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Ficoll-Paque Pharmacia pbmcs
Platelets Are Critical for the Production of IL-1 Cytokines by Human Primary Monocytes (A) Representative FACS dot plots of CD41 and <t>CD14</t> expression in human <t>PBMCs,</t> standard (Std-Mo), or platelet-depleted (PD-Mo) CD14 + monocytes (see STAR Methods ). (B) Quantification of platelets (CD41 + CD14 − ), platelet-monocyte aggregates (CD41 + CD14 + ), and platelet-free monocytes (CD41 − CD14 + ) in PBMCs and isolated monocytes. (C) IL-1β and TNF-α levels in cell-free supernatants of Std-Mo versus PD-Mo, or in platelet-depleted monocytes replenished with autologous platelets (PD-Mo + Plts, 50:1 Ptl:Cell), and stimulated as indicated. (D and E) IL-1β levels released by non-canonically activated Std-Mo, PD-Mo, or PD-Mo + Plts, stimulated with LPS (1 μg mL −1 ) for 16 h (D) or by inflammasome-activated THP-1 s ± platelets (E). Data is presented as floating bars (with mean and minimum to maximum values) and pooled from independent experiments. Each symbol represents the average from technical triplicates per blood donor. Data in (D) shows bar graph with Mean + SD, from a representative of two independent experiments. See also Figure S2 .
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Images

1) Product Images from "Platelets Fuel the Inflammasome Activation of Innate Immune Cells"

Article Title: Platelets Fuel the Inflammasome Activation of Innate Immune Cells

Journal: Cell Reports

doi: 10.1016/j.celrep.2020.107615

Platelets Are Critical for the Production of IL-1 Cytokines by Human Primary Monocytes (A) Representative FACS dot plots of CD41 and CD14 expression in human PBMCs, standard (Std-Mo), or platelet-depleted (PD-Mo) CD14 + monocytes (see STAR Methods ). (B) Quantification of platelets (CD41 + CD14 − ), platelet-monocyte aggregates (CD41 + CD14 + ), and platelet-free monocytes (CD41 − CD14 + ) in PBMCs and isolated monocytes. (C) IL-1β and TNF-α levels in cell-free supernatants of Std-Mo versus PD-Mo, or in platelet-depleted monocytes replenished with autologous platelets (PD-Mo + Plts, 50:1 Ptl:Cell), and stimulated as indicated. (D and E) IL-1β levels released by non-canonically activated Std-Mo, PD-Mo, or PD-Mo + Plts, stimulated with LPS (1 μg mL −1 ) for 16 h (D) or by inflammasome-activated THP-1 s ± platelets (E). Data is presented as floating bars (with mean and minimum to maximum values) and pooled from independent experiments. Each symbol represents the average from technical triplicates per blood donor. Data in (D) shows bar graph with Mean + SD, from a representative of two independent experiments. See also Figure S2 .
Figure Legend Snippet: Platelets Are Critical for the Production of IL-1 Cytokines by Human Primary Monocytes (A) Representative FACS dot plots of CD41 and CD14 expression in human PBMCs, standard (Std-Mo), or platelet-depleted (PD-Mo) CD14 + monocytes (see STAR Methods ). (B) Quantification of platelets (CD41 + CD14 − ), platelet-monocyte aggregates (CD41 + CD14 + ), and platelet-free monocytes (CD41 − CD14 + ) in PBMCs and isolated monocytes. (C) IL-1β and TNF-α levels in cell-free supernatants of Std-Mo versus PD-Mo, or in platelet-depleted monocytes replenished with autologous platelets (PD-Mo + Plts, 50:1 Ptl:Cell), and stimulated as indicated. (D and E) IL-1β levels released by non-canonically activated Std-Mo, PD-Mo, or PD-Mo + Plts, stimulated with LPS (1 μg mL −1 ) for 16 h (D) or by inflammasome-activated THP-1 s ± platelets (E). Data is presented as floating bars (with mean and minimum to maximum values) and pooled from independent experiments. Each symbol represents the average from technical triplicates per blood donor. Data in (D) shows bar graph with Mean + SD, from a representative of two independent experiments. See also Figure S2 .

Techniques Used: FACS, Expressing, Isolation

2) Product Images from "Bovine Leukemia Virus Structural Gene Vectors Are Immunogenic and Lack Pathogenicity in a Rabbit Model"

Article Title: Bovine Leukemia Virus Structural Gene Vectors Are Immunogenic and Lack Pathogenicity in a Rabbit Model

Journal: Journal of Virology

doi:

Semiquantitative PCR analysis to evaluate differences in proviral load. (A) PCR to detect BLV pol in PBMC from treated rabbits. Rabbit PBMC were harvested at 1, 4, and 10 months postinoculation and subjected to PCR with BLV pol primers KB560 and KB561 (10 × 10 4 PBMC) or β- actin to control for sample variation (2 × 10 4 PBMC). Each panel is labeled with month of sample harvest. Lanes are labeled with the source of PBMC DNA by rabbit number and treatment, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). In lower panels, the corresponding rabbit samples are designated by the matching parallel black lines. Positions of BLV pol amplicon (591 bp) and β- actin amplicon (594 bp) are designated. *, not determined (ND) because sample was not available; †, not determined because the animal died before harvest. (B) PCR standard curve. PBMC lysate harvested at 1 month postinoculation from BLV rabbit 44-10 was serially diluted in a range of 5 × 10 2 to 5 × 10 4 PBMC and subjected to PCR with BLV pol primers KB560 and KB561. Each lane is labeled with the number of cells used for PCR amplification, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). The arrow on the left indicates the position of BLV pol amplicon (591 bp), and the lines on the right indicate positions of 676- and 571-bp DNA size markers.
Figure Legend Snippet: Semiquantitative PCR analysis to evaluate differences in proviral load. (A) PCR to detect BLV pol in PBMC from treated rabbits. Rabbit PBMC were harvested at 1, 4, and 10 months postinoculation and subjected to PCR with BLV pol primers KB560 and KB561 (10 × 10 4 PBMC) or β- actin to control for sample variation (2 × 10 4 PBMC). Each panel is labeled with month of sample harvest. Lanes are labeled with the source of PBMC DNA by rabbit number and treatment, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). In lower panels, the corresponding rabbit samples are designated by the matching parallel black lines. Positions of BLV pol amplicon (591 bp) and β- actin amplicon (594 bp) are designated. *, not determined (ND) because sample was not available; †, not determined because the animal died before harvest. (B) PCR standard curve. PBMC lysate harvested at 1 month postinoculation from BLV rabbit 44-10 was serially diluted in a range of 5 × 10 2 to 5 × 10 4 PBMC and subjected to PCR with BLV pol primers KB560 and KB561. Each lane is labeled with the number of cells used for PCR amplification, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). The arrow on the left indicates the position of BLV pol amplicon (591 bp), and the lines on the right indicate positions of 676- and 571-bp DNA size markers.

