pbmcs  (Lonza)


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    Human PBMC Human Peripheral Blood Mononuclear Cells Cryopreserved
    Description:
    Cryopreserved ampule of Human Peripheral Blood Mononuclear Cells PBMC containing 50 million cells
    Catalog Number:
    cc-2702
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    Category:
    Primary and Stem Cells
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    Structured Review

    Lonza pbmcs
    Modulation of cytokine secretion by Bmem cells. ( A ) Transiently <t>transfected</t> <t>PBMCs</t> with HCK p59 or FGR in the presence of FCRL4 wt or “empty vector” control were stimulated with CpG at 5 μg/ml or PBS as negative control for 48 h,
    Cryopreserved ampule of Human Peripheral Blood Mononuclear Cells PBMC containing 50 million cells
    https://www.bioz.com/result/pbmcs/product/Lonza
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pbmcs - by Bioz Stars, 2021-03
    86/100 stars

    Images

    1) Product Images from "Involvement of the HCK and FGR src-Family Kinases in FCRL4-Mediated Immune Regulation"

    Article Title: Involvement of the HCK and FGR src-Family Kinases in FCRL4-Mediated Immune Regulation

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1401533

    Modulation of cytokine secretion by Bmem cells. ( A ) Transiently transfected PBMCs with HCK p59 or FGR in the presence of FCRL4 wt or “empty vector” control were stimulated with CpG at 5 μg/ml or PBS as negative control for 48 h,
    Figure Legend Snippet: Modulation of cytokine secretion by Bmem cells. ( A ) Transiently transfected PBMCs with HCK p59 or FGR in the presence of FCRL4 wt or “empty vector” control were stimulated with CpG at 5 μg/ml or PBS as negative control for 48 h,

    Techniques Used: Transfection, Plasmid Preparation, Negative Control

    2) Product Images from "Optimizing the Procedure to Manufacture Clinical-Grade NK Cells for Adoptive Immunotherapy"

    Article Title: Optimizing the Procedure to Manufacture Clinical-Grade NK Cells for Adoptive Immunotherapy

    Journal: Cancers

    doi: 10.3390/cancers13030577

    Cytotoxicity of NKAE cells expanded from PPBMC or CD45RA+ cells using K562mbIL15 or K562mbIL21 as aAPC. No differences in antitumor ability were observed among the different NKAE cell products. Error bars show mean ± SEM. E:T ratio was 2:1. Geometrical symbols represent individual data of NKAE obtained from different donors in the conditions specified in the X axis. Dots: PBMC+K562mbIL15; Squares: PBMC+ K562mbIL21, triangles: CD45RA+ K562mbIL15, inverted triangles: CD45RA+ K562mbIL21.
    Figure Legend Snippet: Cytotoxicity of NKAE cells expanded from PPBMC or CD45RA+ cells using K562mbIL15 or K562mbIL21 as aAPC. No differences in antitumor ability were observed among the different NKAE cell products. Error bars show mean ± SEM. E:T ratio was 2:1. Geometrical symbols represent individual data of NKAE obtained from different donors in the conditions specified in the X axis. Dots: PBMC+K562mbIL15; Squares: PBMC+ K562mbIL21, triangles: CD45RA+ K562mbIL15, inverted triangles: CD45RA+ K562mbIL21.

    Techniques Used:

    3) Product Images from "Cord Blood Platelet Rich Plasma Derivatives for Clinical Applications in Non-transfusion Medicine"

    Article Title: Cord Blood Platelet Rich Plasma Derivatives for Clinical Applications in Non-transfusion Medicine

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.00942

    Expression of CD107a after PMA and ionomycin stimulation on NK, NKT, and T cells from healthy donors is reduced by incubation with, CB-PPP, CB-PL, and CB-PR. Data represents analysis of CD3– CD56 dim, CD3– CD56 bright NK cells, CD3+ CD56+ NKT cells and CD3+ CD56– T cells in adult donor PBMCs ( n = 4) after incubation with CB-PRP derived samples or complete media only. Results show percentage of maximum expression relative to complete media for each cell type following 2 h stimulation with PMA and ionomycin. CB-PRP preparations investigated were cord blood platelet poor plasma (CB-PPP; n = 10), cord blood platelet lysate (CB-PL; n = 10), or cord blood platelet releasate (CB-PR; n = 10). PBMCs were incubated with each preparation diluted 50:50 with media and supplemented with IL-2, or with complete media and IL-2 only for 48 h prior to antibody staining and flow cytometry analysis. Each experiment was repeated with four different PBMC donors and data points represent donor means. Statistical analysis was performed using non-parametric one-way ANOVA (Kruskal-Wallis test with Dunn's post-hoc test for unpaired samples and Friedman's for paired samples). * p
    Figure Legend Snippet: Expression of CD107a after PMA and ionomycin stimulation on NK, NKT, and T cells from healthy donors is reduced by incubation with, CB-PPP, CB-PL, and CB-PR. Data represents analysis of CD3– CD56 dim, CD3– CD56 bright NK cells, CD3+ CD56+ NKT cells and CD3+ CD56– T cells in adult donor PBMCs ( n = 4) after incubation with CB-PRP derived samples or complete media only. Results show percentage of maximum expression relative to complete media for each cell type following 2 h stimulation with PMA and ionomycin. CB-PRP preparations investigated were cord blood platelet poor plasma (CB-PPP; n = 10), cord blood platelet lysate (CB-PL; n = 10), or cord blood platelet releasate (CB-PR; n = 10). PBMCs were incubated with each preparation diluted 50:50 with media and supplemented with IL-2, or with complete media and IL-2 only for 48 h prior to antibody staining and flow cytometry analysis. Each experiment was repeated with four different PBMC donors and data points represent donor means. Statistical analysis was performed using non-parametric one-way ANOVA (Kruskal-Wallis test with Dunn's post-hoc test for unpaired samples and Friedman's for paired samples). * p

    Techniques Used: Expressing, Incubation, Derivative Assay, Staining, Flow Cytometry

    Viability of CD3- CD56 dim, CD3- CD56 bright NK cells, CD3+ CD56+ NKT cells, and CD3+ CD56- T cells from adult donor PBMCs ( n = 3) after incubation with cord blood plasma preparations or complete media only. Results show percentage of live cells (Annexin V negative, 7-AAD negative) for each cell type. Cord blood plasma preparations investigated were cord blood platelet poor plasma (CB-PPP; n = 10), cord blood platelet lysate (CB-PL; n = 10), or cord blood platelet releasate (CB-PR; n = 10). PBMCs were incubated with each preparation diluted 50:50 with media and supplemented with IL-2, or with complete media and IL-2 only for 48 h prior to antibody staining and flow cytometry analysis. Each experiment was repeated with four different PBMC donors and data points represent donor means. Statistical analysis was performed using non-parametric one-way ANOVA (Kruskal-Wallis test with Dunn's post-hoc test for unpaired samples and Friedman's for paired samples). * p
    Figure Legend Snippet: Viability of CD3- CD56 dim, CD3- CD56 bright NK cells, CD3+ CD56+ NKT cells, and CD3+ CD56- T cells from adult donor PBMCs ( n = 3) after incubation with cord blood plasma preparations or complete media only. Results show percentage of live cells (Annexin V negative, 7-AAD negative) for each cell type. Cord blood plasma preparations investigated were cord blood platelet poor plasma (CB-PPP; n = 10), cord blood platelet lysate (CB-PL; n = 10), or cord blood platelet releasate (CB-PR; n = 10). PBMCs were incubated with each preparation diluted 50:50 with media and supplemented with IL-2, or with complete media and IL-2 only for 48 h prior to antibody staining and flow cytometry analysis. Each experiment was repeated with four different PBMC donors and data points represent donor means. Statistical analysis was performed using non-parametric one-way ANOVA (Kruskal-Wallis test with Dunn's post-hoc test for unpaired samples and Friedman's for paired samples). * p

    Techniques Used: Incubation, Staining, Flow Cytometry

    Expression of NKG2D on NK, NKT and T cells from healthy donors is reduced by incubation with, CB-PPP, CB-PL, and CB-PR. Data represents analysis of CD3– CD56 dim, CD3– CD56 bright NK cells, CD3+ CD56+ NKT cells and CD3+ CD56– T cells in adult donor PBMCs ( n = 4) after incubation with CB-PRP preparations or complete media only. Results show percentage of maximum expression relative to complete media for each cell type from unstimulated cultures. CB-PRP preparations investigated were cord blood platelet poor plasma (CB-PPP; n = 10), cord blood platelet lysate (CB-PL; n = 10), or cord blood platelet releasate (CB-PR; n = 10). PBMCs were incubated with each preparation diluted 50:50 with media and supplemented with IL-2, or with complete media and IL-2 only for 48 h prior to antibody staining and flow cytometry analysis. Each experiment was repeated with four different PBMC donors and data points represent donor means. Statistical analysis was performed using non-parametric one-way ANOVA (Kruskal-Wallis test with Dunn's post-hoc test for unpaired samples and Friedman's for paired samples). * p
    Figure Legend Snippet: Expression of NKG2D on NK, NKT and T cells from healthy donors is reduced by incubation with, CB-PPP, CB-PL, and CB-PR. Data represents analysis of CD3– CD56 dim, CD3– CD56 bright NK cells, CD3+ CD56+ NKT cells and CD3+ CD56– T cells in adult donor PBMCs ( n = 4) after incubation with CB-PRP preparations or complete media only. Results show percentage of maximum expression relative to complete media for each cell type from unstimulated cultures. CB-PRP preparations investigated were cord blood platelet poor plasma (CB-PPP; n = 10), cord blood platelet lysate (CB-PL; n = 10), or cord blood platelet releasate (CB-PR; n = 10). PBMCs were incubated with each preparation diluted 50:50 with media and supplemented with IL-2, or with complete media and IL-2 only for 48 h prior to antibody staining and flow cytometry analysis. Each experiment was repeated with four different PBMC donors and data points represent donor means. Statistical analysis was performed using non-parametric one-way ANOVA (Kruskal-Wallis test with Dunn's post-hoc test for unpaired samples and Friedman's for paired samples). * p

