Structured Review

Promega pbmc
Endogenous platelet-activating factor (PAF) contributes to the pro-inflammatory response elicited by lipopolysaccharide (LPS) and exogenous PAF potentiates LPS-induced inflammation in chicken primary macrophages. After stimulation with E. coli LPS (10 ng/ml) in the presence or absence of PCA 4248 (10 µM) or human PAF-acetylhydrolase (PAF-AH) (10 µg/ml) for 6 h, B13/B13 histocompatible chicken-derived bone marrow-derived macrophage <t>(BMDM)</t> supernatants were assessed for the presence of (A) nitric oxide (NO), and cell lysates were used to analyze gene expression of (B) iNOS/ NOS2 , (C) COX-2/ PTGS2 , and (D) IL-1β/ IL1B by quantitative real-time PCR. Data are expressed as relative normalized expression (as compared to vehicle control group). (E) PAF (0.1–10 µM) alone or together with LPS was added to BMDM, and NO production in the supernatants was assessed after 6 h. (F) NO production by BMDM derived from the bone marrow of the outbred PA12 chickens following exposure to PAF (10 µM) and/or LPS in the presence or absence of PCA 4248 (10 µM) for 6 h. (G) NO production by peripheral blood mononuclear cells <t>(PBMC)</t> from B13/B13 histocompatible chickens following exposure to PAF (10 µM) and/or LPS in the presence or absence of PCA 4248 (10 µM) for 6 h. Values are mean ± SEM. * P
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1) Product Images from "Characterization of the Phospholipid Platelet-Activating Factor As a Mediator of Inflammation in Chickens"

Article Title: Characterization of the Phospholipid Platelet-Activating Factor As a Mediator of Inflammation in Chickens

Journal: Frontiers in Veterinary Science

doi: 10.3389/fvets.2017.00226

Endogenous platelet-activating factor (PAF) contributes to the pro-inflammatory response elicited by lipopolysaccharide (LPS) and exogenous PAF potentiates LPS-induced inflammation in chicken primary macrophages. After stimulation with E. coli LPS (10 ng/ml) in the presence or absence of PCA 4248 (10 µM) or human PAF-acetylhydrolase (PAF-AH) (10 µg/ml) for 6 h, B13/B13 histocompatible chicken-derived bone marrow-derived macrophage (BMDM) supernatants were assessed for the presence of (A) nitric oxide (NO), and cell lysates were used to analyze gene expression of (B) iNOS/ NOS2 , (C) COX-2/ PTGS2 , and (D) IL-1β/ IL1B by quantitative real-time PCR. Data are expressed as relative normalized expression (as compared to vehicle control group). (E) PAF (0.1–10 µM) alone or together with LPS was added to BMDM, and NO production in the supernatants was assessed after 6 h. (F) NO production by BMDM derived from the bone marrow of the outbred PA12 chickens following exposure to PAF (10 µM) and/or LPS in the presence or absence of PCA 4248 (10 µM) for 6 h. (G) NO production by peripheral blood mononuclear cells (PBMC) from B13/B13 histocompatible chickens following exposure to PAF (10 µM) and/or LPS in the presence or absence of PCA 4248 (10 µM) for 6 h. Values are mean ± SEM. * P
Figure Legend Snippet: Endogenous platelet-activating factor (PAF) contributes to the pro-inflammatory response elicited by lipopolysaccharide (LPS) and exogenous PAF potentiates LPS-induced inflammation in chicken primary macrophages. After stimulation with E. coli LPS (10 ng/ml) in the presence or absence of PCA 4248 (10 µM) or human PAF-acetylhydrolase (PAF-AH) (10 µg/ml) for 6 h, B13/B13 histocompatible chicken-derived bone marrow-derived macrophage (BMDM) supernatants were assessed for the presence of (A) nitric oxide (NO), and cell lysates were used to analyze gene expression of (B) iNOS/ NOS2 , (C) COX-2/ PTGS2 , and (D) IL-1β/ IL1B by quantitative real-time PCR. Data are expressed as relative normalized expression (as compared to vehicle control group). (E) PAF (0.1–10 µM) alone or together with LPS was added to BMDM, and NO production in the supernatants was assessed after 6 h. (F) NO production by BMDM derived from the bone marrow of the outbred PA12 chickens following exposure to PAF (10 µM) and/or LPS in the presence or absence of PCA 4248 (10 µM) for 6 h. (G) NO production by peripheral blood mononuclear cells (PBMC) from B13/B13 histocompatible chickens following exposure to PAF (10 µM) and/or LPS in the presence or absence of PCA 4248 (10 µM) for 6 h. Values are mean ± SEM. * P