Techniques Used: Polymerase Chain Reaction, Labeling, Positive Control, Marker, Amplification

3) Product Images from "High-Dose Dexamethasone Alters the Increase in Interleukin-16 Level in Adult Immune Thrombocytopenia"

Article Title: High-Dose Dexamethasone Alters the Increase in Interleukin-16 Level in Adult Immune Thrombocytopenia

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.00451

Relative mRNA expression of pro-IL16, caspase-3 and T-bet in ITP patients with active disease and healthy controls. mRNA expression in bone marrow mononuclear cells (BMMCs) and peripheral blood mononuclear cells (PBMCs) from ITP patients and healthy controls were quantified by real-time PCR. Furthermore, correlations between plasma IL-16 and mRNA expression levels of pro-IL16, caspase-3 and T-bet in active ITP patients were calculated. (A) Relative mRNA expression of pro-IL16, caspase-3 in ITP patients with active disease and healthy controls in BMMCs; *** P
Figure Legend Snippet: Relative mRNA expression of pro-IL16, caspase-3 and T-bet in ITP patients with active disease and healthy controls. mRNA expression in bone marrow mononuclear cells (BMMCs) and peripheral blood mononuclear cells (PBMCs) from ITP patients and healthy controls were quantified by real-time PCR. Furthermore, correlations between plasma IL-16 and mRNA expression levels of pro-IL16, caspase-3 and T-bet in active ITP patients were calculated. (A) Relative mRNA expression of pro-IL16, caspase-3 in ITP patients with active disease and healthy controls in BMMCs; *** P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

4) Product Images from "No detectable alloreactive transcriptional responses during donor-multiplexed single-cell RNA sequencing of peripheral blood mononuclear cells"

Article Title: No detectable alloreactive transcriptional responses during donor-multiplexed single-cell RNA sequencing of peripheral blood mononuclear cells

Journal: bioRxiv

doi: 10.1101/2020.02.12.946509

Mixing blood-type-mismatched PBMCs does not cause allogenic response during multiplexed scRNA-seq sample preparation (A) Sample classification results plotted as densities in PBMC gene expression space (top left) grouped according to unmixed donor A PBMCs (bottom left), mixed Donor A PBMCs (bottom right), and Donors A-C PBMCs (top right). Discordant region representing donor-specific expression profiles highlighted with white arrow. (B) Evenly-subsetted CD4+ T-cell gene expression space grouped according to donor ID (e.g., A, B, C) and mixing status (e.g., - = unmixed, + = mixed). n = 1,336 CD4+ T-cells. (C) Quantitative inter-sample comparison using Jensen-Shannon Divergence (JSD) and hierarchical clustering demonstrates similarity between evenly-subsetted CD4+ T-cells from distinct healthy donors regardless of mixing.
Figure Legend Snippet: Mixing blood-type-mismatched PBMCs does not cause allogenic response during multiplexed scRNA-seq sample preparation (A) Sample classification results plotted as densities in PBMC gene expression space (top left) grouped according to unmixed donor A PBMCs (bottom left), mixed Donor A PBMCs (bottom right), and Donors A-C PBMCs (top right). Discordant region representing donor-specific expression profiles highlighted with white arrow. (B) Evenly-subsetted CD4+ T-cell gene expression space grouped according to donor ID (e.g., A, B, C) and mixing status (e.g., - = unmixed, + = mixed). n = 1,336 CD4+ T-cells. (C) Quantitative inter-sample comparison using Jensen-Shannon Divergence (JSD) and hierarchical clustering demonstrates similarity between evenly-subsetted CD4+ T-cells from distinct healthy donors regardless of mixing.

Techniques Used: Sample Prep, Expressing

Schematic overview of experimental design. PBMCs from 8 healthy blood-type-mismatched donors (tubes on left, table on top) were barcoded with MULTI-seq LMOs (black double-helix hybridized to red DNA barcode) and BD single-cell multiplexing kit (SCMK) antibodies (black antibody conjugated to teal DNA barcode). Cells were then strategically pooled to directly assess whether mixing blood-type-mismatched PBMCs during scRNA-seq causes alloreactivity.
Figure Legend Snippet: Schematic overview of experimental design. PBMCs from 8 healthy blood-type-mismatched donors (tubes on left, table on top) were barcoded with MULTI-seq LMOs (black double-helix hybridized to red DNA barcode) and BD single-cell multiplexing kit (SCMK) antibodies (black antibody conjugated to teal DNA barcode). Cells were then strategically pooled to directly assess whether mixing blood-type-mismatched PBMCs during scRNA-seq causes alloreactivity.

Techniques Used: Multiplexing

Trima-associated gene expression signatures (A) Identification of gene expression patterns across all PBMC cell-types (left) linked to method of PBMC isolation (e.g., Ficoll or Trima; right). n = 15,340 cells. (B) Identification of Trima-specific marker genes in classical monocytes (left) and NK cells (right). n = 2,303 classical monocytes, n = 1,545 NK cells. Marker gene expression values are depicted as log1p-normalized counts.
Figure Legend Snippet: Trima-associated gene expression signatures (A) Identification of gene expression patterns across all PBMC cell-types (left) linked to method of PBMC isolation (e.g., Ficoll or Trima; right). n = 15,340 cells. (B) Identification of Trima-specific marker genes in classical monocytes (left) and NK cells (right). n = 2,303 classical monocytes, n = 1,545 NK cells. Marker gene expression values are depicted as log1p-normalized counts.

Techniques Used: Expressing, Isolation, Marker

Exploring alloreactivity in CD4+ T-cell subset and classical monocytes (A) Marker genes used for CD4+ T-cell subtype annotations (left) in Ficoll PBMCs. n = 6,879 CD4+ T-cells. (B) Evenly-subsetted classical monocyte gene expression space grouped according to donor ID (e.g., A, B, C) and mixing status (e.g., - = unmixed, + = mixed). n = 384 classical monocytes. (C) Quantitative inter-sample comparison using Jensen-Shannon Divergence (JSD) and hierarchical clustering demonstrates similarity between evenly-subsetted classical monocytes from distinct healthy donors regardless of mixing. (D) Expression of genes known to be up-regulated (e.g., IFNG and CD40LG) or down-regulated (e.g., DUSP1 and FOS) in lymphocytes during an allogenic response.
Figure Legend Snippet: Exploring alloreactivity in CD4+ T-cell subset and classical monocytes (A) Marker genes used for CD4+ T-cell subtype annotations (left) in Ficoll PBMCs. n = 6,879 CD4+ T-cells. (B) Evenly-subsetted classical monocyte gene expression space grouped according to donor ID (e.g., A, B, C) and mixing status (e.g., - = unmixed, + = mixed). n = 384 classical monocytes. (C) Quantitative inter-sample comparison using Jensen-Shannon Divergence (JSD) and hierarchical clustering demonstrates similarity between evenly-subsetted classical monocytes from distinct healthy donors regardless of mixing. (D) Expression of genes known to be up-regulated (e.g., IFNG and CD40LG) or down-regulated (e.g., DUSP1 and FOS) in lymphocytes during an allogenic response.

Techniques Used: Marker, Expressing

MULTI-seq and SCMK classifications largely match in silico genotyping, with lower SCMK classification efficiency and bias against activated CD4+ T-cells. (A) Sample classification results from three demultiplexing pipelines (e.g., deMULTIplex, souporcell, and demuxEM) projected onto MULTI-seq (top) and SCMK (bottom) sample barcode space. n = 4,032 cells from microfluidic lane #3. (B) Frequencies of classified and unclassified cells across all PBMC cell types following SCMK sample demultiplexing. (C) Localization of SCMK unclassified cells in CD4+ T-cell gene expression space. n = 6,879 CD4+ T-cells.
Figure Legend Snippet: MULTI-seq and SCMK classifications largely match in silico genotyping, with lower SCMK classification efficiency and bias against activated CD4+ T-cells. (A) Sample classification results from three demultiplexing pipelines (e.g., deMULTIplex, souporcell, and demuxEM) projected onto MULTI-seq (top) and SCMK (bottom) sample barcode space. n = 4,032 cells from microfluidic lane #3. (B) Frequencies of classified and unclassified cells across all PBMC cell types following SCMK sample demultiplexing. (C) Localization of SCMK unclassified cells in CD4+ T-cell gene expression space. n = 6,879 CD4+ T-cells.

