytokine among CD4 + T cells. Data are issued from 2 experiments performed in patient C1 and 1 experiment from D1 and G1.

Techniques Used: Functional Assay, Flow Cytometry, Cytometry, Expressing, Derivative Assay, Luminex

38) Product Images from "Concordant Proficiency in Measurement of T-Cell Immunity in Human Immunodeficiency Virus Vaccine Clinical Trials by Peripheral Blood Mononuclear Cell and Enzyme-Linked Immunospot Assays in Laboratories from Three Continents ▿"

Article Title: Concordant Proficiency in Measurement of T-Cell Immunity in Human Immunodeficiency Virus Vaccine Clinical Trials by Peripheral Blood Mononuclear Cell and Enzyme-Linked Immunospot Assays in Laboratories from Three Continents ▿

Journal:

doi: 10.1128/CVI.00326-08

Laboratory ELISPOT spot-forming unit (SFU) counts for each donor PBMC sample in response to specific stimuli in panels 1 to 3. SFU counts shown for CEF and CMV peptides and PHA were determined by subtracting background values and are presented per 10
Figure Legend Snippet: Laboratory ELISPOT spot-forming unit (SFU) counts for each donor PBMC sample in response to specific stimuli in panels 1 to 3. SFU counts shown for CEF and CMV peptides and PHA were determined by subtracting background values and are presented per 10

Techniques Used: Enzyme-linked Immunospot

The CV between replicate wells for two of the laboratories that participated in all three panels are shown, in relation to grouped ELISPOT SFC counts for CEF and CMV peptides and PHA. The dotted line represents a CV of 70%. Counts were determined
Figure Legend Snippet: The CV between replicate wells for two of the laboratories that participated in all three panels are shown, in relation to grouped ELISPOT SFC counts for CEF and CMV peptides and PHA. The dotted line represents a CV of 70%. Counts were determined

Techniques Used: Enzyme-linked Immunospot

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    Millipore phytohaemagglutinin pha
    ASC functions after paracrine interaction with <t>PHA-stimulated</t> <t>PBMCs.</t> (a) ASC viability: percentage of viable, apoptotic, and necrotic cells in monoculture and after coculture during 72 h at 20% and 5% O 2 . (b) ASC proliferation: the change in cell number in monoculture and after coculture during 72 h at 20% and 5% O 2 . (c) Matrix mineralization of ASCs at 20% (1) and 5% O 2 (2), alizarin red staining: bar 200 μ m. (d) Osteodifferentiation: matrix mineralization in monocultured and cocultured ASCs. ∗ Significant difference between ASC under 20% and 5% O 2 in monoculture. # Significant difference between ASC under 20% and 5% O 2 in coculture.
    Phytohaemagglutinin Pha, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    LL T-cells display restricted peptide recognition. S.I. of 1 × 10 4 T-cells isolated from 8 LL donors upon stimulation with either whole hHSP60 protein or hHSP60 peptide pools (indicated by Roman numerals, all at 10 μg/mL). <t>PHA</t> and monoclonal <t>anti-CD3/anti-CD28</t> antibodies (αCD3/αCD28) were used as positive control. Antigen was omitted in the negative control (Control). Data are shown as stimulation index (S.I. ± SEM). All assays were carried out in triplicates.
    Pha, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ASC functions after paracrine interaction with PHA-stimulated PBMCs. (a) ASC viability: percentage of viable, apoptotic, and necrotic cells in monoculture and after coculture during 72 h at 20% and 5% O 2 . (b) ASC proliferation: the change in cell number in monoculture and after coculture during 72 h at 20% and 5% O 2 . (c) Matrix mineralization of ASCs at 20% (1) and 5% O 2 (2), alizarin red staining: bar 200 μ m. (d) Osteodifferentiation: matrix mineralization in monocultured and cocultured ASCs. ∗ Significant difference between ASC under 20% and 5% O 2 in monoculture. # Significant difference between ASC under 20% and 5% O 2 in coculture.