    Techniques Used: Expressing, Incubation, Staining, Flow Cytometry

    4) Product Images from "Alternative splicing of SLAMF6 in human T cells creates a co-stimulatory isoform that counteracts the inhibitory effect of the full-length receptor"

    Article Title: Alternative splicing of SLAMF6 in human T cells creates a co-stimulatory isoform that counteracts the inhibitory effect of the full-length receptor

    Journal: bioRxiv

    doi: 10.1101/2020.08.21.262238

    SLAMF6 splice switching with antisense oligonucleotides leads to improved anti-tumor T cell response a , Schematic diagram of the antisense-oligos (ASOs) targeting the splicing site of SLAMF6 in exon2. b , RNA expression of SLAMF6 isoforms in WT Jurkat cells electroporated with scrambled ASO or ASO targeting the splice site. c , ELISA for IL-2 in WT Jurkat cells activated for 48 h after electroporation with scrambled ASO or increasing concentration of ASO targeting the splice site. d , Quantitative RT-PCR of WT Jurkat cells electroporated with ASO or control ASO and activated for 6 h. Data were normalized to HPRT expression for each manipulation. Further normalization for each transcript comparing to control ASO (0 h). e , ELISA for IFN-γ in anti-CD3 plate-bound activated PBMCs, 24h after electroporation with ASO, or control ASO. f-j , TILs were electroporated with ASO or control ASO. Twenty-four h after electroporation, the cells were washed and mixed at a 1:1 ratio with 526 mel cells and immediately injected subcutaneously into the back of nude (athymic Foxn1-/-) mice (N=7). f , Schematic diagram showing the experimental layout. g , RNA expression of SLAMF6 isoforms in TILs electroporated with scrambled ASOs or ASOs targeting the splice site. h , Spider plot showing tumor volume [calculated as L (length) x W (width) 2 x 0.5]. i , Tumor volume (Mean+SEM) until day 29, when the first mouse was sacrificed. j , Mean+SEM tumor volume on day 29. One-way ANOVA. *, P
    Figure Legend Snippet: SLAMF6 splice switching with antisense oligonucleotides leads to improved anti-tumor T cell response a , Schematic diagram of the antisense-oligos (ASOs) targeting the splicing site of SLAMF6 in exon2. b , RNA expression of SLAMF6 isoforms in WT Jurkat cells electroporated with scrambled ASO or ASO targeting the splice site. c , ELISA for IL-2 in WT Jurkat cells activated for 48 h after electroporation with scrambled ASO or increasing concentration of ASO targeting the splice site. d , Quantitative RT-PCR of WT Jurkat cells electroporated with ASO or control ASO and activated for 6 h. Data were normalized to HPRT expression for each manipulation. Further normalization for each transcript comparing to control ASO (0 h). e , ELISA for IFN-γ in anti-CD3 plate-bound activated PBMCs, 24h after electroporation with ASO, or control ASO. f-j , TILs were electroporated with ASO or control ASO. Twenty-four h after electroporation, the cells were washed and mixed at a 1:1 ratio with 526 mel cells and immediately injected subcutaneously into the back of nude (athymic Foxn1-/-) mice (N=7). f , Schematic diagram showing the experimental layout. g , RNA expression of SLAMF6 isoforms in TILs electroporated with scrambled ASOs or ASOs targeting the splice site. h , Spider plot showing tumor volume [calculated as L (length) x W (width) 2 x 0.5]. i , Tumor volume (Mean+SEM) until day 29, when the first mouse was sacrificed. j , Mean+SEM tumor volume on day 29. One-way ANOVA. *, P

    Techniques Used: RNA Expression, Allele-specific Oligonucleotide, Enzyme-linked Immunosorbent Assay, Electroporation, Concentration Assay, Quantitative RT-PCR, Expressing, Injection, Mouse Assay

    5) Product Images from "HLA-DR15 Molecules Jointly Shape an Autoreactive T Cell Repertoire in Multiple Sclerosis"

    Article Title: HLA-DR15 Molecules Jointly Shape an Autoreactive T Cell Repertoire in Multiple Sclerosis

    Journal: Cell

    doi: 10.1016/j.cell.2020.09.054

    Responses of Autoreactive CD4 + TCCs to RASGRP2 and HLA-DR-SPs (A) Dose-response curves of TCC14 to RASGRP2 (78–87) and HLA-DR-SPs. (B) Irradiated BLS-DR2b cells were incubated with peptides for 12 h and then co-cultured with TCC14. Shown are expression levels of CD69 and CD25 on TCC14 at different time points after co-culture. ΔMFI indicates the mean fluorescence intensity (MFI) value above the no-peptide control. (C–E) CD45RA − PBMCs from 3 HLA-DR15 + RRMS_NAT patients were stimulated with RASGRP2 (78–87) to generate TCCs. Proliferation of memory CD4 + T cells was analyzed on day 12 (C). Acquired TCCs were co-cultured with irradiated autologous PBMCs as APCs and stimulated with RASGRP2 (78–87) or DRB1 (57–70) , and proliferation was detected on day 3. The TCCs responding to RASGRP2 (78–87) and DRB1 (57–70) are highlighted in blue (D). Five new 1159AG_TCCs that responded to RASGRP2 (78–87) and DRB1 (57–70) were generated from RRMS_NAT-2. Their corresponding TCRVβ sequence and functional phenotype are shown (E). (F) Restriction of the RASGRP2 (78–87) -specific 1159AG_TCCs was tested with irradiated BLS-DR2b cells and stimulation with RASGRP2 (78–87) . (G) Th1/Th2/Th17-related cytokines in supernatants of 1159AG_TCCs after co-culture with irradiated BLS-DR2b cells and stimulation with RASGRP2 (78–87) for 3 days. (H and I) 1159AG_TCCs were co-cultured with irradiated BLS-DR2b cells and stimulated with RASGRP2 (78–87) or DRB1/5 (184–199) for 3 days. Shown is proliferation of 1159AG_TCCs (H) and IFN-γ in supernatants (I). Data are expressed as mean ± SEM, and p values were determined by unpaired t test. See also Figures S4 and S5 and Table S1 .
    Figure Legend Snippet: Responses of Autoreactive CD4 + TCCs to RASGRP2 and HLA-DR-SPs (A) Dose-response curves of TCC14 to RASGRP2 (78–87) and HLA-DR-SPs. (B) Irradiated BLS-DR2b cells were incubated with peptides for 12 h and then co-cultured with TCC14. Shown are expression levels of CD69 and CD25 on TCC14 at different time points after co-culture. ΔMFI indicates the mean fluorescence intensity (MFI) value above the no-peptide control. (C–E) CD45RA − PBMCs from 3 HLA-DR15 + RRMS_NAT patients were stimulated with RASGRP2 (78–87) to generate TCCs. Proliferation of memory CD4 + T cells was analyzed on day 12 (C). Acquired TCCs were co-cultured with irradiated autologous PBMCs as APCs and stimulated with RASGRP2 (78–87) or DRB1 (57–70) , and proliferation was detected on day 3. The TCCs responding to RASGRP2 (78–87) and DRB1 (57–70) are highlighted in blue (D). Five new 1159AG_TCCs that responded to RASGRP2 (78–87) and DRB1 (57–70) were generated from RRMS_NAT-2. Their corresponding TCRVβ sequence and functional phenotype are shown (E). (F) Restriction of the RASGRP2 (78–87) -specific 1159AG_TCCs was tested with irradiated BLS-DR2b cells and stimulation with RASGRP2 (78–87) . (G) Th1/Th2/Th17-related cytokines in supernatants of 1159AG_TCCs after co-culture with irradiated BLS-DR2b cells and stimulation with RASGRP2 (78–87) for 3 days. (H and I) 1159AG_TCCs were co-cultured with irradiated BLS-DR2b cells and stimulated with RASGRP2 (78–87) or DRB1/5 (184–199) for 3 days. Shown is proliferation of 1159AG_TCCs (H) and IFN-γ in supernatants (I). Data are expressed as mean ± SEM, and p values were determined by unpaired t test. See also Figures S4 and S5 and Table S1 .

