pbmc  (Lonza)


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    Name:
    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
    Price:
    None
    Category:
    Primary and Stem Cells
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    Structured Review

    Lonza pbmc
    Characterization of Mtb antigen-specific memory CD4 + T cells in LTBI and BCG individuals using multiparameter flow <t>cytometry.</t> (A) Immunophenotyping of memory CD4 + T cells from LTBI and BCG individuals. Data from two representative donors are shown. The indicated cell surface markers on CW-stimulated <t>PBMC</t> gated on total CD4 + T cells were assessed by flow cytometry (black) and on IFN-γ producing cells were assayed by intracellular staining (red). (B) Graphical summary of immunophenotypic analyses. The expression of each marker is expressed as a percentage of the total antigen-specific IFN-γ + CD4 + T cells in LTBI (n = 18) and BCG (n = 18) groups (C). Expression of CD27 on antigen-specific memory CD4 + T cells from LTBI (n = 18) and BCG (n = 18). The percentage of antigen-specific CD4 + T cells is significantly different in LTBI and BCG groups (p
    Cryopreserved ampule of Human Peripheral Blood Mononuclear Cells PBMC containing 50 million cells
    https://www.bioz.com/result/pbmc/product/Lonza
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pbmc - by Bioz Stars, 2021-03
    86/100 stars

    Images

    1) Product Images from "Distinct Effector Memory CD4+ T Cell Signatures in Latent Mycobacterium tuberculosis Infection, BCG Vaccination and Clinically Resolved Tuberculosis"

    Article Title: Distinct Effector Memory CD4+ T Cell Signatures in Latent Mycobacterium tuberculosis Infection, BCG Vaccination and Clinically Resolved Tuberculosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036046

    Characterization of Mtb antigen-specific memory CD4 + T cells in LTBI and BCG individuals using multiparameter flow cytometry. (A) Immunophenotyping of memory CD4 + T cells from LTBI and BCG individuals. Data from two representative donors are shown. The indicated cell surface markers on CW-stimulated PBMC gated on total CD4 + T cells were assessed by flow cytometry (black) and on IFN-γ producing cells were assayed by intracellular staining (red). (B) Graphical summary of immunophenotypic analyses. The expression of each marker is expressed as a percentage of the total antigen-specific IFN-γ + CD4 + T cells in LTBI (n = 18) and BCG (n = 18) groups (C). Expression of CD27 on antigen-specific memory CD4 + T cells from LTBI (n = 18) and BCG (n = 18). The percentage of antigen-specific CD4 + T cells is significantly different in LTBI and BCG groups (p
    Figure Legend Snippet: Characterization of Mtb antigen-specific memory CD4 + T cells in LTBI and BCG individuals using multiparameter flow cytometry. (A) Immunophenotyping of memory CD4 + T cells from LTBI and BCG individuals. Data from two representative donors are shown. The indicated cell surface markers on CW-stimulated PBMC gated on total CD4 + T cells were assessed by flow cytometry (black) and on IFN-γ producing cells were assayed by intracellular staining (red). (B) Graphical summary of immunophenotypic analyses. The expression of each marker is expressed as a percentage of the total antigen-specific IFN-γ + CD4 + T cells in LTBI (n = 18) and BCG (n = 18) groups (C). Expression of CD27 on antigen-specific memory CD4 + T cells from LTBI (n = 18) and BCG (n = 18). The percentage of antigen-specific CD4 + T cells is significantly different in LTBI and BCG groups (p

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

    2) Product Images from "Peripheral Blood TIM-3 Positive NK and CD8+ T Cells throughout Pregnancy: TIM-3/Galectin-9 Interaction and Its Possible Role during Pregnancy"

    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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0092371

    CD107a expression by peripheral blood mononuclear cell subsets throughout pregnancy and in non-pregnant women. The expression of CD107a by TIM-3 negative and TIM-3 positive cytotoxic T cells and NK cells in the peripheral blood of normal pregnant women during each trimester of pregnancy and in non-pregnant women. The solid bars represent medians, the boxes indicate the interquartile ranges and the lines show the most extreme observations. Differences were considered statistically significant for P-values ≤0.05.
    Figure Legend Snippet: CD107a expression by peripheral blood mononuclear cell subsets throughout pregnancy and in non-pregnant women. The expression of CD107a by TIM-3 negative and TIM-3 positive cytotoxic T cells and NK cells in the peripheral blood of normal pregnant women during each trimester of pregnancy and in non-pregnant women. The solid bars represent medians, the boxes indicate the interquartile ranges and the lines show the most extreme observations. Differences were considered statistically significant for P-values ≤0.05.

    Techniques Used: Expressing

    CD107a expression by peripheral blood mononuclear cell subsets throughout pregnancy and in non-pregnant women. The expression of CD107a by TIM-3 negative and TIM-3 positive CD56dim and CD56bright cells in the peripheral blood of normal pregnant women during each trimester of pregnancy and in non-pregnant women. The solid bars represent medians, the boxes indicate the interquartile ranges and the lines show the most extreme observations. Differences were considered statistically significant for P-values ≤0.05.
    Figure Legend Snippet: CD107a expression by peripheral blood mononuclear cell subsets throughout pregnancy and in non-pregnant women. The expression of CD107a by TIM-3 negative and TIM-3 positive CD56dim and CD56bright cells in the peripheral blood of normal pregnant women during each trimester of pregnancy and in non-pregnant women. The solid bars represent medians, the boxes indicate the interquartile ranges and the lines show the most extreme observations. Differences were considered statistically significant for P-values ≤0.05.