Techniques Used: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction

Components of the platelet-activating factor (PAF) system are expressed in chickens, and exogenous PAF induces intracellular calcium increase in chicken macrophages. (A) The gene expressions of PAF receptor (PAFR)/ PTAFR and (B) LPCAT2/ LPCAT2 were evaluated by quantitative real-time PCR (qRT-PCR) in unstimulated chicken tissues and macrophages [HD11 macrophage-like cells, bone marrow-derived macrophages (BMDM) and peripheral blood mononuclear cells (PBMC)]. Amplicon size (in base pairs, bp) was confirmed in 2% agarose gels. (C) PAFR/ PTAFR gene expression was evaluated by qRT-PCR in purified bone marrow cells following chCSF-1 medium complementation for BMDM differentiation from days 0 to 7. Data are expressed as relative normalized expression (as compared to two housekeeping genes). Values are mean ± SEM. In (D,E) the HD11 cell line was stimulated with PAF and/or PAFR antagonists (PCA 4248 and WEB 2086), and the increase in intracellular calcium signal was recorded over time (216 s, 3 s intervals) using a fluorescent probe. RLU, relative luminescence units; AUC, area under the curve.
Figure Legend Snippet: Components of the platelet-activating factor (PAF) system are expressed in chickens, and exogenous PAF induces intracellular calcium increase in chicken macrophages. (A) The gene expressions of PAF receptor (PAFR)/ PTAFR and (B) LPCAT2/ LPCAT2 were evaluated by quantitative real-time PCR (qRT-PCR) in unstimulated chicken tissues and macrophages [HD11 macrophage-like cells, bone marrow-derived macrophages (BMDM) and peripheral blood mononuclear cells (PBMC)]. Amplicon size (in base pairs, bp) was confirmed in 2% agarose gels. (C) PAFR/ PTAFR gene expression was evaluated by qRT-PCR in purified bone marrow cells following chCSF-1 medium complementation for BMDM differentiation from days 0 to 7. Data are expressed as relative normalized expression (as compared to two housekeeping genes). Values are mean ± SEM. In (D,E) the HD11 cell line was stimulated with PAF and/or PAFR antagonists (PCA 4248 and WEB 2086), and the increase in intracellular calcium signal was recorded over time (216 s, 3 s intervals) using a fluorescent probe. RLU, relative luminescence units; AUC, area under the curve.

Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Derivative Assay, Amplification, Expressing, Purification

2) Product Images from "Autophagy Plays a Critical Role in ChLym-1-Induced Cytotoxicity of Non-Hodgkin's Lymphoma Cells"

Article Title: Autophagy Plays a Critical Role in ChLym-1-Induced Cytotoxicity of Non-Hodgkin's Lymphoma Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0072478