Techniques Used: In Silico, Expressing

5) Product Images from "Bovine Leukemia Virus Structural Gene Vectors Are Immunogenic and Lack Pathogenicity in a Rabbit Model"

Article Title: Bovine Leukemia Virus Structural Gene Vectors Are Immunogenic and Lack Pathogenicity in a Rabbit Model

Journal: Journal of Virology

doi:

Semiquantitative PCR analysis to evaluate differences in proviral load. (A) PCR to detect BLV pol in PBMC from treated rabbits. Rabbit PBMC were harvested at 1, 4, and 10 months postinoculation and subjected to PCR with BLV pol primers KB560 and KB561 (10 × 10 4 PBMC) or β- actin to control for sample variation (2 × 10 4 PBMC). Each panel is labeled with month of sample harvest. Lanes are labeled with the source of PBMC DNA by rabbit number and treatment, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). In lower panels, the corresponding rabbit samples are designated by the matching parallel black lines. Positions of BLV pol amplicon (591 bp) and β- actin amplicon (594 bp) are designated. *, not determined (ND) because sample was not available; †, not determined because the animal died before harvest. (B) PCR standard curve. PBMC lysate harvested at 1 month postinoculation from BLV rabbit 44-10 was serially diluted in a range of 5 × 10 2 to 5 × 10 4 PBMC and subjected to PCR with BLV pol primers KB560 and KB561. Each lane is labeled with the number of cells used for PCR amplification, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). The arrow on the left indicates the position of BLV pol amplicon (591 bp), and the lines on the right indicate positions of 676- and 571-bp DNA size markers.
Figure Legend Snippet: Semiquantitative PCR analysis to evaluate differences in proviral load. (A) PCR to detect BLV pol in PBMC from treated rabbits. Rabbit PBMC were harvested at 1, 4, and 10 months postinoculation and subjected to PCR with BLV pol primers KB560 and KB561 (10 × 10 4 PBMC) or β- actin to control for sample variation (2 × 10 4 PBMC). Each panel is labeled with month of sample harvest. Lanes are labeled with the source of PBMC DNA by rabbit number and treatment, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). In lower panels, the corresponding rabbit samples are designated by the matching parallel black lines. Positions of BLV pol amplicon (591 bp) and β- actin amplicon (594 bp) are designated. *, not determined (ND) because sample was not available; †, not determined because the animal died before harvest. (B) PCR standard curve. PBMC lysate harvested at 1 month postinoculation from BLV rabbit 44-10 was serially diluted in a range of 5 × 10 2 to 5 × 10 4 PBMC and subjected to PCR with BLV pol primers KB560 and KB561. Each lane is labeled with the number of cells used for PCR amplification, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). The arrow on the left indicates the position of BLV pol amplicon (591 bp), and the lines on the right indicate positions of 676- and 571-bp DNA size markers.

Techniques Used: Polymerase Chain Reaction, Labeling, Positive Control, Marker, Amplification

6) Product Images from "Targeted nanoparticle delivery overcomes off-target immunostimulatory effects of oligonucleotides and improves therapeutic efficacy in chronic lymphocytic leukemia"

Article Title: Targeted nanoparticle delivery overcomes off-target immunostimulatory effects of oligonucleotides and improves therapeutic efficacy in chronic lymphocytic leukemia

Journal: Blood

doi: 10.1182/blood-2012-01-407742

B cell–specific delivery of the payload and lack of immunostimulation by RIT-INP in whole blood from CLL patients. (A) Nonselectivity of free FAM-ODN (1μM) in PBMCs. (B) Selectivity of RIT-INP–encapsulated FAM-ODN (1μM)
Figure Legend Snippet: B cell–specific delivery of the payload and lack of immunostimulation by RIT-INP in whole blood from CLL patients. (A) Nonselectivity of free FAM-ODN (1μM) in PBMCs. (B) Selectivity of RIT-INP–encapsulated FAM-ODN (1μM)

Techniques Used:

7) Product Images from "Functional comparison of PBMCs isolated by Cell Preparation Tubes (CPT) vs. Lymphoprep Tubes"

Article Title: Functional comparison of PBMCs isolated by Cell Preparation Tubes (CPT) vs. Lymphoprep Tubes

Journal: BMC Immunology

doi: 10.1186/s12865-020-00345-0

Equivalent PBMC yield and post-thaw recovery using CPT or LP Tubes. a-e Three healthy donor blood draws of six tubes each were split between three operators for parallel PBMC isolation using CPT and LP Tube methods. Total PBMCs were counted and assessed for viability by acridine iodine and propidium iodide staining using a Cellometer K2 immediately after isolation (pre-freeze), and by near-IR viability staining using a BD LSRII flow cytometer after cryopreservation and recovery (post-thaw). a The yield of PBMCs per mL of input blood pre-freeze. b The yield of PBMCs per mL of input blood post-thaw. c Percent of cell recovery post-thaw. d Cell viability pre-freeze. e Cell viability post-thaw. Horizontal lines indicate mean +/− SEM. Donors 1,2, 3 are depicted by red, black, and blue dots respectively. Three dots are shown per donor which represent single samples processed by three different operators. For statistical analysis for all figures see Table 1
Figure Legend Snippet: Equivalent PBMC yield and post-thaw recovery using CPT or LP Tubes. a-e Three healthy donor blood draws of six tubes each were split between three operators for parallel PBMC isolation using CPT and LP Tube methods. Total PBMCs were counted and assessed for viability by acridine iodine and propidium iodide staining using a Cellometer K2 immediately after isolation (pre-freeze), and by near-IR viability staining using a BD LSRII flow cytometer after cryopreservation and recovery (post-thaw). a The yield of PBMCs per mL of input blood pre-freeze. b The yield of PBMCs per mL of input blood post-thaw. c Percent of cell recovery post-thaw. d Cell viability pre-freeze. e Cell viability post-thaw. Horizontal lines indicate mean +/− SEM. Donors 1,2, 3 are depicted by red, black, and blue dots respectively. Three dots are shown per donor which represent single samples processed by three different operators. For statistical analysis for all figures see Table 1

Techniques Used: Isolation, Staining, Flow Cytometry

CPT and LP methods maintain equivalent T cell functionality in isolated PBMCs. a Gating to identify CD4+ and CD8+ T cells (upper) and biaxial plots showing intracellular cytokine production for IL-17A, IFNγ, and IL-4 in CD4+ and CD8+ T cells after stimulation with CEF, PMA-I, or unstimulated control (unstim). One representative PBMC sample is shown. b, c Quantification of indicated intracellular cytokine production in CD4+ and CD8+ T cells from PBMCs isolated by CPT or LP methods following stimulation with b CEF or c PMA-I. All data generated by flow cytometry; values shown are normalized to unstimulated control (value subtracted by unstimulated control), horizontal lines indicate mean +/− SEM. Donors 1,2, 3 are depicted by red, black, and blue dots respectively. Three dots are shown per donor which represent single samples processed by three different operators
Figure Legend Snippet: CPT and LP methods maintain equivalent T cell functionality in isolated PBMCs. a Gating to identify CD4+ and CD8+ T cells (upper) and biaxial plots showing intracellular cytokine production for IL-17A, IFNγ, and IL-4 in CD4+ and CD8+ T cells after stimulation with CEF, PMA-I, or unstimulated control (unstim). One representative PBMC sample is shown. b, c Quantification of indicated intracellular cytokine production in CD4+ and CD8+ T cells from PBMCs isolated by CPT or LP methods following stimulation with b CEF or c PMA-I. All data generated by flow cytometry; values shown are normalized to unstimulated control (value subtracted by unstimulated control), horizontal lines indicate mean +/− SEM. Donors 1,2, 3 are depicted by red, black, and blue dots respectively. Three dots are shown per donor which represent single samples processed by three different operators

Techniques Used: Isolation, Generated, Flow Cytometry

Similar distribution of immune cell subsets in PBMCs isolated with CPT or LP Tubes. a Gates used to define ten major indicated cell populations in flow cytometry data from one representative PBMC sample (donor 3). b-k Frequencies of cell populations (% of parent gate) in PBMCs isolated by CPT or LP methods determined by flow cytometry for b Lymphocytes, c B cells, d T cells, e CD4+ memory T cells, f CD4+ naïve T cells, g CD4- Naïve T cells, h CD4- memory T cells, i NKT cells, j NK cells, and k monocytes. Note: Lymphocytes used as parent gate for B cells statistics. Horizontal lines indicate mean +/− SEM. Donors 1,2, 3 are depicted by red, black, and blue dots respectively. Three dots are shown per donor which represent single samples processed by three different operators
Figure Legend Snippet: Similar distribution of immune cell subsets in PBMCs isolated with CPT or LP Tubes. a Gates used to define ten major indicated cell populations in flow cytometry data from one representative PBMC sample (donor 3). b-k Frequencies of cell populations (% of parent gate) in PBMCs isolated by CPT or LP methods determined by flow cytometry for b Lymphocytes, c B cells, d T cells, e CD4+ memory T cells, f CD4+ naïve T cells, g CD4- Naïve T cells, h CD4- memory T cells, i NKT cells, j NK cells, and k monocytes. Note: Lymphocytes used as parent gate for B cells statistics. Horizontal lines indicate mean +/− SEM. Donors 1,2, 3 are depicted by red, black, and blue dots respectively. Three dots are shown per donor which represent single samples processed by three different operators