    Techniques Used: Irradiation, Incubation, Cell Culture, Expressing, Co-Culture Assay, Fluorescence, Generated, Sequencing, Functional Assay

    Increased Reactivity of Memory CD4 + T Cells against HLA-DR-SPs in MS Patients (A) Responses of CD45RA − PBMCs of HLA-DR15 + HDs (n = 8) and MS patients (RRMS, n = 4; RRMS_NAT, n = 10) against the five most common HLA-DR-SPs alone or as pool. 10–15 replicate wells were tested for proliferation, and responses are depicted as the stimulatory index (SI). Individual wells are represented by dots for HDs (left) or MS patients (right). The purple dotted line indicates SI = 2, and SI ≥ 2 was considered positive. Responses to control CEF II peptides are shown at the bottom. (B) Proliferations of CD45RA − PBMCs from HDs and MS patients after stimulation with pooled HLA-DR-SPs in the presence of a blocking anti-HLA-DR Ab. (C) CD45RA − PBMCs from HLA-DR15 + MS patients were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with pooled HLA-DR-SPs. After 7 days, cells were analyzed, and proportions of memory CD4 + T cells in the proliferating (CFSE dim ) and highly proliferating (CFSE low ) compartments are shown in the pie charts. (D) Blood CD4 + T cells and monocytes were purified from PBMCs of HLA-DR15 + MS patients and co-cultured with pooled HLA-DR-SPs for 7 days. Proliferation of naive CD4 + T cells (CD45RA + ) and memory CD4 + T cells (CD45RA − ) was detected. (E) Th1/Th2/Th17-related cytokines in supernatants of CD45RA − PBMCs after stimulation with pooled HLA-DR-SPs. (F) IFN-γ, IL-17A, and IL-17F in supernatants of co-cultured blood CD4 + T cells and monocytes after stimulation with pooled HLA-DR-SPs. (G) Expression levels of CCR6 and CXCR3 on non-proliferating (CFSE high ) and proliferating (CFSE dim ) memory CD4 + T cells after stimulation with pooled HLA-DR-SPs for 7 days. Data are expressed as mean ± SEM, and p values were determined by unpaired t test. See also Figure S4 and Tables S1 and S5 .
    Figure Legend Snippet: Increased Reactivity of Memory CD4 + T Cells against HLA-DR-SPs in MS Patients (A) Responses of CD45RA − PBMCs of HLA-DR15 + HDs (n = 8) and MS patients (RRMS, n = 4; RRMS_NAT, n = 10) against the five most common HLA-DR-SPs alone or as pool. 10–15 replicate wells were tested for proliferation, and responses are depicted as the stimulatory index (SI). Individual wells are represented by dots for HDs (left) or MS patients (right). The purple dotted line indicates SI = 2, and SI ≥ 2 was considered positive. Responses to control CEF II peptides are shown at the bottom. (B) Proliferations of CD45RA − PBMCs from HDs and MS patients after stimulation with pooled HLA-DR-SPs in the presence of a blocking anti-HLA-DR Ab. (C) CD45RA − PBMCs from HLA-DR15 + MS patients were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with pooled HLA-DR-SPs. After 7 days, cells were analyzed, and proportions of memory CD4 + T cells in the proliferating (CFSE dim ) and highly proliferating (CFSE low ) compartments are shown in the pie charts. (D) Blood CD4 + T cells and monocytes were purified from PBMCs of HLA-DR15 + MS patients and co-cultured with pooled HLA-DR-SPs for 7 days. Proliferation of naive CD4 + T cells (CD45RA + ) and memory CD4 + T cells (CD45RA − ) was detected. (E) Th1/Th2/Th17-related cytokines in supernatants of CD45RA − PBMCs after stimulation with pooled HLA-DR-SPs. (F) IFN-γ, IL-17A, and IL-17F in supernatants of co-cultured blood CD4 + T cells and monocytes after stimulation with pooled HLA-DR-SPs. (G) Expression levels of CCR6 and CXCR3 on non-proliferating (CFSE high ) and proliferating (CFSE dim ) memory CD4 + T cells after stimulation with pooled HLA-DR-SPs for 7 days. Data are expressed as mean ± SEM, and p values were determined by unpaired t test. See also Figure S4 and Tables S1 and S5 .

    Techniques Used: Blocking Assay, Labeling, Purification, Cell Culture, Expressing

    HLA-DR-SPs Mainly Activate Memory CD4 + T Cells in HLA-DR15 + MS Patients, Related to Figures 3 and 5 and Table S1 (A) Correlation of the total number of HLA-DR-SPs presented by the two HLA-DR15 molecules on B cells and the degree of autoproliferation in MS patients (RRMS, n = 3; RRMS_NAT, n = 3; Spearman’s rank correlation test). (B) PBMCs were separated into CD45RA + cells and CD45RA − cells by magnetic cell isolation using CD45RA microbeads. Cell subsets in untouched PBMCs, CD45RA + PBMCs, and CD45RA − PBMCs were analyzed by flow cytometry. (C) Comparison of the autoproliferation between untouched PBMCs and CD45RA − PBMCs from HLA-DR15 + HDs (n = 6), RRMS (n = 4), or RRMS_NAT (n = 6). Data are expressed as mean, and p values were determined by paired t test. (D) CD45RA − PBMCs from HLA-DR15 + HDs (n = 5) and MS patients (n = 5) were stimulated with Tetanus Toxin peptides, TT (830-844) and TT (947-967) , which could be presented by DR2a and/or DR2b as previously reported. For each sample, each stimulation was performed with 10 replicate wells. Proliferations of CD45RA − PBMCs were detected after 7 days by 3 H-thymidine incorporation assay, and the proliferation strength is depicted as SI. The p values were determined by unpaired t test. (E) CD45RA − PBMCs from HLA-DR15 + RRMS (n = 4) and RRMS_NAT (n = 10) patients were stimulated with the most common HLA-DR-SPs either alone or as pool. Proliferations of CD45RA − PBMCs were detected by 3 H-thymidine incorporation assay after 7 days, and proliferation strength is depicted as counts per minute (cpm). 10-15 replicate wells per condition are indicated by individual dots. The blue dotted line indicates the mean value of the no peptide control. The purple dotted line indicates the mean value plus three standard deviations of the no peptide control. Values above the purple dotted line were considered positive.
    Figure Legend Snippet: HLA-DR-SPs Mainly Activate Memory CD4 + T Cells in HLA-DR15 + MS Patients, Related to Figures 3 and 5 and Table S1 (A) Correlation of the total number of HLA-DR-SPs presented by the two HLA-DR15 molecules on B cells and the degree of autoproliferation in MS patients (RRMS, n = 3; RRMS_NAT, n = 3; Spearman’s rank correlation test). (B) PBMCs were separated into CD45RA + cells and CD45RA − cells by magnetic cell isolation using CD45RA microbeads. Cell subsets in untouched PBMCs, CD45RA + PBMCs, and CD45RA − PBMCs were analyzed by flow cytometry. (C) Comparison of the autoproliferation between untouched PBMCs and CD45RA − PBMCs from HLA-DR15 + HDs (n = 6), RRMS (n = 4), or RRMS_NAT (n = 6). Data are expressed as mean, and p values were determined by paired t test. (D) CD45RA − PBMCs from HLA-DR15 + HDs (n = 5) and MS patients (n = 5) were stimulated with Tetanus Toxin peptides, TT (830-844) and TT (947-967) , which could be presented by DR2a and/or DR2b as previously reported. For each sample, each stimulation was performed with 10 replicate wells. Proliferations of CD45RA − PBMCs were detected after 7 days by 3 H-thymidine incorporation assay, and the proliferation strength is depicted as SI. The p values were determined by unpaired t test. (E) CD45RA − PBMCs from HLA-DR15 + RRMS (n = 4) and RRMS_NAT (n = 10) patients were stimulated with the most common HLA-DR-SPs either alone or as pool. Proliferations of CD45RA − PBMCs were detected by 3 H-thymidine incorporation assay after 7 days, and proliferation strength is depicted as counts per minute (cpm). 10-15 replicate wells per condition are indicated by individual dots. The blue dotted line indicates the mean value of the no peptide control. The purple dotted line indicates the mean value plus three standard deviations of the no peptide control. Values above the purple dotted line were considered positive.

    Techniques Used: Cell Isolation, Flow Cytometry, Thymidine Incorporation Assay

    6) Product Images from "Increased Plasma Soluble Interleukin-2 Receptor Alpha Levels in Patients With Long-Term Type 1 Diabetes With Vascular Complications Associated With IL2RA and PTPN2 Gene Polymorphisms"

    Article Title: Increased Plasma Soluble Interleukin-2 Receptor Alpha Levels in Patients With Long-Term Type 1 Diabetes With Vascular Complications Associated With IL2RA and PTPN2 Gene Polymorphisms

    Journal: Frontiers in Endocrinology

    doi: 10.3389/fendo.2020.575469

    Patients with type 1 diabetes (T1D) with complications display a shift from naïve T cells to effector T cells. (A) Flow cytometry screening of PBMCs from PROLONG patients revealed a significant decrease of CD8 + naïve T (T N ) cells (CD3 + CD4 + CD45RA + CCR7 + CCR5 - ) in progressors (P) compared to non-progressors (NP). (B) Progressors displayed elevated CD8 + effector T (T EFF ) cells (CD3 + CD4 + CD45RA + CCR7 - ) simultaneously (p = ns). (C) CD4 + T N cells were also declined in patients with T1D with complications as compared to NPs (p = ns). (D) In progressors CD4 + T EFF cells were significantly elevated compared to NPs. (For the comparison between the different groups, multiple linear regression was applied and adjusted for the age and sex covariates. *p
    Figure Legend Snippet: Patients with type 1 diabetes (T1D) with complications display a shift from naïve T cells to effector T cells. (A) Flow cytometry screening of PBMCs from PROLONG patients revealed a significant decrease of CD8 + naïve T (T N ) cells (CD3 + CD4 + CD45RA + CCR7 + CCR5 - ) in progressors (P) compared to non-progressors (NP). (B) Progressors displayed elevated CD8 + effector T (T EFF ) cells (CD3 + CD4 + CD45RA + CCR7 - ) simultaneously (p = ns). (C) CD4 + T N cells were also declined in patients with T1D with complications as compared to NPs (p = ns). (D) In progressors CD4 + T EFF cells were significantly elevated compared to NPs. (For the comparison between the different groups, multiple linear regression was applied and adjusted for the age and sex covariates. *p