    Techniques Used: Expressing

    3) Product Images from "Dimethyl fumarate–induced lymphopenia in MS due to differential T-cell subset apoptosis"

    Article Title: Dimethyl fumarate–induced lymphopenia in MS due to differential T-cell subset apoptosis

    Journal: Neurology® Neuroimmunology & Neuroinflammation

    doi: 10.1212/NXI.0000000000000340

    DMF causes T-cell apoptosis in vitro with a preferential effect on CD8 + T cells Healthy control (n = 10) PBMC were cultured with the addition of DMF, MMF, vehicle alone, or medium alone. Total CD3 + , CD4 + , and CD8 + T-cell subsets showed a dose-dependent decrease in survival after the addition of DMF, whereas MMF and vehicle alone had no effect (A). The proportion of apoptotic cells within CD3 + , CD4 + , and CD8 + subsets increased with increasing DMF exposure (B). Relative to viability in untreated cultures, there was significantly greater DMF-induced loss of viability among CD8 + vs CD4 + T cells following a 25μM DMF exposure (C). The pattern of DMF-induced apoptosis of CD3 + , CD4 + , and CD8 + T cells was also seen in purified T-cell cultures (n = 4) (D). Statistical analyses used were a 2-way repeated measures ANOVA with adjustment for multiple comparisons using Dunnett test (A, B, and D) and a paired t test (C). ANOVA = analysis of variance; DMF = dimethyl fumarate; MMF = monomethyl fumarate; PBMC = peripheral blood mononuclear cells; UnTx = untreated; Veh = vehicle alone.
    Figure Legend Snippet: DMF causes T-cell apoptosis in vitro with a preferential effect on CD8 + T cells Healthy control (n = 10) PBMC were cultured with the addition of DMF, MMF, vehicle alone, or medium alone. Total CD3 + , CD4 + , and CD8 + T-cell subsets showed a dose-dependent decrease in survival after the addition of DMF, whereas MMF and vehicle alone had no effect (A). The proportion of apoptotic cells within CD3 + , CD4 + , and CD8 + subsets increased with increasing DMF exposure (B). Relative to viability in untreated cultures, there was significantly greater DMF-induced loss of viability among CD8 + vs CD4 + T cells following a 25μM DMF exposure (C). The pattern of DMF-induced apoptosis of CD3 + , CD4 + , and CD8 + T cells was also seen in purified T-cell cultures (n = 4) (D). Statistical analyses used were a 2-way repeated measures ANOVA with adjustment for multiple comparisons using Dunnett test (A, B, and D) and a paired t test (C). ANOVA = analysis of variance; DMF = dimethyl fumarate; MMF = monomethyl fumarate; PBMC = peripheral blood mononuclear cells; UnTx = untreated; Veh = vehicle alone.

    Techniques Used: In Vitro, Cell Culture, Purification

    Memory T-cell subsets and conventional T-cell subsets are most susceptible to in vitro DMF-induced apoptosis Healthy control (n = 10) peripheral blood mononuclear cells were cultured with the addition of DMF, MMF, vehicle alone, or medium alone. All CD8 + (A) and CD4 + (B) naive and memory subsets underwent a degree of DMF-induced apoptosis, whereas MMF and vehicle alone had no effect. Relative to viability in untreated cultures, there was significantly greater DMF-induced loss of cell viability among memory vs naive CD8 + (C) and CD4 + T cells (D) following a 25μM DMF exposure, as well as among conventional vs regulatory CD4 + T cells following a 50μM DMF exposure (E). Statistical analyses used were repeated measures 2-way ANOVA with adjustment for multiple comparisons using Dunnett test (A and B), repeated measures 1-way ANOVA with Sidak multiple comparisons test (C and D), and a paired t test (E). ANOVA = analysis of variance; DMF = dimethyl fumarate; MMF = monomethyl fumarate; TCM = central memory T-cells; Tconv = conventional T-cells; TEM = effector memory T-cells; TEMRA = terminally differentiated effector memory T-cells; TN = naïve T-cells; Treg = regulatory T-cells; UnTx = untreated; Veh = vehicle alone.
    Figure Legend Snippet: Memory T-cell subsets and conventional T-cell subsets are most susceptible to in vitro DMF-induced apoptosis Healthy control (n = 10) peripheral blood mononuclear cells were cultured with the addition of DMF, MMF, vehicle alone, or medium alone. All CD8 + (A) and CD4 + (B) naive and memory subsets underwent a degree of DMF-induced apoptosis, whereas MMF and vehicle alone had no effect. Relative to viability in untreated cultures, there was significantly greater DMF-induced loss of cell viability among memory vs naive CD8 + (C) and CD4 + T cells (D) following a 25μM DMF exposure, as well as among conventional vs regulatory CD4 + T cells following a 50μM DMF exposure (E). Statistical analyses used were repeated measures 2-way ANOVA with adjustment for multiple comparisons using Dunnett test (A and B), repeated measures 1-way ANOVA with Sidak multiple comparisons test (C and D), and a paired t test (E). ANOVA = analysis of variance; DMF = dimethyl fumarate; MMF = monomethyl fumarate; TCM = central memory T-cells; Tconv = conventional T-cells; TEM = effector memory T-cells; TEMRA = terminally differentiated effector memory T-cells; TN = naïve T-cells; Treg = regulatory T-cells; UnTx = untreated; Veh = vehicle alone.

    Techniques Used: In Vitro, Cell Culture, Transmission Electron Microscopy

    4) Product Images from "Assessing Anti-HCMV Cell Mediated Immune Responses in Transplant Recipients and Healthy Controls Using a Novel Functional Assay"

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

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2020.00275

    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
    Figure Legend 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

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

    Related Articles

    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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    • 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
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    human pbmcs - by Bioz Stars, 2021-03
    94/100 stars
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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.
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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