Techniques Used: Isolation, Flow Cytometry

Schematic of the PBMC sample collection and processing. Six aliquots of blood (three for CPT processing and three for LP tube processing) were drawn from three donors. A CPT and LP tube from each donor was processed in parallel by three unique operators. Performance of PBMC isolation methods were measured by their cell yields, viabilities, and recovery both fresh and after cryopreservation and thawing. PBMCs were also stained with surface and intracellular antibodies to assess abundance of cell subsets and functional T cell response following stimulation
Figure Legend Snippet: Schematic of the PBMC sample collection and processing. Six aliquots of blood (three for CPT processing and three for LP tube processing) were drawn from three donors. A CPT and LP tube from each donor was processed in parallel by three unique operators. Performance of PBMC isolation methods were measured by their cell yields, viabilities, and recovery both fresh and after cryopreservation and thawing. PBMCs were also stained with surface and intracellular antibodies to assess abundance of cell subsets and functional T cell response following stimulation

Techniques Used: Isolation, Staining, Functional Assay

CPT and LP methods maintain equivalent T cell functionality in isolated PBMCs. a Gating to identify CD4+ and CD8+ T cells (upper) and biaxial plots showing intracellular cytokine production for IL-17A, IFNγ, and IL-4 in CD4+ and CD8+ T cells after stimulation with CEF, PMA-I, or unstimulated control (unstim). One representative PBMC sample is shown. b, c Quantification of indicated intracellular cytokine production in CD4+ and CD8+ T cells from PBMCs isolated by CPT or LP methods following stimulation with b CEF or c PMA-I. All data generated by flow cytometry; values shown are normalized to unstimulated control (value subtracted by unstimulated control), horizontal lines indicate mean +/− SEM. Donors 1,2, 3 are depicted by red, black, and blue dots respectively. Three dots are shown per donor which represent single samples processed by three different operators
Figure Legend Snippet: CPT and LP methods maintain equivalent T cell functionality in isolated PBMCs. a Gating to identify CD4+ and CD8+ T cells (upper) and biaxial plots showing intracellular cytokine production for IL-17A, IFNγ, and IL-4 in CD4+ and CD8+ T cells after stimulation with CEF, PMA-I, or unstimulated control (unstim). One representative PBMC sample is shown. b, c Quantification of indicated intracellular cytokine production in CD4+ and CD8+ T cells from PBMCs isolated by CPT or LP methods following stimulation with b CEF or c PMA-I. All data generated by flow cytometry; values shown are normalized to unstimulated control (value subtracted by unstimulated control), horizontal lines indicate mean +/− SEM. Donors 1,2, 3 are depicted by red, black, and blue dots respectively. Three dots are shown per donor which represent single samples processed by three different operators

Techniques Used: Isolation, Generated, Flow Cytometry

8) Product Images from "Functional comparison of PBMCs isolated by Cell Preparation Tubes (CPT) vs. Lymphoprep Tubes"

Article Title: Functional comparison of PBMCs isolated by Cell Preparation Tubes (CPT) vs. Lymphoprep Tubes

Journal: BMC Immunology

doi: 10.1186/s12865-020-00345-0

Equivalent PBMC yield and post-thaw recovery using CPT or LP Tubes. a-e Three healthy donor blood draws of six tubes each were split between three operators for parallel PBMC isolation using CPT and LP Tube methods. Total PBMCs were counted and assessed for viability by acridine iodine and propidium iodide staining using a Cellometer K2 immediately after isolation (pre-freeze), and by near-IR viability staining using a BD LSRII flow cytometer after cryopreservation and recovery (post-thaw). a The yield of PBMCs per mL of input blood pre-freeze. b The yield of PBMCs per mL of input blood post-thaw. c Percent of cell recovery post-thaw. d Cell viability pre-freeze. e Cell viability post-thaw. Horizontal lines indicate mean +/− SEM. Donors 1,2, 3 are depicted by red, black, and blue dots respectively. Three dots are shown per donor which represent single samples processed by three different operators. For statistical analysis for all figures see Table 1
Figure Legend Snippet: Equivalent PBMC yield and post-thaw recovery using CPT or LP Tubes. a-e Three healthy donor blood draws of six tubes each were split between three operators for parallel PBMC isolation using CPT and LP Tube methods. Total PBMCs were counted and assessed for viability by acridine iodine and propidium iodide staining using a Cellometer K2 immediately after isolation (pre-freeze), and by near-IR viability staining using a BD LSRII flow cytometer after cryopreservation and recovery (post-thaw). a The yield of PBMCs per mL of input blood pre-freeze. b The yield of PBMCs per mL of input blood post-thaw. c Percent of cell recovery post-thaw. d Cell viability pre-freeze. e Cell viability post-thaw. Horizontal lines indicate mean +/− SEM. Donors 1,2, 3 are depicted by red, black, and blue dots respectively. Three dots are shown per donor which represent single samples processed by three different operators. For statistical analysis for all figures see Table 1

Techniques Used: Isolation, Staining, Flow Cytometry

CPT and LP methods maintain equivalent T cell functionality in isolated PBMCs. a Gating to identify CD4+ and CD8+ T cells (upper) and biaxial plots showing intracellular cytokine production for IL-17A, IFNγ, and IL-4 in CD4+ and CD8+ T cells after stimulation with CEF, PMA-I, or unstimulated control (unstim). One representative PBMC sample is shown. b, c Quantification of indicated intracellular cytokine production in CD4+ and CD8+ T cells from PBMCs isolated by CPT or LP methods following stimulation with b CEF or c PMA-I. All data generated by flow cytometry; values shown are normalized to unstimulated control (value subtracted by unstimulated control), horizontal lines indicate mean +/− SEM. Donors 1,2, 3 are depicted by red, black, and blue dots respectively. Three dots are shown per donor which represent single samples processed by three different operators
Figure Legend Snippet: CPT and LP methods maintain equivalent T cell functionality in isolated PBMCs. a Gating to identify CD4+ and CD8+ T cells (upper) and biaxial plots showing intracellular cytokine production for IL-17A, IFNγ, and IL-4 in CD4+ and CD8+ T cells after stimulation with CEF, PMA-I, or unstimulated control (unstim). One representative PBMC sample is shown. b, c Quantification of indicated intracellular cytokine production in CD4+ and CD8+ T cells from PBMCs isolated by CPT or LP methods following stimulation with b CEF or c PMA-I. All data generated by flow cytometry; values shown are normalized to unstimulated control (value subtracted by unstimulated control), horizontal lines indicate mean +/− SEM. Donors 1,2, 3 are depicted by red, black, and blue dots respectively. Three dots are shown per donor which represent single samples processed by three different operators