    Techniques Used: Flow Cytometry

    7) Product Images from "A Truncation Variant of the Cation Channel P2RX5 Is Upregulated during T Cell Activation"

    Article Title: A Truncation Variant of the Cation Channel P2RX5 Is Upregulated during T Cell Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0104692

    T cell activation alters mRNA expression of specific ion channel subunits. A, B, Bar diagram summarizing n-fold increase or decrease in intensity of hybridization signals obtained by probing oligonucleotide-based arrays with Cy3 and Cy5 labeled cDNA derived from mRNA isolated from non-activated and PHA-L activated PBMCs, respectively. Gray bars – custom-made array (n = 14) ( Tables S1 , S2 ); black bars – Affimetrix array (human U133A 2.0; n = 6). Error bars are SEM. Channel subunit genes are indicated on the left. Nomenclature is from http://www.ncbi.nlm.nih.gov/omim . Brackets - selectivity of corresponding ion channel. cat – cation; n.p. – not present in array; n.d. – not determined. CD25 (IL2RA) served as control. C, Bar diagram summarizing qPCR results for changes in mRNA expression of purified CD4 + and CD8 + T cells activated with anti-CD3/CD28 antibody-coated beads (n = 5–8). D, qPCR analysis of P2RX5 mRNA expression in activated CD4 + T cells in absence (▴) or presence (□) of cycloheximide (n = 3). Error bars are SEM.
    Figure Legend Snippet: T cell activation alters mRNA expression of specific ion channel subunits. A, B, Bar diagram summarizing n-fold increase or decrease in intensity of hybridization signals obtained by probing oligonucleotide-based arrays with Cy3 and Cy5 labeled cDNA derived from mRNA isolated from non-activated and PHA-L activated PBMCs, respectively. Gray bars – custom-made array (n = 14) ( Tables S1 , S2 ); black bars – Affimetrix array (human U133A 2.0; n = 6). Error bars are SEM. Channel subunit genes are indicated on the left. Nomenclature is from http://www.ncbi.nlm.nih.gov/omim . Brackets - selectivity of corresponding ion channel. cat – cation; n.p. – not present in array; n.d. – not determined. CD25 (IL2RA) served as control. C, Bar diagram summarizing qPCR results for changes in mRNA expression of purified CD4 + and CD8 + T cells activated with anti-CD3/CD28 antibody-coated beads (n = 5–8). D, qPCR analysis of P2RX5 mRNA expression in activated CD4 + T cells in absence (▴) or presence (□) of cycloheximide (n = 3). Error bars are SEM.

    Techniques Used: Activation Assay, Expressing, Hybridization, Labeling, Derivative Assay, Isolation, Real-time Polymerase Chain Reaction, Purification

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    Cytotoxicity Assay:

    Article Title: Peripheral Blood TIM-3 Positive NK and CD8+ T Cells throughout Pregnancy: TIM-3/Galectin-9 Interaction and Its Possible Role during Pregnancy
    Article Snippet: Labeled cells were analyzed with a FACSCalibur flow cytometer (BD Immunocytometry Systems, Erembodegen, Belgium) equipped with the CellQuest software program (BD Biosciences, San Diego, CA, USA) for data acquisition and analysis. .. CD107a cytotoxicity assay To determine CD107a surface expression by cytotoxic T cells and NK cells, PBMC were incubated for 4 h at 37°C in an atmosphere containing 5% CO2 in the presence of FITC-conjugated anti-human CD107a monoclonal antibody in RPMI 1640 medium containing 10% fetal bovine serum, penicillin and streptomycin (Lonza, Switzerland), ionomycin (Sigma–Aldrich, Hungary) and phorbol myristate acetate (Sigma–Aldrich, Hungary). .. After stimulation the cells were washed and resuspended in PBS then stained with antibodies to Tc cell and NK cell markers (APC-conjugated anti-human CD8 or APC-conjugated anti-human CD56) together with PE-conjugated anti-human TIM-3 antibody for 30 min at room temperature.

    Expressing:

    Article Title: Peripheral Blood TIM-3 Positive NK and CD8+ T Cells throughout Pregnancy: TIM-3/Galectin-9 Interaction and Its Possible Role during Pregnancy
    Article Snippet: Labeled cells were analyzed with a FACSCalibur flow cytometer (BD Immunocytometry Systems, Erembodegen, Belgium) equipped with the CellQuest software program (BD Biosciences, San Diego, CA, USA) for data acquisition and analysis. .. CD107a cytotoxicity assay To determine CD107a surface expression by cytotoxic T cells and NK cells, PBMC were incubated for 4 h at 37°C in an atmosphere containing 5% CO2 in the presence of FITC-conjugated anti-human CD107a monoclonal antibody in RPMI 1640 medium containing 10% fetal bovine serum, penicillin and streptomycin (Lonza, Switzerland), ionomycin (Sigma–Aldrich, Hungary) and phorbol myristate acetate (Sigma–Aldrich, Hungary). .. After stimulation the cells were washed and resuspended in PBS then stained with antibodies to Tc cell and NK cell markers (APC-conjugated anti-human CD8 or APC-conjugated anti-human CD56) together with PE-conjugated anti-human TIM-3 antibody for 30 min at room temperature.

    Incubation:

    Article Title: Peripheral Blood TIM-3 Positive NK and CD8+ T Cells throughout Pregnancy: TIM-3/Galectin-9 Interaction and Its Possible Role during Pregnancy
    Article Snippet: Labeled cells were analyzed with a FACSCalibur flow cytometer (BD Immunocytometry Systems, Erembodegen, Belgium) equipped with the CellQuest software program (BD Biosciences, San Diego, CA, USA) for data acquisition and analysis. .. CD107a cytotoxicity assay To determine CD107a surface expression by cytotoxic T cells and NK cells, PBMC were incubated for 4 h at 37°C in an atmosphere containing 5% CO2 in the presence of FITC-conjugated anti-human CD107a monoclonal antibody in RPMI 1640 medium containing 10% fetal bovine serum, penicillin and streptomycin (Lonza, Switzerland), ionomycin (Sigma–Aldrich, Hungary) and phorbol myristate acetate (Sigma–Aldrich, Hungary). .. After stimulation the cells were washed and resuspended in PBS then stained with antibodies to Tc cell and NK cell markers (APC-conjugated anti-human CD8 or APC-conjugated anti-human CD56) together with PE-conjugated anti-human TIM-3 antibody for 30 min at room temperature.

    Isolation:

    Article Title: Conditioned media from endothelial progenitor cells cultured in simulated microgravity promote angiogenesis and bone fracture healing
    Article Snippet: .. Briefly, peripheral blood mononuclear cells were isolated using Ficoll density gradient centrifugation according to the manufacturer’s protocol, followed by washing with phosphate-buffered saline (PBS). .. The cells were plated in culture dishes pre-coated with fibronectin (5 μg/cm2 ) (Merck, Darmstadt, Germany) and cultured in endothelial basal medium 2 (EBM-2; Lonza, Basel, Switzerland) supplemented with EBM-2-MV-SingleQuots (Lonza).

    Article Title: Porcupine Is Not Required for the Production of the Majority of Wnts from Primary Human Astrocytes and CD8+ T Cells
    Article Snippet: L-Wnt3a (ATCC CRL-2647) were cultured in complete DMEM with 0.4 mg/ml G-418. .. Culture and Activation of Human Peripheral Blood Mononuclear Cells (PBMCs) and Isolation of CD8+ T cells PBMCs were isolated from venous blood of healthy donors using lymphocyte separation medium (Lonza Biologics, Portsmouth, NH) and density centrifugation. .. PBMCs were then cultured at 1×106 cells mL of RPMI 1640, 10% heat-inactivated fetal bovine serum (FBS), 200 mM L-glutamine, and 1% penicillin/streptomycin (Lonza Biologics, Portsmouth, NH).

    Article Title: Efficient Generation of Isogenic Primary Human Myeloid Cells using CRISPR-Cas9 Ribonucleoproteins
    Article Snippet: Further, we confirmed consistently robust knockout across biological replicates at the genetic level by Tracking of Indels by DEcomposition (TIDE) analysis , showing that guides against CXCR4 and CCR5 led to disruption in greater than 90% of alleles ( ) . .. This protocol led to reproducible knockout when starting with CD14+ monocytes from freshly isolated peripheral blood mononuclear cells (PBMC), from cryopreserved PBMC, or from isolated-then-cryopreserved CD14+ monocytes , allowing for a flexible workflow and enabling iterative experiments on consistent biological samples. .. Collectively, these data demonstrate optimized knockout of targeted genes in primary human myeloid cells using CRISPR-Cas9 RNPs.