    Journal: Stem Cells International

    Article Title: Tissue-Related Hypoxia Attenuates Proinflammatory Effects of Allogeneic PBMCs on Adipose-Derived Stromal Cells In Vitro

    doi: 10.1155/2016/4726267

    Figure Lengend Snippet: ASC functions after paracrine interaction with PHA-stimulated PBMCs. (a) ASC viability: percentage of viable, apoptotic, and necrotic cells in monoculture and after coculture during 72 h at 20% and 5% O 2 . (b) ASC proliferation: the change in cell number in monoculture and after coculture during 72 h at 20% and 5% O 2 . (c) Matrix mineralization of ASCs at 20% (1) and 5% O 2 (2), alizarin red staining: bar 200 μ m. (d) Osteodifferentiation: matrix mineralization in monocultured and cocultured ASCs. ∗ Significant difference between ASC under 20% and 5% O 2 in monoculture. # Significant difference between ASC under 20% and 5% O 2 in coculture.

    Article Snippet: PBMCs were activated with 10 μ g/mL phytohaemagglutinin (PHA) (L8754-5MG, Sigma-Aldrich, USA).

    Techniques: Staining

    ASC immunophenotype after monoculture and transwell coculture with PHA-stimulated PBMCs under 20% and 5% O 2 . (a) Representative histograms of ASC surface marker expression. The white filled histograms indicate the positively stained cells while the grey filled histograms indicate the isotype-matched antibody controls. (b) CD90 mean fluorescence intensity (MFI) on cocultured ASCs versus ASCs in monoculture. (c) The proportion of CD54-positive ASCs in monoculture and transwell coculture with PBMCs. (d) CD54 MFI on cocultured ASCs versus ASCs in monoculture. ∗ Significant difference between monoculture and coculture at 20% O 2 ( p

    Journal: Stem Cells International

    Article Title: Tissue-Related Hypoxia Attenuates Proinflammatory Effects of Allogeneic PBMCs on Adipose-Derived Stromal Cells In Vitro

    doi: 10.1155/2016/4726267

    Figure Lengend Snippet: ASC immunophenotype after monoculture and transwell coculture with PHA-stimulated PBMCs under 20% and 5% O 2 . (a) Representative histograms of ASC surface marker expression. The white filled histograms indicate the positively stained cells while the grey filled histograms indicate the isotype-matched antibody controls. (b) CD90 mean fluorescence intensity (MFI) on cocultured ASCs versus ASCs in monoculture. (c) The proportion of CD54-positive ASCs in monoculture and transwell coculture with PBMCs. (d) CD54 MFI on cocultured ASCs versus ASCs in monoculture. ∗ Significant difference between monoculture and coculture at 20% O 2 ( p

    Article Snippet: PBMCs were activated with 10 μ g/mL phytohaemagglutinin (PHA) (L8754-5MG, Sigma-Aldrich, USA).

    Techniques: Marker, Expressing, Staining, Fluorescence

    Cellular organelle state and ROS level in ASCs after coculture with PHA-stimulated PBMCs versus ASCs in monoculture. Transmembrane mitochondrial potential (a), lysosome activity (b), endoplasmic reticulum activity (c), and ROS production (d). ∗ Significant difference between monocultured and cocultured ASCs at 20% O 2 ( p

    Journal: Stem Cells International

    Article Title: Tissue-Related Hypoxia Attenuates Proinflammatory Effects of Allogeneic PBMCs on Adipose-Derived Stromal Cells In Vitro

    doi: 10.1155/2016/4726267

    Figure Lengend Snippet: Cellular organelle state and ROS level in ASCs after coculture with PHA-stimulated PBMCs versus ASCs in monoculture. Transmembrane mitochondrial potential (a), lysosome activity (b), endoplasmic reticulum activity (c), and ROS production (d). ∗ Significant difference between monocultured and cocultured ASCs at 20% O 2 ( p

    Article Snippet: PBMCs were activated with 10 μ g/mL phytohaemagglutinin (PHA) (L8754-5MG, Sigma-Aldrich, USA).

    Techniques: Activity Assay

    ASC morphology in monoculture and after transwell coculture with PHA-stimulated PBMCs under 20% and 5% O 2 . Phase contrast: bar 100 μ m.

    Journal: Stem Cells International

    Article Title: Tissue-Related Hypoxia Attenuates Proinflammatory Effects of Allogeneic PBMCs on Adipose-Derived Stromal Cells In Vitro

    doi: 10.1155/2016/4726267

    Figure Lengend Snippet: ASC morphology in monoculture and after transwell coculture with PHA-stimulated PBMCs under 20% and 5% O 2 . Phase contrast: bar 100 μ m.