Techniques Used: Isolation, Generated, Flow Cytometry

Similar distribution of immune cell subsets in PBMCs isolated with CPT or LP Tubes. a Gates used to define ten major indicated cell populations in flow cytometry data from one representative PBMC sample (donor 3). b-k Frequencies of cell populations (% of parent gate) in PBMCs isolated by CPT or LP methods determined by flow cytometry for b Lymphocytes, c B cells, d T cells, e CD4+ memory T cells, f CD4+ naïve T cells, g CD4- Naïve T cells, h CD4- memory T cells, i NKT cells, j NK cells, and k monocytes. Note: Lymphocytes used as parent gate for B cells statistics. Horizontal lines indicate mean +/− SEM. Donors 1,2, 3 are depicted by red, black, and blue dots respectively. Three dots are shown per donor which represent single samples processed by three different operators
Figure Legend Snippet: Similar distribution of immune cell subsets in PBMCs isolated with CPT or LP Tubes. a Gates used to define ten major indicated cell populations in flow cytometry data from one representative PBMC sample (donor 3). b-k Frequencies of cell populations (% of parent gate) in PBMCs isolated by CPT or LP methods determined by flow cytometry for b Lymphocytes, c B cells, d T cells, e CD4+ memory T cells, f CD4+ naïve T cells, g CD4- Naïve T cells, h CD4- memory T cells, i NKT cells, j NK cells, and k monocytes. Note: Lymphocytes used as parent gate for B cells statistics. Horizontal lines indicate mean +/− SEM. Donors 1,2, 3 are depicted by red, black, and blue dots respectively. Three dots are shown per donor which represent single samples processed by three different operators

Techniques Used: Isolation, Flow Cytometry

Schematic of the PBMC sample collection and processing. Six aliquots of blood (three for CPT processing and three for LP tube processing) were drawn from three donors. A CPT and LP tube from each donor was processed in parallel by three unique operators. Performance of PBMC isolation methods were measured by their cell yields, viabilities, and recovery both fresh and after cryopreservation and thawing. PBMCs were also stained with surface and intracellular antibodies to assess abundance of cell subsets and functional T cell response following stimulation
Figure Legend Snippet: Schematic of the PBMC sample collection and processing. Six aliquots of blood (three for CPT processing and three for LP tube processing) were drawn from three donors. A CPT and LP tube from each donor was processed in parallel by three unique operators. Performance of PBMC isolation methods were measured by their cell yields, viabilities, and recovery both fresh and after cryopreservation and thawing. PBMCs were also stained with surface and intracellular antibodies to assess abundance of cell subsets and functional T cell response following stimulation

Techniques Used: Isolation, Staining, Functional Assay

CPT and LP methods maintain equivalent T cell functionality in isolated PBMCs. a Gating to identify CD4+ and CD8+ T cells (upper) and biaxial plots showing intracellular cytokine production for IL-17A, IFNγ, and IL-4 in CD4+ and CD8+ T cells after stimulation with CEF, PMA-I, or unstimulated control (unstim). One representative PBMC sample is shown. b, c Quantification of indicated intracellular cytokine production in CD4+ and CD8+ T cells from PBMCs isolated by CPT or LP methods following stimulation with b CEF or c PMA-I. All data generated by flow cytometry; values shown are normalized to unstimulated control (value subtracted by unstimulated control), horizontal lines indicate mean +/− SEM. Donors 1,2, 3 are depicted by red, black, and blue dots respectively. Three dots are shown per donor which represent single samples processed by three different operators
Figure Legend Snippet: CPT and LP methods maintain equivalent T cell functionality in isolated PBMCs. a Gating to identify CD4+ and CD8+ T cells (upper) and biaxial plots showing intracellular cytokine production for IL-17A, IFNγ, and IL-4 in CD4+ and CD8+ T cells after stimulation with CEF, PMA-I, or unstimulated control (unstim). One representative PBMC sample is shown. b, c Quantification of indicated intracellular cytokine production in CD4+ and CD8+ T cells from PBMCs isolated by CPT or LP methods following stimulation with b CEF or c PMA-I. All data generated by flow cytometry; values shown are normalized to unstimulated control (value subtracted by unstimulated control), horizontal lines indicate mean +/− SEM. Donors 1,2, 3 are depicted by red, black, and blue dots respectively. Three dots are shown per donor which represent single samples processed by three different operators

Techniques Used: Isolation, Generated, Flow Cytometry

9) Product Images from "Long-term infection with retroviral structural gene vector provides protection against bovine leukemia virus disease in rabbits"

Article Title: Long-term infection with retroviral structural gene vector provides protection against bovine leukemia virus disease in rabbits

Journal: Virology

doi: 10.1016/j.virol.2004.09.001

Semi-quantitative PCR analysis of BLV pol detects differences in proviral load between BLV SGV and BLV-rabbits. PBMC were harvested at month indicated after challenge with 1 × 10 8 FLK/BLV cells from indicated rabbits that had been infected with BLV SGV for 24 months or from age-matched naïve controls. DNA from 50,000 PMBC was subjected to nested PCR and gel electrophoresis and stained with ethidium bromide. The position of the 591-bp amplicon is designated by the labeled arrow. In parallel, DNA from 20,000 PBMC was subjected to PCR with primers complementary to β- actin . The position of the 594-bp amplicon is designated by the labeled arrow and the signals were quantified by densitometry.
Figure Legend Snippet: Semi-quantitative PCR analysis of BLV pol detects differences in proviral load between BLV SGV and BLV-rabbits. PBMC were harvested at month indicated after challenge with 1 × 10 8 FLK/BLV cells from indicated rabbits that had been infected with BLV SGV for 24 months or from age-matched naïve controls. DNA from 50,000 PMBC was subjected to nested PCR and gel electrophoresis and stained with ethidium bromide. The position of the 591-bp amplicon is designated by the labeled arrow. In parallel, DNA from 20,000 PBMC was subjected to PCR with primers complementary to β- actin . The position of the 594-bp amplicon is designated by the labeled arrow and the signals were quantified by densitometry.

Techniques Used: Real-time Polymerase Chain Reaction, Infection, Nested PCR, Nucleic Acid Electrophoresis, Staining, Amplification, Labeling, Polymerase Chain Reaction

Related Articles

Diagnostic Assay:

Article Title: High-Dose Dexamethasone Alters the Increase in Interleukin-16 Level in Adult Immune Thrombocytopenia
Article Snippet: .. PBMCs or BMMCs were isolated by density gradient centrifugation using Ficoll-Paque (Pharmacia Diagnostic, Uppsala, Sweden) and stored at −80°C until RNA isolation. .. Enzyme-Linked Immunosorbent Assays Cell-free plasma IL-16 was measured using commercial Quantikine enzyme-linked immunosorbent assay (ELISA) kits (R & D systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

RNA Extraction:

Article Title: Activation of macrophages by lysophosphatidic acid through the lysophosphatidic acid receptor 1 as a novel mechanism in multiple sclerosis pathogenesis
Article Snippet: .. Isolation of PBMC for RNA extraction PBMCs were isolated from whole blood by standard Ficoll®-Paque density gradient centrifugation. ..

In Vitro:

Article Title: Induction of IgG2 and IgG4 B‐cell memory following sublingual immunotherapy for ryegrass pollen allergy, et al. Induction of IgG2 and IgG4 B‐cell memory following sublingual immunotherapy for ryegrass pollen allergy
Article Snippet: .. 2.5 In vitro RGP stimulation of PBMC, Treg staining, and measurement of cytokines PBMC were isolated by Ficoll‐paque density centrifugation. .. Fresh PBMC were used for in vitro culture, and the remaining cells stored in liquid nitrogen.

Isolation:

Article Title: Activation of macrophages by lysophosphatidic acid through the lysophosphatidic acid receptor 1 as a novel mechanism in multiple sclerosis pathogenesis
Article Snippet: .. Isolation of PBMC for RNA extraction PBMCs were isolated from whole blood by standard Ficoll®-Paque density gradient centrifugation. ..

Article Title: Induction of IgG2 and IgG4 B‐cell memory following sublingual immunotherapy for ryegrass pollen allergy, et al. Induction of IgG2 and IgG4 B‐cell memory following sublingual immunotherapy for ryegrass pollen allergy
Article Snippet: .. 2.5 In vitro RGP stimulation of PBMC, Treg staining, and measurement of cytokines PBMC were isolated by Ficoll‐paque density centrifugation. .. Fresh PBMC were used for in vitro culture, and the remaining cells stored in liquid nitrogen.