    Gradient Centrifugation:

    Article Title: Conditioned media from endothelial progenitor cells cultured in simulated microgravity promote angiogenesis and bone fracture healing
    Article Snippet: .. Briefly, peripheral blood mononuclear cells were isolated using Ficoll density gradient centrifugation according to the manufacturer’s protocol, followed by washing with phosphate-buffered saline (PBS). .. The cells were plated in culture dishes pre-coated with fibronectin (5 μg/cm2 ) (Merck, Darmstadt, Germany) and cultured in endothelial basal medium 2 (EBM-2; Lonza, Basel, Switzerland) supplemented with EBM-2-MV-SingleQuots (Lonza).

    Cell Stimulation:

    Article Title: Distinct Effector Memory CD4+ T Cell Signatures in Latent Mycobacterium tuberculosis Infection, BCG Vaccination and Clinically Resolved Tuberculosis
    Article Snippet: Responses were scored as positive if the test wells contained a mean of at least 10 SFCs more than the mean of the negative control wells. .. Cell stimulation, staining and flow cytometry Before stimulation, cryopreserved PBMC were thawed and resuspended overnight at 37°C, 5% CO2 in RPMI-1640 medium (Lonza, Walkersville, MD) containing 10% FBS, 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. .. 1×106 PBMC were each stimulated with CW antigens (10 µg/ml) and ESAT-6 and CFP-10 peptides (10 µg/ml) at 37°C, 5% CO2 for 2 h followed by the addition of Brefeldin A (10 µg/ml) (BD Biosciences, San Diego, CA) and further incubated for 16 h. PBMC were washed, surface-stained with appropriate antibodies, permeabilized with Cytofix/Cytoperm Kit (BD Biosciences), stained intracellularly with appropriate antibodies: IFN-γ (clone B27), TNF-α (clone Mab11), IL-2 (clone MQ1-17H12), Ki-67 (clone B56), Bcl-2 (clone Bcl-2/100) and fixed with 1% paraformaldehyde before acquisition on a FACSCalibur (BD BioSciences) or LSR-II system (BD Biosciences).

    Staining:

    Article Title: Distinct Effector Memory CD4+ T Cell Signatures in Latent Mycobacterium tuberculosis Infection, BCG Vaccination and Clinically Resolved Tuberculosis
    Article Snippet: Responses were scored as positive if the test wells contained a mean of at least 10 SFCs more than the mean of the negative control wells. .. Cell stimulation, staining and flow cytometry Before stimulation, cryopreserved PBMC were thawed and resuspended overnight at 37°C, 5% CO2 in RPMI-1640 medium (Lonza, Walkersville, MD) containing 10% FBS, 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. .. 1×106 PBMC were each stimulated with CW antigens (10 µg/ml) and ESAT-6 and CFP-10 peptides (10 µg/ml) at 37°C, 5% CO2 for 2 h followed by the addition of Brefeldin A (10 µg/ml) (BD Biosciences, San Diego, CA) and further incubated for 16 h. PBMC were washed, surface-stained with appropriate antibodies, permeabilized with Cytofix/Cytoperm Kit (BD Biosciences), stained intracellularly with appropriate antibodies: IFN-γ (clone B27), TNF-α (clone Mab11), IL-2 (clone MQ1-17H12), Ki-67 (clone B56), Bcl-2 (clone Bcl-2/100) and fixed with 1% paraformaldehyde before acquisition on a FACSCalibur (BD BioSciences) or LSR-II system (BD Biosciences).

    Flow Cytometry:

    Article Title: Distinct Effector Memory CD4+ T Cell Signatures in Latent Mycobacterium tuberculosis Infection, BCG Vaccination and Clinically Resolved Tuberculosis
    Article Snippet: Responses were scored as positive if the test wells contained a mean of at least 10 SFCs more than the mean of the negative control wells. .. Cell stimulation, staining and flow cytometry Before stimulation, cryopreserved PBMC were thawed and resuspended overnight at 37°C, 5% CO2 in RPMI-1640 medium (Lonza, Walkersville, MD) containing 10% FBS, 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. .. 1×106 PBMC were each stimulated with CW antigens (10 µg/ml) and ESAT-6 and CFP-10 peptides (10 µg/ml) at 37°C, 5% CO2 for 2 h followed by the addition of Brefeldin A (10 µg/ml) (BD Biosciences, San Diego, CA) and further incubated for 16 h. PBMC were washed, surface-stained with appropriate antibodies, permeabilized with Cytofix/Cytoperm Kit (BD Biosciences), stained intracellularly with appropriate antibodies: IFN-γ (clone B27), TNF-α (clone Mab11), IL-2 (clone MQ1-17H12), Ki-67 (clone B56), Bcl-2 (clone Bcl-2/100) and fixed with 1% paraformaldehyde before acquisition on a FACSCalibur (BD BioSciences) or LSR-II system (BD Biosciences).

    Article Title: Postnatal Expansion, Maturation and Functionality of MR1T Cells in Humans
    Article Snippet: .. Flow cytometry assays To perform ICS, cryopreserved PBMC were thawed in the presence of DNAse, resuspended, in 10% heat inactivated human serum with RPMI (Lonza) at a concentration of 2 × 10^7/ml. .. PBMC (2 × 106 cells/well) were stimulated with anti-CD28 (1 μg/ml) and anti-CD49d (1 μg/ml) (FastImmune, BD Biosciences) and 1e5 M. smegmatis-infected or uninfected A549 cells in RPMI/10% Human Serum for 18 h at 37°C with 5% C02.

    Cytometry:

    Article Title: Distinct Effector Memory CD4+ T Cell Signatures in Latent Mycobacterium tuberculosis Infection, BCG Vaccination and Clinically Resolved Tuberculosis
    Article Snippet: Responses were scored as positive if the test wells contained a mean of at least 10 SFCs more than the mean of the negative control wells. .. Cell stimulation, staining and flow cytometry Before stimulation, cryopreserved PBMC were thawed and resuspended overnight at 37°C, 5% CO2 in RPMI-1640 medium (Lonza, Walkersville, MD) containing 10% FBS, 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. .. 1×106 PBMC were each stimulated with CW antigens (10 µg/ml) and ESAT-6 and CFP-10 peptides (10 µg/ml) at 37°C, 5% CO2 for 2 h followed by the addition of Brefeldin A (10 µg/ml) (BD Biosciences, San Diego, CA) and further incubated for 16 h. PBMC were washed, surface-stained with appropriate antibodies, permeabilized with Cytofix/Cytoperm Kit (BD Biosciences), stained intracellularly with appropriate antibodies: IFN-γ (clone B27), TNF-α (clone Mab11), IL-2 (clone MQ1-17H12), Ki-67 (clone B56), Bcl-2 (clone Bcl-2/100) and fixed with 1% paraformaldehyde before acquisition on a FACSCalibur (BD BioSciences) or LSR-II system (BD Biosciences).

    Activation Assay:

    Article Title: Porcupine Is Not Required for the Production of the Majority of Wnts from Primary Human Astrocytes and CD8+ T Cells
    Article Snippet: L-Wnt3a (ATCC CRL-2647) were cultured in complete DMEM with 0.4 mg/ml G-418. .. Culture and Activation of Human Peripheral Blood Mononuclear Cells (PBMCs) and Isolation of CD8+ T cells PBMCs were isolated from venous blood of healthy donors using lymphocyte separation medium (Lonza Biologics, Portsmouth, NH) and density centrifugation. .. PBMCs were then cultured at 1×106 cells mL of RPMI 1640, 10% heat-inactivated fetal bovine serum (FBS), 200 mM L-glutamine, and 1% penicillin/streptomycin (Lonza Biologics, Portsmouth, NH).

    Centrifugation:

    Article Title: Porcupine Is Not Required for the Production of the Majority of Wnts from Primary Human Astrocytes and CD8+ T Cells
    Article Snippet: L-Wnt3a (ATCC CRL-2647) were cultured in complete DMEM with 0.4 mg/ml G-418. .. Culture and Activation of Human Peripheral Blood Mononuclear Cells (PBMCs) and Isolation of CD8+ T cells PBMCs were isolated from venous blood of healthy donors using lymphocyte separation medium (Lonza Biologics, Portsmouth, NH) and density centrifugation. .. PBMCs were then cultured at 1×106 cells mL of RPMI 1640, 10% heat-inactivated fetal bovine serum (FBS), 200 mM L-glutamine, and 1% penicillin/streptomycin (Lonza Biologics, Portsmouth, NH).

    Concentration Assay:

    Article Title: Postnatal Expansion, Maturation and Functionality of MR1T Cells in Humans
    Article Snippet: .. Flow cytometry assays To perform ICS, cryopreserved PBMC were thawed in the presence of DNAse, resuspended, in 10% heat inactivated human serum with RPMI (Lonza) at a concentration of 2 × 10^7/ml. .. PBMC (2 × 106 cells/well) were stimulated with anti-CD28 (1 μg/ml) and anti-CD49d (1 μg/ml) (FastImmune, BD Biosciences) and 1e5 M. smegmatis-infected or uninfected A549 cells in RPMI/10% Human Serum for 18 h at 37°C with 5% C02.

