peripheral blood mononuclear cells mncs  (Lonza)


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    Name:
    Human Peripheral Blood Mononuclear Cells
    Description:
    Mononuclear cells from human peripheral blood cryopreserved 10 million cells
    Catalog Number:
    4w-270
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    None
    Category:
    Primary and Stem Cells
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    Lonza peripheral blood mononuclear cells mncs
    Mononuclear cells from human peripheral blood cryopreserved 10 million cells
    https://www.bioz.com/result/peripheral blood mononuclear cells mncs/product/Lonza
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    peripheral blood mononuclear cells mncs - by Bioz Stars, 2021-03
    94/100 stars

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    Co-Culture Assay:

    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
    Article Snippet: ECC1 is an endometrial primary cell line, responsive to hormones and to C. trachomatis infections, was previously used in infectivity assays and similar co-culture models [ , ]. .. 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 . .. In addition, control plates with either ECC1 cells alone or human PBMCs alone were used.

    Isolation:

    Article Title: P2X7 Receptor Induces Tumor Necrosis Factor-α Converting Enzyme Activation and Release to Boost TNF-α Production
    Article Snippet: .. Human peripheral blood mononuclear cells were isolated following standard procedure ( ) and cultured for 16 h in RPMI 1640 medium (Lonza) with 10% of FCS, 2 mM Glutamax, and 100 U/ml penicillin–streptomycin (Life Technologies). .. After monocyte adherence, cells were washed and primed for 4 h with LPS (10 ng/ml), and then cells were washed or not with physiological buffer and incubated in the same buffer at 37°C with 3 mM of ATP for 20 min.

    Article Title: Kv1.3 Channel Blockade Modulates the Effector Function of B Cells in Granulomatosis with Polyangiitis
    Article Snippet: Induction and Measurement of Total and PR3-ANCA-Specific IgG Cell culture and quantification of total and PR3-ANCA IgG was performed as previously described by our group ( ). .. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from GPA patients, who produce ANCA upon in vitro induction , and stored in RPMI 1640 (Lonza, Basel, Switzerland) supplemented with 50 µg/mL gentamycin (GIBCO, Life Technologies, Grand Island, NY, USA), 10% fetal calf serum (FCS, Lonza), and 10% dimethyl sulfoxide (DMSO). ..

    Cell Culture:

    Article Title: P2X7 Receptor Induces Tumor Necrosis Factor-α Converting Enzyme Activation and Release to Boost TNF-α Production
    Article Snippet: .. Human peripheral blood mononuclear cells were isolated following standard procedure ( ) and cultured for 16 h in RPMI 1640 medium (Lonza) with 10% of FCS, 2 mM Glutamax, and 100 U/ml penicillin–streptomycin (Life Technologies). .. After monocyte adherence, cells were washed and primed for 4 h with LPS (10 ng/ml), and then cells were washed or not with physiological buffer and incubated in the same buffer at 37°C with 3 mM of ATP for 20 min.

    other:

    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
    Article Snippet: In addition, control plates with either ECC1 cells alone or human PBMCs alone were used.

    Article Title: Vasoreparative Dysfunction of CD34+ Cells in Diabetic Individuals Involves Hypoxic Desensitization and Impaired Autocrine/Paracrine Mechanisms
    Article Snippet: Culturing ECFCs and eEPCs For ECFCs, peripheral blood MNCs were suspended in EBM-2 medium supplemented with EGM-2MV single quotes (Lonza).

    In Vitro:

    Article Title: Kv1.3 Channel Blockade Modulates the Effector Function of B Cells in Granulomatosis with Polyangiitis
    Article Snippet: Induction and Measurement of Total and PR3-ANCA-Specific IgG Cell culture and quantification of total and PR3-ANCA IgG was performed as previously described by our group ( ). .. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from GPA patients, who produce ANCA upon in vitro induction , and stored in RPMI 1640 (Lonza, Basel, Switzerland) supplemented with 50 µg/mL gentamycin (GIBCO, Life Technologies, Grand Island, NY, USA), 10% fetal calf serum (FCS, Lonza), and 10% dimethyl sulfoxide (DMSO). ..