Article Title: Bovine Leukemia Virus Structural Gene Vectors Are Immunogenic and Lack Pathogenicity in a Rabbit Model
Article Snippet: .. To prepare PBMCs samples for PCR, PBMCs were isolated by Ficoll-Paque (Pharmacia) gradient centrifugation and washed three times with phosphate-buffered saline. .. PBMCs (107 ) were lysed in 1 ml of PCR buffer (50 mM KCl, 10 mM Tris [pH 8.3], 2.5 mM MgCl2 , 0.45% Nonidet P-40, 0.45% Tween 20) containing 200 μg of proteinase K (Calbiochem-Behring, La Jolla, Calif.) per ml.

Article Title: Platelets Fuel the Inflammasome Activation of Innate Immune Cells
Article Snippet: .. Isolation of CD14+ human Monocytes Venous blood was collected in S-Monovette® K3EDTA tubes and PBMCs were obtained by density gradient centrifugation in Ficoll-Paque PLUS. .. Monocytes were isolated from PBMCs using the EasySepTM Human Monocyte Isolation Kit according to the manufacturer instructions (STEMCELL Technologies™).

Article Title: High-Dose Dexamethasone Alters the Increase in Interleukin-16 Level in Adult Immune Thrombocytopenia
Article Snippet: .. PBMCs or BMMCs were isolated by density gradient centrifugation using Ficoll-Paque (Pharmacia Diagnostic, Uppsala, Sweden) and stored at −80°C until RNA isolation. .. Enzyme-Linked Immunosorbent Assays Cell-free plasma IL-16 was measured using commercial Quantikine enzyme-linked immunosorbent assay (ELISA) kits (R & D systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Centrifugation:

Article Title: Induction of IgG2 and IgG4 B‐cell memory following sublingual immunotherapy for ryegrass pollen allergy, et al. Induction of IgG2 and IgG4 B‐cell memory following sublingual immunotherapy for ryegrass pollen allergy
Article Snippet: .. 2.5 In vitro RGP stimulation of PBMC, Treg staining, and measurement of cytokines PBMC were isolated by Ficoll‐paque density centrifugation. .. Fresh PBMC were used for in vitro culture, and the remaining cells stored in liquid nitrogen.

Article Title: On the origin of low‐density neutrophils, et al. On the origin of low‐density neutrophils
Article Snippet: .. After phenotyping the lower and normal density neutrophils, a difference in numbers of CD16dim /CD62Lhigh neutrophils between the PBMC and the granulocyte fraction after Ficoll‐Paque centrifugation (median of 42.0% in LDNs vs. 11.9% in NDNs) was again apparent (Fig. ). ..

Polymerase Chain Reaction:

Article Title: Bovine Leukemia Virus Structural Gene Vectors Are Immunogenic and Lack Pathogenicity in a Rabbit Model
Article Snippet: .. To prepare PBMCs samples for PCR, PBMCs were isolated by Ficoll-Paque (Pharmacia) gradient centrifugation and washed three times with phosphate-buffered saline. .. PBMCs (107 ) were lysed in 1 ml of PCR buffer (50 mM KCl, 10 mM Tris [pH 8.3], 2.5 mM MgCl2 , 0.45% Nonidet P-40, 0.45% Tween 20) containing 200 μg of proteinase K (Calbiochem-Behring, La Jolla, Calif.) per ml.

Staining:

Article Title: Induction of IgG2 and IgG4 B‐cell memory following sublingual immunotherapy for ryegrass pollen allergy, et al. Induction of IgG2 and IgG4 B‐cell memory following sublingual immunotherapy for ryegrass pollen allergy
Article Snippet: .. 2.5 In vitro RGP stimulation of PBMC, Treg staining, and measurement of cytokines PBMC were isolated by Ficoll‐paque density centrifugation. .. Fresh PBMC were used for in vitro culture, and the remaining cells stored in liquid nitrogen.

Gradient Centrifugation:

Article Title: Activation of macrophages by lysophosphatidic acid through the lysophosphatidic acid receptor 1 as a novel mechanism in multiple sclerosis pathogenesis
Article Snippet: .. Isolation of PBMC for RNA extraction PBMCs were isolated from whole blood by standard Ficoll®-Paque density gradient centrifugation. ..

Article Title: Bovine Leukemia Virus Structural Gene Vectors Are Immunogenic and Lack Pathogenicity in a Rabbit Model
Article Snippet: .. To prepare PBMCs samples for PCR, PBMCs were isolated by Ficoll-Paque (Pharmacia) gradient centrifugation and washed three times with phosphate-buffered saline. .. PBMCs (107 ) were lysed in 1 ml of PCR buffer (50 mM KCl, 10 mM Tris [pH 8.3], 2.5 mM MgCl2 , 0.45% Nonidet P-40, 0.45% Tween 20) containing 200 μg of proteinase K (Calbiochem-Behring, La Jolla, Calif.) per ml.

Article Title: Platelets Fuel the Inflammasome Activation of Innate Immune Cells
Article Snippet: .. Isolation of CD14+ human Monocytes Venous blood was collected in S-Monovette® K3EDTA tubes and PBMCs were obtained by density gradient centrifugation in Ficoll-Paque PLUS. .. Monocytes were isolated from PBMCs using the EasySepTM Human Monocyte Isolation Kit according to the manufacturer instructions (STEMCELL Technologies™).

Article Title: High-Dose Dexamethasone Alters the Increase in Interleukin-16 Level in Adult Immune Thrombocytopenia
Article Snippet: .. PBMCs or BMMCs were isolated by density gradient centrifugation using Ficoll-Paque (Pharmacia Diagnostic, Uppsala, Sweden) and stored at −80°C until RNA isolation. .. Enzyme-Linked Immunosorbent Assays Cell-free plasma IL-16 was measured using commercial Quantikine enzyme-linked immunosorbent assay (ELISA) kits (R & D systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Article Title: No detectable alloreactive transcriptional responses during donor-multiplexed single-cell RNA sequencing of peripheral blood mononuclear cells
Article Snippet: .. Trima apheresis introduces biologically-relevant confounders into PBMC scRNA-seq data The PBMCs that were used in this study came from whole blood that was processed using Ficoll-Paque density gradient centrifugation. .. Notably, these samples either underwent (donors D-H) or did not undergo (donors A-C) apheresis using Trima filtration, a method to enhance leukocyte yield during sample preparation , .

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    Ficoll-Paque Pharmacia pbmc isolation
    Ex vivo production of IL-1β ( a ) and IFN-γ ( b ) by <t>PBMCs</t> stimulated with LPS, heat-killed S. aureus ( S.a ), heat-killed C. albicans ( C.a ), and sonicated M. tuberculosis ( M.tb ) at baseline, and at 1 and 4 days after vaccination with TFV or BCG vaccine. Wilcoxon signed-rank test comparing values before and after vaccination with the same stimulus; N = 29, ^ p = 0.06, * p
    Pbmc Isolation, supplied by Ficoll-Paque Pharmacia, 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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    Platelets Are Critical for the Production of IL-1 Cytokines by Human Primary Monocytes (A) Representative FACS dot plots of CD41 and <t>CD14</t> expression in human <t>PBMCs,</t> standard (Std-Mo), or platelet-depleted (PD-Mo) CD14 + monocytes (see STAR Methods ). (B) Quantification of platelets (CD41 + CD14 − ), platelet-monocyte aggregates (CD41 + CD14 + ), and platelet-free monocytes (CD41 − CD14 + ) in PBMCs and isolated monocytes. (C) IL-1β and TNF-α levels in cell-free supernatants of Std-Mo versus PD-Mo, or in platelet-depleted monocytes replenished with autologous platelets (PD-Mo + Plts, 50:1 Ptl:Cell), and stimulated as indicated. (D and E) IL-1β levels released by non-canonically activated Std-Mo, PD-Mo, or PD-Mo + Plts, stimulated with LPS (1 μg mL −1 ) for 16 h (D) or by inflammasome-activated THP-1 s ± platelets (E). Data is presented as floating bars (with mean and minimum to maximum values) and pooled from independent experiments. Each symbol represents the average from technical triplicates per blood donor. Data in (D) shows bar graph with Mean + SD, from a representative of two independent experiments. See also Figure S2 .
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    Ficoll-Paque Pharmacia orov infection pbmc
    <t>OROV</t> infection of <t>PBMCs</t> induces the expression of cytokines and innate immune response genes. Cell lysates from PBMCs obtained from healthy donors ( n = 3) and infected with OROV at MOI 10 were processed for RNA extraction and RT-PCR. ( A ) Scheme of the RNA PAMPs recognition pathways analyzed. ( B – E ) SYBR Green qRT-PCR data analysis of gene expression are shown in fold change (2^∆∆ C T). Graphics show results from two independent experiments. Bars represent mean ± SEM. All times post-infection were compared to the time 0 h samples (noninfected) by two-way ANOVA and Dunnett’s multiple comparisons test. **** p -value
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    Ex vivo production of IL-1β ( a ) and IFN-γ ( b ) by PBMCs stimulated with LPS, heat-killed S. aureus ( S.a ), heat-killed C. albicans ( C.a ), and sonicated M. tuberculosis ( M.tb ) at baseline, and at 1 and 4 days after vaccination with TFV or BCG vaccine. Wilcoxon signed-rank test comparing values before and after vaccination with the same stimulus; N = 29, ^ p = 0.06, * p