    Knock-Out:

    Article Title: Efficient Generation of Isogenic Primary Human Myeloid Cells using CRISPR-Cas9 Ribonucleoproteins
    Article Snippet: Further, we confirmed consistently robust knockout across biological replicates at the genetic level by Tracking of Indels by DEcomposition (TIDE) analysis , showing that guides against CXCR4 and CCR5 led to disruption in greater than 90% of alleles ( ) . .. This protocol led to reproducible knockout when starting with CD14+ monocytes from freshly isolated peripheral blood mononuclear cells (PBMC), from cryopreserved PBMC, or from isolated-then-cryopreserved CD14+ monocytes , allowing for a flexible workflow and enabling iterative experiments on consistent biological samples. .. Collectively, these data demonstrate optimized knockout of targeted genes in primary human myeloid cells using CRISPR-Cas9 RNPs.

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    Lonza human pbmcs
    C. trachomatis infectivity from a cell culture of <t>ECC1,</t> human <t>PBMCs</t> and a co-culture of ECC1 and PBMCs, with or without azithromycin treatment. ECC1/PBMCs/co-culture of ECC1 and PBMCs were infected with C. trachomatis in MOI of 0.1. Cultures were either treated with azithromycin at 20 h PI, or not. The Chlamydia infected cells and culture supernatant were harvested a 44 h PI, sonicated and used to infect a new ECC1 monolayer for enumeration of recoverable IFUs. Data are presented as mean ± SD IFU/m ( n = 9) determinations
    Human Pbmcs, supplied by Lonza, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pbmc  (Lonza)
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    Lonza pbmc
    Functional analysis of MAIT cells in individuals of different ages. <t>PBMC</t> or CBMC from the US cohort were incubated overnight with M. smegmatis -infected A549 cells or uninfected A549 cells and then stained with the MR1-5-OP-RU or MR1/6FP tetramers, followed by staining with a live/dead discriminator and antibodies to TCRγδ, CD3, CD4, CD8, TRAV1-2, CD26 and CD161. <t>ICS</t> was then performed and the cells stained for TNF. A . Dot plots showing representative co-staining of live, TCRγδ - CD3 + TRAV1-2 + CD26 + CD161 + cells with MR1-5-OP-RU and TNF in M. smegmatis stimulated or unstimulated samples. The gating strategy is shown in Figure S3. Examples of the TNF response in a neonate, infant and adult are shown. B . Frequencies of functional, phenotypically-defined (CD3 + TRAV1-2 + CD26 + CD161 + ) MAIT cells that are MR1-5-OP-RU + or MR1-5-OP-RU - as a proportion of CD3 + TCRγδ - T cells. Horizontal lines depict the median and the error bars the 95% confidence interval. Wilcoxon-rank sum was used to test differences within the same cohort.
    Pbmc, supplied by Lonza, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbmc/product/Lonza
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    C. trachomatis infectivity from a cell culture of ECC1, human PBMCs and a co-culture of ECC1 and PBMCs, with or without azithromycin treatment. ECC1/PBMCs/co-culture of ECC1 and PBMCs were infected with C. trachomatis in MOI of 0.1. Cultures were either treated with azithromycin at 20 h PI, or not. The Chlamydia infected cells and culture supernatant were harvested a 44 h PI, sonicated and used to infect a new ECC1 monolayer for enumeration of recoverable IFUs. Data are presented as mean ± SD IFU/m ( n = 9) determinations

    Journal: BMC Infectious Diseases

    Article Title: High expression of IDO1 and TGF-β1 during recurrence and post infection clearance with Chlamydia trachomatis, are independent of host IFN-γ response

    doi: 10.1186/s12879-019-3843-4

    Figure Lengend Snippet: C. trachomatis infectivity from a cell culture of ECC1, human PBMCs and a co-culture of ECC1 and PBMCs, with or without azithromycin treatment. ECC1/PBMCs/co-culture of ECC1 and PBMCs were infected with C. trachomatis in MOI of 0.1. Cultures were either treated with azithromycin at 20 h PI, or not. The Chlamydia infected cells and culture supernatant were harvested a 44 h PI, sonicated and used to infect a new ECC1 monolayer for enumeration of recoverable IFUs. Data are presented as mean ± SD IFU/m ( n = 9) determinations

    Article Snippet: Experiments were conducted in 48-well plates, where co-culture of 40,000 ECC1 cells/well and 8 × 105 female human PBMCs (Lonza, Australia) cells/well were seeded with RPMI-1640 medium (Sigma-Aldrich, Australia) containing 10% heat inactivated FCS (Life Technologies, Australia), 120 μg/ml streptomycin (Sigma-Aldrich, Australia), 50 μg/ml Gentamycin (Gibco, Australia), 37 °C, 5% CO2 .

    Techniques: Infection, Cell Culture, Co-Culture Assay, Sonication

    Immunological, bacterial and biochemical factors that are associated with initial or repeated C. trachomatis infections in women. In the figure, the chlamydial developmental cycle is described. Infection is initiated with the chlamydial EBs that convert into RBs, multiply and convert back to EBs. Then, the infectious progeny is released from the host cell to initiate an additional cycle. Upon infection, immune cells are recruited to the infected area, among them CD4+ expressing Th1 cells that produce IFN-γ. IFN-γ induces the production of IDO1 that catabolizes tryptophan into kynurenine, depleting the host tryptophan pools. This triggers the tryptophan auxotroph Chlamydia to enter its persistence form, or in severe tryptophan starvation, to its death. Vaginal tract microbiota has an important role in health and disease. Among these bacterial communities, CST IV was associated with current or previous Chlamydia infection, low tryptophan levels and high kynurenine/tryptophan ratios. On the other hand, vaginal Lactobacillus crispatus was shown to inhibit chlamydial growth. Initial and repeated Chlamydia infections were associated with high kynurenine/tryptophan ratios. Repeated Chlamydia infection was shown to be associated with high kynurenine levels. Although low tryptophan levels were found to inhibit Chlamydia in vitro and were associated with natural clearance in vivo, tryptophan depletion is also related to the inhibition of Th1 immunity. Kynurenine and IDO1 are also known to inhibit T cells and local immunity. IDO1 and TGF-β1 are known to synergistically activate tolerogenic effect in pDCs. Azithromycin was shown to be effective in killing Chlamydia , however, also eliciting an anti-inflammatory response. Chlamydia infection clearance post azithromycin treatment in women was found to elicit IDO1, TGF-β1 and FoxP3 regulatory immune response. Repeated Chlamydia infection in women had similar effects on the expression levels of these genes, and might have been triggered by high kynurenine/tryptophan ratios. Chlamydia infection in in vitro ECC1 and PBMCs co-culture was found to elicit IDO1, TGF-β1 and FoxP3 as well

    Journal: BMC Infectious Diseases

    Article Title: High expression of IDO1 and TGF-β1 during recurrence and post infection clearance with Chlamydia trachomatis, are independent of host IFN-γ response

    doi: 10.1186/s12879-019-3843-4

    Figure Lengend Snippet: Immunological, bacterial and biochemical factors that are associated with initial or repeated C. trachomatis infections in women. In the figure, the chlamydial developmental cycle is described. Infection is initiated with the chlamydial EBs that convert into RBs, multiply and convert back to EBs. Then, the infectious progeny is released from the host cell to initiate an additional cycle. Upon infection, immune cells are recruited to the infected area, among them CD4+ expressing Th1 cells that produce IFN-γ. IFN-γ induces the production of IDO1 that catabolizes tryptophan into kynurenine, depleting the host tryptophan pools. This triggers the tryptophan auxotroph Chlamydia to enter its persistence form, or in severe tryptophan starvation, to its death. Vaginal tract microbiota has an important role in health and disease. Among these bacterial communities, CST IV was associated with current or previous Chlamydia infection, low tryptophan levels and high kynurenine/tryptophan ratios. On the other hand, vaginal Lactobacillus crispatus was shown to inhibit chlamydial growth. Initial and repeated Chlamydia infections were associated with high kynurenine/tryptophan ratios. Repeated Chlamydia infection was shown to be associated with high kynurenine levels. Although low tryptophan levels were found to inhibit Chlamydia in vitro and were associated with natural clearance in vivo, tryptophan depletion is also related to the inhibition of Th1 immunity. Kynurenine and IDO1 are also known to inhibit T cells and local immunity. IDO1 and TGF-β1 are known to synergistically activate tolerogenic effect in pDCs. Azithromycin was shown to be effective in killing Chlamydia , however, also eliciting an anti-inflammatory response. Chlamydia infection clearance post azithromycin treatment in women was found to elicit IDO1, TGF-β1 and FoxP3 regulatory immune response. Repeated Chlamydia infection in women had similar effects on the expression levels of these genes, and might have been triggered by high kynurenine/tryptophan ratios. Chlamydia infection in in vitro ECC1 and PBMCs co-culture was found to elicit IDO1, TGF-β1 and FoxP3 as well

    Article Snippet: Experiments were conducted in 48-well plates, where co-culture of 40,000 ECC1 cells/well and 8 × 105 female human PBMCs (Lonza, Australia) cells/well were seeded with RPMI-1640 medium (Sigma-Aldrich, Australia) containing 10% heat inactivated FCS (Life Technologies, Australia), 120 μg/ml streptomycin (Sigma-Aldrich, Australia), 50 μg/ml Gentamycin (Gibco, Australia), 37 °C, 5% CO2 .