    Article Snippet: PBMCs were activated with 10 μ g/mL phytohaemagglutinin (PHA) (L8754-5MG, Sigma-Aldrich, USA).

    Techniques:

    Characterisation of differential gene expression in ASCs after paracrine interaction with PHA-stimulated allogeneic PBMCs. (a) Venn diagram, showing the number of ASC genes commonly or differentially expressed at 20% and 5% O 2 . (b) The number of differentially expressed genes in Gene Ontology (GO) groups (biological function).

    Journal: Stem Cells International

    Article Title: Tissue-Related Hypoxia Attenuates Proinflammatory Effects of Allogeneic PBMCs on Adipose-Derived Stromal Cells In Vitro

    doi: 10.1155/2016/4726267

    Figure Lengend Snippet: Characterisation of differential gene expression in ASCs after paracrine interaction with PHA-stimulated allogeneic PBMCs. (a) Venn diagram, showing the number of ASC genes commonly or differentially expressed at 20% and 5% O 2 . (b) The number of differentially expressed genes in Gene Ontology (GO) groups (biological function).

    Article Snippet: PBMCs were activated with 10 μ g/mL phytohaemagglutinin (PHA) (L8754-5MG, Sigma-Aldrich, USA).

    Techniques: Expressing

    LL T-cells display restricted peptide recognition. S.I. of 1 × 10 4 T-cells isolated from 8 LL donors upon stimulation with either whole hHSP60 protein or hHSP60 peptide pools (indicated by Roman numerals, all at 10 μg/mL). PHA and monoclonal anti-CD3/anti-CD28 antibodies (αCD3/αCD28) were used as positive control. Antigen was omitted in the negative control (Control). Data are shown as stimulation index (S.I. ± SEM). All assays were carried out in triplicates.

    Journal: Journal of Autoimmunity

    Article Title: Autoreactive HSP60 epitope-specific T-cells in early human atherosclerotic lesions

    doi: 10.1016/j.jaut.2012.07.006

    Figure Lengend Snippet: LL T-cells display restricted peptide recognition. S.I. of 1 × 10 4 T-cells isolated from 8 LL donors upon stimulation with either whole hHSP60 protein or hHSP60 peptide pools (indicated by Roman numerals, all at 10 μg/mL). PHA and monoclonal anti-CD3/anti-CD28 antibodies (αCD3/αCD28) were used as positive control. Antigen was omitted in the negative control (Control). Data are shown as stimulation index (S.I. ± SEM). All assays were carried out in triplicates.

    Article Snippet: Anti-human anti-CD3/anti-CD28 antibodies (1 μg/mL each) and PHA (5 μg/mL) (Sigma–Aldrich, USA) were used as positive controls.

    Techniques: Isolation, Positive Control, Negative Control

    EL T-cells proliferate in response to whole hHSP60 protein and recognize hHSP60-derived peptide pools. The proliferative cellular response of 1 × 10 4 intralesional T-cells isolated from 7 EL donors was evaluated upon stimulation with either whole hHSP60 or hHSP60 peptide pools (indicated by Roman numerals, all at 10 μg/mL). PHA and monoclonal anti-CD3/anti-CD28 antibodies (αCD3/αCD28) were used for positive controls. Antigen was omitted in the negative control (Control). Data are shown as stimulation index (S.I. ± SEM). All assays were carried out in triplicates.

    Journal: Journal of Autoimmunity

    Article Title: Autoreactive HSP60 epitope-specific T-cells in early human atherosclerotic lesions

    doi: 10.1016/j.jaut.2012.07.006

    Figure Lengend Snippet: EL T-cells proliferate in response to whole hHSP60 protein and recognize hHSP60-derived peptide pools. The proliferative cellular response of 1 × 10 4 intralesional T-cells isolated from 7 EL donors was evaluated upon stimulation with either whole hHSP60 or hHSP60 peptide pools (indicated by Roman numerals, all at 10 μg/mL). PHA and monoclonal anti-CD3/anti-CD28 antibodies (αCD3/αCD28) were used for positive controls. Antigen was omitted in the negative control (Control). Data are shown as stimulation index (S.I. ± SEM). All assays were carried out in triplicates.

    Article Snippet: Anti-human anti-CD3/anti-CD28 antibodies (1 μg/mL each) and PHA (5 μg/mL) (Sigma–Aldrich, USA) were used as positive controls.

    Techniques: Derivative Assay, Isolation, Negative Control