    Journal: European Journal of Clinical Microbiology & Infectious Diseases

    Article Title: Differential effects of BCG vaccine on immune responses induced by vi polysaccharide typhoid fever vaccination: an explorative randomized trial

    doi: 10.1007/s10096-020-03813-y

    Figure Lengend Snippet: Ex vivo production of IL-1β ( a ) and IFN-γ ( b ) by PBMCs stimulated with LPS, heat-killed S. aureus ( S.a ), heat-killed C. albicans ( C.a ), and sonicated M. tuberculosis ( M.tb ) at baseline, and at 1 and 4 days after vaccination with TFV or BCG vaccine. Wilcoxon signed-rank test comparing values before and after vaccination with the same stimulus; N = 29, ^ p = 0.06, * p

    Article Snippet: PBMC isolation and stimulation PBMCs were isolated using density-gradient separation over Ficoll-Paque (GE Healthcare, UK ).

    Techniques: Ex Vivo, Sonication

    Ex vivo production of IL-6 ( a ), IL-10 ( b ), IFN-γ ( c ), and IL-22 ( d ) by PBMCs stimulated with LPS, heat-killed S. aureus ( S.a ), heat-killed C. albicans ( C.a ), and sonicated M. tuberculosis ( M.tb ), before TFV or BCG vaccination (baseline) and at 2 weeks and 3 months after TFV vaccination. Wilcoxon signed-rank test comparing values before and after vaccination with the same stimulus; N = 29, * p

    Journal: European Journal of Clinical Microbiology & Infectious Diseases

    Article Title: Differential effects of BCG vaccine on immune responses induced by vi polysaccharide typhoid fever vaccination: an explorative randomized trial

    doi: 10.1007/s10096-020-03813-y

    Figure Lengend Snippet: Ex vivo production of IL-6 ( a ), IL-10 ( b ), IFN-γ ( c ), and IL-22 ( d ) by PBMCs stimulated with LPS, heat-killed S. aureus ( S.a ), heat-killed C. albicans ( C.a ), and sonicated M. tuberculosis ( M.tb ), before TFV or BCG vaccination (baseline) and at 2 weeks and 3 months after TFV vaccination. Wilcoxon signed-rank test comparing values before and after vaccination with the same stimulus; N = 29, * p

    Article Snippet: PBMC isolation and stimulation PBMCs were isolated using density-gradient separation over Ficoll-Paque (GE Healthcare, UK ).

    Techniques: Ex Vivo, Sonication

    Platelets Are Critical for the Production of IL-1 Cytokines by Human Primary Monocytes (A) Representative FACS dot plots of CD41 and CD14 expression in human PBMCs, standard (Std-Mo), or platelet-depleted (PD-Mo) CD14 + monocytes (see STAR Methods ). (B) Quantification of platelets (CD41 + CD14 − ), platelet-monocyte aggregates (CD41 + CD14 + ), and platelet-free monocytes (CD41 − CD14 + ) in PBMCs and isolated monocytes. (C) IL-1β and TNF-α levels in cell-free supernatants of Std-Mo versus PD-Mo, or in platelet-depleted monocytes replenished with autologous platelets (PD-Mo + Plts, 50:1 Ptl:Cell), and stimulated as indicated. (D and E) IL-1β levels released by non-canonically activated Std-Mo, PD-Mo, or PD-Mo + Plts, stimulated with LPS (1 μg mL −1 ) for 16 h (D) or by inflammasome-activated THP-1 s ± platelets (E). Data is presented as floating bars (with mean and minimum to maximum values) and pooled from independent experiments. Each symbol represents the average from technical triplicates per blood donor. Data in (D) shows bar graph with Mean + SD, from a representative of two independent experiments. See also Figure S2 .

    Journal: Cell Reports

    Article Title: Platelets Fuel the Inflammasome Activation of Innate Immune Cells

    doi: 10.1016/j.celrep.2020.107615

    Figure Lengend Snippet: Platelets Are Critical for the Production of IL-1 Cytokines by Human Primary Monocytes (A) Representative FACS dot plots of CD41 and CD14 expression in human PBMCs, standard (Std-Mo), or platelet-depleted (PD-Mo) CD14 + monocytes (see STAR Methods ). (B) Quantification of platelets (CD41 + CD14 − ), platelet-monocyte aggregates (CD41 + CD14 + ), and platelet-free monocytes (CD41 − CD14 + ) in PBMCs and isolated monocytes. (C) IL-1β and TNF-α levels in cell-free supernatants of Std-Mo versus PD-Mo, or in platelet-depleted monocytes replenished with autologous platelets (PD-Mo + Plts, 50:1 Ptl:Cell), and stimulated as indicated. (D and E) IL-1β levels released by non-canonically activated Std-Mo, PD-Mo, or PD-Mo + Plts, stimulated with LPS (1 μg mL −1 ) for 16 h (D) or by inflammasome-activated THP-1 s ± platelets (E). Data is presented as floating bars (with mean and minimum to maximum values) and pooled from independent experiments. Each symbol represents the average from technical triplicates per blood donor. Data in (D) shows bar graph with Mean + SD, from a representative of two independent experiments. See also Figure S2 .

    Article Snippet: Isolation of CD14+ human Monocytes Venous blood was collected in S-Monovette® K3EDTA tubes and PBMCs were obtained by density gradient centrifugation in Ficoll-Paque PLUS.

    Techniques: FACS, Expressing, Isolation

    Unstimulated neutrophils display a spectrum of densities . Neutrophils were first isolated using Ficoll density centrifugation. Thereafter isolated neutrophils were centrifuged on top of Percoll with different densities. ( A ) Depicted is the percentage of total neutrophils in the “PBMC layer” after density centrifugation with different densities of Percoll ( n = 9). ( B ) The lag time in which higher density neutrophils (HDNs) and lower density neutrophils (LDNs) contain growth of GFP labeled S. Aureus ( n = 7). ( C ) The percentage of the original population of lymphocytes that divided after stimulation in the absence of neutrophils, in the presence of all neutrophils and in the presence of either LDN or HDN of the same donor ( n = 7). ( D ) Division index (average number of cell divisions that a cell in the original population has undergone) of lymphocytes under the same conditions as ( C ). ( E ) Proliferation index (the total number of divisions divided by the number of cells that went into division) of lymphocytes under the same conditions as ( C ). For all graphs median ± IQR% is shown. Data are analyzed using Friedman test without correction for multiple comparisons. ( C‐E ) all conditions were tested, but only statistically significant results are indicated