    Techniques: Infection, Expressing, In Vitro, In Vivo, Inhibition, Co-Culture Assay

    IDO1, TGF-β1, FoxP3 and IFN-γ expression levels as a response to C. trachomatis infection, with or without azithromycin treatment. Transcript levels of IDO1, TGF-β1, FoxP3 and IFN-γ (2 - ΔCT ) were measured from cell cultures of ( a ) human endometrial cell line ECC1, ( b ) PBMCs of C. trachomatis negative female, and ( c ) co-culture of ECC1 and PBMCs. Transcript levels were compared between treatments; without Chlamydia infection or azithromycin treatment (−CT –AZ), no Chlamydia infection with azithromycin (−CT + AZ), with Chlamydia infection no azithromycin (+CT –AZ) and with Chlamydia infection and azithromycin treatment (+CT + AZ). Cells were infected with C. trachomatis at MOI of 0.1. Azithromycin was added to the culture at 20 h PI, or at 44 h post seeding in control treatments. Total RNA was isolated from cultures at 44 h PI. Data are presented as mean ± SD. Significant differences are indicated in the graph ( p

    Journal: BMC Infectious Diseases

    Article Title: High expression of IDO1 and TGF-β1 during recurrence and post infection clearance with Chlamydia trachomatis, are independent of host IFN-γ response

    doi: 10.1186/s12879-019-3843-4

    Figure Lengend Snippet: IDO1, TGF-β1, FoxP3 and IFN-γ expression levels as a response to C. trachomatis infection, with or without azithromycin treatment. Transcript levels of IDO1, TGF-β1, FoxP3 and IFN-γ (2 - ΔCT ) were measured from cell cultures of ( a ) human endometrial cell line ECC1, ( b ) PBMCs of C. trachomatis negative female, and ( c ) co-culture of ECC1 and PBMCs. Transcript levels were compared between treatments; without Chlamydia infection or azithromycin treatment (−CT –AZ), no Chlamydia infection with azithromycin (−CT + AZ), with Chlamydia infection no azithromycin (+CT –AZ) and with Chlamydia infection and azithromycin treatment (+CT + AZ). Cells were infected with C. trachomatis at MOI of 0.1. Azithromycin was added to the culture at 20 h PI, or at 44 h post seeding in control treatments. Total RNA was isolated from cultures at 44 h PI. Data are presented as mean ± SD. Significant differences are indicated in the graph ( p

    Article Snippet: Experiments were conducted in 48-well plates, where co-culture of 40,000 ECC1 cells/well and 8 × 105 female human PBMCs (Lonza, Australia) cells/well were seeded with RPMI-1640 medium (Sigma-Aldrich, Australia) containing 10% heat inactivated FCS (Life Technologies, Australia), 120 μg/ml streptomycin (Sigma-Aldrich, Australia), 50 μg/ml Gentamycin (Gibco, Australia), 37 °C, 5% CO2 .

    Techniques: Expressing, Infection, Co-Culture Assay, Isolation

    Bacillus Calmette–Guerin (BCG)-specific T SCM are associated with long-term CD4 + T cell proliferation after vaccination. Whole blood from 1-year-old infants ( n = 23) was stimulated with BCG for 12 h to measure the frequencies of cytokine-producing T SCM (CD45RA + , CCR7 + ), T CM (CD45RA − , CCR7 + ), and T EFF (CD45RA − , CCR7 − ). In parallel, whole blood was stimulated with BCG for 7 days, and the frequency of proliferating CD4 + cells was assessed by upregulation of Ki-67. Correlations between the frequencies of BCG-specific CD4 + memory T cell subsets and those of proliferating CD4 + T cells were calculated by Spearman test 10 months postvaccination.

    Journal: Frontiers in Immunology

    Article Title: Functional, Antigen-Specific Stem Cell Memory (TSCM) CD4+ T Cells Are Induced by Human Mycobacterium tuberculosis Infection

    doi: 10.3389/fimmu.2018.00324

    Figure Lengend Snippet: Bacillus Calmette–Guerin (BCG)-specific T SCM are associated with long-term CD4 + T cell proliferation after vaccination. Whole blood from 1-year-old infants ( n = 23) was stimulated with BCG for 12 h to measure the frequencies of cytokine-producing T SCM (CD45RA + , CCR7 + ), T CM (CD45RA − , CCR7 + ), and T EFF (CD45RA − , CCR7 − ). In parallel, whole blood was stimulated with BCG for 7 days, and the frequency of proliferating CD4 + cells was assessed by upregulation of Ki-67. Correlations between the frequencies of BCG-specific CD4 + memory T cell subsets and those of proliferating CD4 + T cells were calculated by Spearman test 10 months postvaccination.

    Article Snippet: Blood Processing and Stimulation for Intracellular Cytokine Staining Assay Peripheral blood mononuclear cells from adults were isolated by density gradient centrifugation (Ficoll histopaque, Lonza) from blood collected in sodium (Na)-heparin tubes (Greiner Bio-one) or heparinized blood bags.

    Techniques:

    Effects of Lactobacillus paracasei KW3110 on cell viability under blue light exposure conditions. ( A ) Lactobacillus paracasei LW3110 activated human M2 macrophages. Human peripheral blood mononuclear cells (PBMC)-derived M2 macrophages were treated with 10 μg/mL L. paracasei KW3110 or untreated (control) and cultured for 24 h. Interleukin 10 (IL-10) concentration of the culture medium was measured by ELISAs. ( B ) The inhibitory effect of L. paracasei KW3110-stimulated human PBMC-derived M2 macrophage supernatants (KW3110-sup) or vehicle-treated supernatants (vehicle sup) on blue light-induced retinal cell death in ARPE-19 cells as measured by propidium iodide (PI) staining. The number of cells exhibiting PI fluorescence was counted. PI positive cells were expressed as the ratio of PI-positive to Hoechst 33342-positive cells. Assays were performed in triplicate wells. The data shows the mean ± SD for triplicate wells. ** p

    Journal: Nutrients

    Article Title: Effect of Heat-Killed Lactobacillus paracasei KW3110 Ingestion on Ocular Disorders Caused by Visual Display Terminal (VDT) Loads: A Randomized, Double-Blind, Placebo-Controlled Parallel-Group Study

    doi: 10.3390/nu10081058

    Figure Lengend Snippet: Effects of Lactobacillus paracasei KW3110 on cell viability under blue light exposure conditions. ( A ) Lactobacillus paracasei LW3110 activated human M2 macrophages. Human peripheral blood mononuclear cells (PBMC)-derived M2 macrophages were treated with 10 μg/mL L. paracasei KW3110 or untreated (control) and cultured for 24 h. Interleukin 10 (IL-10) concentration of the culture medium was measured by ELISAs. ( B ) The inhibitory effect of L. paracasei KW3110-stimulated human PBMC-derived M2 macrophage supernatants (KW3110-sup) or vehicle-treated supernatants (vehicle sup) on blue light-induced retinal cell death in ARPE-19 cells as measured by propidium iodide (PI) staining. The number of cells exhibiting PI fluorescence was counted. PI positive cells were expressed as the ratio of PI-positive to Hoechst 33342-positive cells. Assays were performed in triplicate wells. The data shows the mean ± SD for triplicate wells. ** p

    Article Snippet: Preparation of Human PBMC-Derived M2 Macrophages and Culture Media Human peripheral blood mononuclear cells (PBMCs) from healthy donors were purchased from Lonza (Basel, Switzerland).

    Techniques: Derivative Assay, Cell Culture, Concentration Assay, Staining, Fluorescence

    Functional analysis of MAIT cells in individuals of different ages. PBMC or CBMC from the US cohort were incubated overnight with M. smegmatis -infected A549 cells or uninfected A549 cells and then stained with the MR1-5-OP-RU or MR1/6FP tetramers, followed by staining with a live/dead discriminator and antibodies to TCRγδ, CD3, CD4, CD8, TRAV1-2, CD26 and CD161. ICS was then performed and the cells stained for TNF. A . Dot plots showing representative co-staining of live, TCRγδ - CD3 + TRAV1-2 + CD26 + CD161 + cells with MR1-5-OP-RU and TNF in M. smegmatis stimulated or unstimulated samples. The gating strategy is shown in Figure S3. Examples of the TNF response in a neonate, infant and adult are shown. B . Frequencies of functional, phenotypically-defined (CD3 + TRAV1-2 + CD26 + CD161 + ) MAIT cells that are MR1-5-OP-RU + or MR1-5-OP-RU - as a proportion of CD3 + TCRγδ - T cells. Horizontal lines depict the median and the error bars the 95% confidence interval. Wilcoxon-rank sum was used to test differences within the same cohort.

    Journal: bioRxiv

    Article Title: Postnatal Expansion, Maturation and Functionality of MR1T Cells in Humans

    doi: 10.1101/2019.12.20.882746

    Figure Lengend Snippet: Functional analysis of MAIT cells in individuals of different ages. PBMC or CBMC from the US cohort were incubated overnight with M. smegmatis -infected A549 cells or uninfected A549 cells and then stained with the MR1-5-OP-RU or MR1/6FP tetramers, followed by staining with a live/dead discriminator and antibodies to TCRγδ, CD3, CD4, CD8, TRAV1-2, CD26 and CD161. ICS was then performed and the cells stained for TNF. A . Dot plots showing representative co-staining of live, TCRγδ - CD3 + TRAV1-2 + CD26 + CD161 + cells with MR1-5-OP-RU and TNF in M. smegmatis stimulated or unstimulated samples. The gating strategy is shown in Figure S3. Examples of the TNF response in a neonate, infant and adult are shown. B . Frequencies of functional, phenotypically-defined (CD3 + TRAV1-2 + CD26 + CD161 + ) MAIT cells that are MR1-5-OP-RU + or MR1-5-OP-RU - as a proportion of CD3 + TCRγδ - T cells. Horizontal lines depict the median and the error bars the 95% confidence interval. Wilcoxon-rank sum was used to test differences within the same cohort.