    Infection:

    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
    Article Snippet: No chlamydial inclusions were found in any of the cultures treated with azithromycin (either ECC1, PBMCs or ECC1 and PBMCs co-culture) (Fig. ) . .. In addition, infection of human PBMCs alone with live C. trachomatis , without the azithromycin treatment, resulted in significantly reduced, yet apparent infectivity (p < 0.0001). .. The number of chlamydial IFUs measured from ECC1 and PBMCs co-culture was significantly reduced in comparison to infected ECC1 culture alone (p < 0.0001) by almost two logs.

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    • human pbmcs  (Lonza)
    94
    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
    https://www.bioz.com/result/human pbmcs/product/Lonza
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human pbmcs - by Bioz Stars, 2021-03
    94/100 stars
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    pbmc  (Lonza)
    95
    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
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pbmc - by Bioz Stars, 2021-03
    95/100 stars
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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:

    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

    Analysis of HCMV specific IFNγ FluoroSpot responses and antiviral activity of HCMV peptide stimulated supernatants with and without IFNγ depletion. (A) IFNγ FluoroSpot responses to HCMV peptide pools covering pp65 and UL144, IE1 and IE2, pp71 and US3, and gB, as well as a polyclonal anti-CD3/CD28 antibody T cell stimulation as a positive control of PBMC from donor CMV1801, calculated as spot-forming units (SFU) per 10 6 PBMC (background corrected). (B) IFNγ FluoroSpot responses to HCMV peptide pools covering pp65 and UL144, IE1 and IE2, pp71 and US3, and gB, as well as a polyclonal anti-CD3/CD28 antibody T cell stimulation as a positive control of PBMC from donor CMV332, calculated as spot-forming units (SFU) per 10 6 PBMC (background corrected). (C) The IFNγ concentration of supernatants following peptide stimulation (black) or after IFNγ depletion by anti-IFNγ-coated FluoroSpot (cyan), measured by ELISA. (D) The effect of IFNγ depletion on the antiviral activity of PBMC from donor CMV1801 stimulated with HCMV peptide pools for pp65/UL144, IE1 2, and pp71/US3 or anti-CD3/CD28 antibody. Bars labeled “IFNγ deplete” were harvested from anti-IFNγ antibody-coated FluoroSpot plates and added to a VDA in parallel with supernatants generated with the same stimulants and PBMC cell number. Significance determined by one-tailed T -test. Key: * p

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Assessing Anti-HCMV Cell Mediated Immune Responses in Transplant Recipients and Healthy Controls Using a Novel Functional Assay

    doi: 10.3389/fcimb.2020.00275

    Figure Lengend Snippet: Analysis of HCMV specific IFNγ FluoroSpot responses and antiviral activity of HCMV peptide stimulated supernatants with and without IFNγ depletion. (A) IFNγ FluoroSpot responses to HCMV peptide pools covering pp65 and UL144, IE1 and IE2, pp71 and US3, and gB, as well as a polyclonal anti-CD3/CD28 antibody T cell stimulation as a positive control of PBMC from donor CMV1801, calculated as spot-forming units (SFU) per 10 6 PBMC (background corrected). (B) IFNγ FluoroSpot responses to HCMV peptide pools covering pp65 and UL144, IE1 and IE2, pp71 and US3, and gB, as well as a polyclonal anti-CD3/CD28 antibody T cell stimulation as a positive control of PBMC from donor CMV332, calculated as spot-forming units (SFU) per 10 6 PBMC (background corrected). (C) The IFNγ concentration of supernatants following peptide stimulation (black) or after IFNγ depletion by anti-IFNγ-coated FluoroSpot (cyan), measured by ELISA. (D) The effect of IFNγ depletion on the antiviral activity of PBMC from donor CMV1801 stimulated with HCMV peptide pools for pp65/UL144, IE1 2, and pp71/US3 or anti-CD3/CD28 antibody. Bars labeled “IFNγ deplete” were harvested from anti-IFNγ antibody-coated FluoroSpot plates and added to a VDA in parallel with supernatants generated with the same stimulants and PBMC cell number. Significance determined by one-tailed T -test. Key: * p

    Article Snippet: Detection of Cytokine Production by FluoroSpot To maximize available cell numbers, 1 × 105 total PBMC were suspended in X-VIVO 15 (Lonza, Slough, UK) supplemented with 5% Human AB serum (Sigma Aldrich).

    Techniques: Activity Assay, Cell Stimulation, Positive Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Labeling, Generated, One-tailed Test