    Journal: Journal of Leukocyte Biology

    Article Title: On the origin of low‐density neutrophils, et al. On the origin of low‐density neutrophils

    doi: 10.1002/JLB.5HR0120-459R

    Figure Lengend Snippet: Unstimulated neutrophils display a spectrum of densities . Neutrophils were first isolated using Ficoll density centrifugation. Thereafter isolated neutrophils were centrifuged on top of Percoll with different densities. ( A ) Depicted is the percentage of total neutrophils in the “PBMC layer” after density centrifugation with different densities of Percoll ( n = 9). ( B ) The lag time in which higher density neutrophils (HDNs) and lower density neutrophils (LDNs) contain growth of GFP labeled S. Aureus ( n = 7). ( C ) The percentage of the original population of lymphocytes that divided after stimulation in the absence of neutrophils, in the presence of all neutrophils and in the presence of either LDN or HDN of the same donor ( n = 7). ( D ) Division index (average number of cell divisions that a cell in the original population has undergone) of lymphocytes under the same conditions as ( C ). ( E ) Proliferation index (the total number of divisions divided by the number of cells that went into division) of lymphocytes under the same conditions as ( C ). For all graphs median ± IQR% is shown. Data are analyzed using Friedman test without correction for multiple comparisons. ( C‐E ) all conditions were tested, but only statistically significant results are indicated

    Article Snippet: After phenotyping the lower and normal density neutrophils, a difference in numbers of CD16dim /CD62Lhigh neutrophils between the PBMC and the granulocyte fraction after Ficoll‐Paque centrifugation (median of 42.0% in LDNs vs. 11.9% in NDNs) was again apparent (Fig. ).

    Techniques: Isolation, Centrifugation, Labeling

    OROV infection of PBMCs induces the expression of cytokines and innate immune response genes. Cell lysates from PBMCs obtained from healthy donors ( n = 3) and infected with OROV at MOI 10 were processed for RNA extraction and RT-PCR. ( A ) Scheme of the RNA PAMPs recognition pathways analyzed. ( B – E ) SYBR Green qRT-PCR data analysis of gene expression are shown in fold change (2^∆∆ C T). Graphics show results from two independent experiments. Bars represent mean ± SEM. All times post-infection were compared to the time 0 h samples (noninfected) by two-way ANOVA and Dunnett’s multiple comparisons test. **** p -value

    Journal: Viruses

    Article Title: Oropouche Virus Infects, Persists and Induces IFN Response in Human Peripheral Blood Mononuclear Cells as Identified by RNA PrimeFlow™ and qRT-PCR Assays

    doi: 10.3390/v12070785

    Figure Lengend Snippet: OROV infection of PBMCs induces the expression of cytokines and innate immune response genes. Cell lysates from PBMCs obtained from healthy donors ( n = 3) and infected with OROV at MOI 10 were processed for RNA extraction and RT-PCR. ( A ) Scheme of the RNA PAMPs recognition pathways analyzed. ( B – E ) SYBR Green qRT-PCR data analysis of gene expression are shown in fold change (2^∆∆ C T). Graphics show results from two independent experiments. Bars represent mean ± SEM. All times post-infection were compared to the time 0 h samples (noninfected) by two-way ANOVA and Dunnett’s multiple comparisons test. **** p -value

    Article Snippet: Human PBMC Culture and OROV Infection PBMC were obtained by gradient centrifugation with Ficoll-Paque 1.077 g/mL, according to the manufacturer’s instructions with some modifications.

    Techniques: Infection, Expressing, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Quantitative RT-PCR

    OROV infection and replication in PBMCs is favored when the type I IFN receptor is blocked and when the cells are treated with Dexamethasone. PBMC were pretreated for 2 h with Dexamethasone (Dexo) 1 µM and human anti-IFNAR antibody 5 ng/mL, and infected with OROV at MOI 1. Cell lysates and supernatants were collected for analysis. ( A ) qRT-PCR of cell lysate and supernatant ( B ) for OROV RNA detection through the time of infection. Data were compared by two-way ANOVA and Tukey’s multiple comparison test. ( C ) Infectious particles released in the supernatant were measured by FFA, 48 hpi. Treatment groups were compared by one-way ANOVA and Dunn’s multiple comparisons test. Bars represent viral load ± SEM. **** p -value

    Journal: Viruses

    Article Title: Oropouche Virus Infects, Persists and Induces IFN Response in Human Peripheral Blood Mononuclear Cells as Identified by RNA PrimeFlow™ and qRT-PCR Assays

    doi: 10.3390/v12070785

    Figure Lengend Snippet: OROV infection and replication in PBMCs is favored when the type I IFN receptor is blocked and when the cells are treated with Dexamethasone. PBMC were pretreated for 2 h with Dexamethasone (Dexo) 1 µM and human anti-IFNAR antibody 5 ng/mL, and infected with OROV at MOI 1. Cell lysates and supernatants were collected for analysis. ( A ) qRT-PCR of cell lysate and supernatant ( B ) for OROV RNA detection through the time of infection. Data were compared by two-way ANOVA and Tukey’s multiple comparison test. ( C ) Infectious particles released in the supernatant were measured by FFA, 48 hpi. Treatment groups were compared by one-way ANOVA and Dunn’s multiple comparisons test. Bars represent viral load ± SEM. **** p -value

    Article Snippet: Human PBMC Culture and OROV Infection PBMC were obtained by gradient centrifugation with Ficoll-Paque 1.077 g/mL, according to the manufacturer’s instructions with some modifications.

    Techniques: Infection, Quantitative RT-PCR, RNA Detection

    Human peripheral blood monocytes and lymphocytes are susceptible to OROV infection but generate low yields of infectious particles. Human peripheral blood mononuclear cells (PBMCs) were obtained from whole blood of healthy donors, infected in vitro and analyzed by different methodologies. ( A ) PBMCs were seeded in 24-well plates and infected with OROV MOI 0.1; 1 and 10. Cell lysates and supernatants were collected to RNA quantification by qRT-PCR ( n = 3). ( B ) The supernatants were also analyzed by FFA assay. Symbols represent the mean of viral load ± SEM. ( C ) PBMCs from healthy donors ( n = 2) were infected with MOI 1, submitted RNA PrimeFlow™ protocol 24 hpi and flow cytometry. CD3 + and CD14 + HLA DR + percentage of events with OROV Grna, and the gating strategy are shown. ( D ) Detection of OROV 48 hpi in cells infected with MOI 2, by confocal microscopy. OROV proteins in red (Alexa Fluor 594); genome (gRNA) in magenta (AlexaFluor 647); antigenome (agRNA) in green (AlexaFluor 488); DAPI (blue). Images with 63x times magnification. Scales at 25 μm.

    Journal: Viruses

    Article Title: Oropouche Virus Infects, Persists and Induces IFN Response in Human Peripheral Blood Mononuclear Cells as Identified by RNA PrimeFlow™ and qRT-PCR Assays

    doi: 10.3390/v12070785

    Figure Lengend Snippet: Human peripheral blood monocytes and lymphocytes are susceptible to OROV infection but generate low yields of infectious particles. Human peripheral blood mononuclear cells (PBMCs) were obtained from whole blood of healthy donors, infected in vitro and analyzed by different methodologies. ( A ) PBMCs were seeded in 24-well plates and infected with OROV MOI 0.1; 1 and 10. Cell lysates and supernatants were collected to RNA quantification by qRT-PCR ( n = 3). ( B ) The supernatants were also analyzed by FFA assay. Symbols represent the mean of viral load ± SEM. ( C ) PBMCs from healthy donors ( n = 2) were infected with MOI 1, submitted RNA PrimeFlow™ protocol 24 hpi and flow cytometry. CD3 + and CD14 + HLA DR + percentage of events with OROV Grna, and the gating strategy are shown. ( D ) Detection of OROV 48 hpi in cells infected with MOI 2, by confocal microscopy. OROV proteins in red (Alexa Fluor 594); genome (gRNA) in magenta (AlexaFluor 647); antigenome (agRNA) in green (AlexaFluor 488); DAPI (blue). Images with 63x times magnification. Scales at 25 μm.

    Article Snippet: Human PBMC Culture and OROV Infection PBMC were obtained by gradient centrifugation with Ficoll-Paque 1.077 g/mL, according to the manufacturer’s instructions with some modifications.

    Techniques: Infection, In Vitro, Quantitative RT-PCR, Flow Cytometry, Confocal Microscopy