    Article Snippet: Flow cytometry assays To perform ICS, cryopreserved PBMC were thawed in the presence of DNAse, resuspended, in 10% heat inactivated human serum with RPMI (Lonza) at a concentration of 2 × 10^7/ml.

    Techniques: Functional Assay, Incubation, Infection, Staining

    Phenotypic analysis of functional MR1T cells at different ages. PBMC or CBMC from the US cohort were incubated overnight with M. smegmatis -infected A549 cells, uninfected A549 cells, or uninfected A549 cells and PMA/ionomycin. All cells were then stained with the MR1-5-OP-RU or MR1-6FP tetramers, followed by a live/dead discriminator and antibodies to TCRγδ, CD3, CD4, CD8, TRAV1-2, CD26 and CD161. ICS was then performed and the cells stained for TNF. Live, TCRγδ - CD3 + MR1-5-OP-RU + cells were gated and the TNF + percentage determined in both the M. smegmatis stimulated and unstimulated conditions. The gating strategy is shown in Figure S3. A . Dot plots showing TNF expression by live, TCRγδ - MR1-5-OP-RU + CD3 + cells in the same representative neonate, infant and adult as Figure 2A . B . Background subtracted frequencies of TNF + MR1-5-OP-RU + CD3 + cells in response to M. smegmatis -infected A549 cells as a percentage of total MR1/5-OP-RU + CD3 + cells are shown. Mann-Whitney u-tests were used to test differences between groups. C . Background subtracted frequencies of TNF + cells to PMA/ionomycin among different T cell subpopulations, 1) MR1T cells (CD3 + TCRγδ - MR1-5-OP-RU + cells); 2) γδ T cells (CD3 + TCRγδ + MR1-5-OP-RU - cells); 3) CD8 + T cells (CD3 + TCRγδ - MR1-5-OP-RU - CD8 + cells); and 4) CD4 + T cells (CD3 + TCRγδ - MR1-5-OP-RU - CD4 + cells). Wilcoxon-rank sum was used to test differences within the same cohort. D . and E . Frequencies of TNF + TRAV1-2 + MR1-5-OP-RU + CD3 + and TNF + TRAV1-2 - MR1-5-OP-RU + CD3 + cells in response to M. smegmatis infected A549 cells ( D ) or PMA/ionomycin stimulation ( E ) minus the background frequencies of TNF + TRAV1-2 + /- MR1-5-OP-RU + CD3 + cells. Wilcoxon-rank sum was used to test differences within the same cohort.

    Journal: bioRxiv

    Article Title: Postnatal Expansion, Maturation and Functionality of MR1T Cells in Humans

    doi: 10.1101/2019.12.20.882746

    Figure Lengend Snippet: Phenotypic analysis of functional MR1T cells at different ages. PBMC or CBMC from the US cohort were incubated overnight with M. smegmatis -infected A549 cells, uninfected A549 cells, or uninfected A549 cells and PMA/ionomycin. All cells were then stained with the MR1-5-OP-RU or MR1-6FP tetramers, followed by a live/dead discriminator and antibodies to TCRγδ, CD3, CD4, CD8, TRAV1-2, CD26 and CD161. ICS was then performed and the cells stained for TNF. Live, TCRγδ - CD3 + MR1-5-OP-RU + cells were gated and the TNF + percentage determined in both the M. smegmatis stimulated and unstimulated conditions. The gating strategy is shown in Figure S3. A . Dot plots showing TNF expression by live, TCRγδ - MR1-5-OP-RU + CD3 + cells in the same representative neonate, infant and adult as Figure 2A . B . Background subtracted frequencies of TNF + MR1-5-OP-RU + CD3 + cells in response to M. smegmatis -infected A549 cells as a percentage of total MR1/5-OP-RU + CD3 + cells are shown. Mann-Whitney u-tests were used to test differences between groups. C . Background subtracted frequencies of TNF + cells to PMA/ionomycin among different T cell subpopulations, 1) MR1T cells (CD3 + TCRγδ - MR1-5-OP-RU + cells); 2) γδ T cells (CD3 + TCRγδ + MR1-5-OP-RU - cells); 3) CD8 + T cells (CD3 + TCRγδ - MR1-5-OP-RU - CD8 + cells); and 4) CD4 + T cells (CD3 + TCRγδ - MR1-5-OP-RU - CD4 + cells). Wilcoxon-rank sum was used to test differences within the same cohort. D . and E . Frequencies of TNF + TRAV1-2 + MR1-5-OP-RU + CD3 + and TNF + TRAV1-2 - MR1-5-OP-RU + CD3 + cells in response to M. smegmatis infected A549 cells ( D ) or PMA/ionomycin stimulation ( E ) minus the background frequencies of TNF + TRAV1-2 + /- MR1-5-OP-RU + CD3 + cells. Wilcoxon-rank sum was used to test differences within the same cohort.

    Article Snippet: Flow cytometry assays To perform ICS, cryopreserved PBMC were thawed in the presence of DNAse, resuspended, in 10% heat inactivated human serum with RPMI (Lonza) at a concentration of 2 × 10^7/ml.

    Techniques: Functional Assay, Incubation, Infection, Staining, Expressing, MANN-WHITNEY

    Frequencies of tetramer-defined MR1T cell and phenotypically defined MAIT cell populations in peripheral blood from individuals of different ages. PBMC or CBMC were stained with a live/dead discriminator, antibodies to CD3, CD4, CD8, TRAV1-2, CD26, CD161, and either the MR1-5-OP-RU or MR1-6FP tetramers. Live, CD3 + lymphocytes were gated and the frequencies of MR1-5-OP-RU + or TRAV1-2 + CD26 + CD161 + cells as a percentage of CD3 + lymphocytes were determined (gating strategy in Figure S2). A . Flow cytometry plots showing CD3 + T cell staining with MR1-5-OP-RU tetramer, MR1-6FP tetramer, TRAV1-2 or CD26/CD161 of samples from a representative US adult, infant and neonate. B . Frequencies of tetramer-defined (CD3 + MR1-5-OP-RU + ) and phenotypically-defined (CD3 + TRAV1-2 + CD26 + CD161 + ) MAIT cells in neonates, 10 week-old infants and adolescents from South Africa, neonates, 12 month-old infants and adults from the United States and infants (0-2 years old), children (2-5 years old) and adults from Uganda (all cohorts, n =10). Mann-Whitney u-tests were used to test differences between groups. Horizontal lines depict the median and the error bars the 95% confidence interval. C . Relative proportions of median phenotypically-defined (CD3 + TRAV1-2 + CD26 + CD161 + ) MAIT cells that are MR1-5-OP-RU + (Blue) and MR1-5-OP-RU - (Red shades) for each age group at each site. In each doughnut chart the MR1-5-OP-RU - population is further divided into CD8 + , CD8 + CD4 + , CD4 + , and CD8 - CD4 - populations.

    Journal: bioRxiv

    Article Title: Postnatal Expansion, Maturation and Functionality of MR1T Cells in Humans

    doi: 10.1101/2019.12.20.882746

    Figure Lengend Snippet: Frequencies of tetramer-defined MR1T cell and phenotypically defined MAIT cell populations in peripheral blood from individuals of different ages. PBMC or CBMC were stained with a live/dead discriminator, antibodies to CD3, CD4, CD8, TRAV1-2, CD26, CD161, and either the MR1-5-OP-RU or MR1-6FP tetramers. Live, CD3 + lymphocytes were gated and the frequencies of MR1-5-OP-RU + or TRAV1-2 + CD26 + CD161 + cells as a percentage of CD3 + lymphocytes were determined (gating strategy in Figure S2). A . Flow cytometry plots showing CD3 + T cell staining with MR1-5-OP-RU tetramer, MR1-6FP tetramer, TRAV1-2 or CD26/CD161 of samples from a representative US adult, infant and neonate. B . Frequencies of tetramer-defined (CD3 + MR1-5-OP-RU + ) and phenotypically-defined (CD3 + TRAV1-2 + CD26 + CD161 + ) MAIT cells in neonates, 10 week-old infants and adolescents from South Africa, neonates, 12 month-old infants and adults from the United States and infants (0-2 years old), children (2-5 years old) and adults from Uganda (all cohorts, n =10). Mann-Whitney u-tests were used to test differences between groups. Horizontal lines depict the median and the error bars the 95% confidence interval. C . Relative proportions of median phenotypically-defined (CD3 + TRAV1-2 + CD26 + CD161 + ) MAIT cells that are MR1-5-OP-RU + (Blue) and MR1-5-OP-RU - (Red shades) for each age group at each site. In each doughnut chart the MR1-5-OP-RU - population is further divided into CD8 + , CD8 + CD4 + , CD4 + , and CD8 - CD4 - populations.

    Article Snippet: Flow cytometry assays To perform ICS, cryopreserved PBMC were thawed in the presence of DNAse, resuspended, in 10% heat inactivated human serum with RPMI (Lonza) at a concentration of 2 × 10^7/ml.

    Techniques: Staining, Flow Cytometry, MANN-WHITNEY