Inhibition of autophagy with PI3K inhibitor 3-MA and lysosome inhibitor NH 4 Cl suppress chLym-1induced apoptosis, antibody-dependent cellular cytotoxicity(ADCC) and complement-dependent cytotoxicity(CDC) in Raji cells. A: 3-MA and NH 4 Cl significantly inhibit apoptosis induced by chLym-1 in Raji cells. Raji cells were treated with 10 µg/ml of chLym-1 or/and autophagy inhibitors for 24 h, while Vehicles were treated with full medium. Thus, FCM was used to detect apoptotic rates of Raji cells. B: ChLym-1-induced ADCC is inhibited by PI3K inhibitor 3-MA. Raji cells, together with PBMCs (effect cells: target cells = 60∶1, 40∶1 and 20∶1), were treated with autophagy inhibitor 3-MA or/and chLym-1 for 5 h. Autophagy inhibitor 3-MA was added 12 h before chLym-1 treatment. Then, ADCC mediated by chLym-1 on Raji cells was measured in a 5 h LDH release assay. C: ChLym-1-induced ADCC is inhibited by Anti-ATG5 RNA inference. Raji cells (non-treated, transferred with control siRNA or transferred with Anti-ATG5 siRNA), together with PBMCs (effect cells: target cells = 60∶1, 40∶1 and 20∶1), were treated with chLym-1 for 5 h. ATG5 RNA-inference is performed 48 h before chLym-1 treatment. Then, ADCC mediated by chLym-1 on Raji cells was measured in a 5 h LDH release assay. D: ChLym-1 mediated CDC is blocked by PI3K inhibitor 3-MA or lysosome inhibitor NH 4 Cl. Raji cells were treated with autophagy inhibitor 3-MA/NH 4 Cl or/and chLym-1 for 1 h. Autophagy inhibitor 3-MA and NH 4 Cl was added 24 h before chLym-1 treatment. Then, CDC mediated by chLym-1 on Raji cells was measured immediately by MTT assay.
Figure Legend Snippet: Inhibition of autophagy with PI3K inhibitor 3-MA and lysosome inhibitor NH 4 Cl suppress chLym-1induced apoptosis, antibody-dependent cellular cytotoxicity(ADCC) and complement-dependent cytotoxicity(CDC) in Raji cells. A: 3-MA and NH 4 Cl significantly inhibit apoptosis induced by chLym-1 in Raji cells. Raji cells were treated with 10 µg/ml of chLym-1 or/and autophagy inhibitors for 24 h, while Vehicles were treated with full medium. Thus, FCM was used to detect apoptotic rates of Raji cells. B: ChLym-1-induced ADCC is inhibited by PI3K inhibitor 3-MA. Raji cells, together with PBMCs (effect cells: target cells = 60∶1, 40∶1 and 20∶1), were treated with autophagy inhibitor 3-MA or/and chLym-1 for 5 h. Autophagy inhibitor 3-MA was added 12 h before chLym-1 treatment. Then, ADCC mediated by chLym-1 on Raji cells was measured in a 5 h LDH release assay. C: ChLym-1-induced ADCC is inhibited by Anti-ATG5 RNA inference. Raji cells (non-treated, transferred with control siRNA or transferred with Anti-ATG5 siRNA), together with PBMCs (effect cells: target cells = 60∶1, 40∶1 and 20∶1), were treated with chLym-1 for 5 h. ATG5 RNA-inference is performed 48 h before chLym-1 treatment. Then, ADCC mediated by chLym-1 on Raji cells was measured in a 5 h LDH release assay. D: ChLym-1 mediated CDC is blocked by PI3K inhibitor 3-MA or lysosome inhibitor NH 4 Cl. Raji cells were treated with autophagy inhibitor 3-MA/NH 4 Cl or/and chLym-1 for 1 h. Autophagy inhibitor 3-MA and NH 4 Cl was added 24 h before chLym-1 treatment. Then, CDC mediated by chLym-1 on Raji cells was measured immediately by MTT assay.

Techniques Used: Inhibition, Lactate Dehydrogenase Assay, MTT Assay

3) Product Images from "NFKB2 mutation in common variable immunodeficiency and isolated adrenocorticotropic hormone deficiency"

Article Title: NFKB2 mutation in common variable immunodeficiency and isolated adrenocorticotropic hormone deficiency

Journal: Medicine

doi: 10.1097/MD.0000000000005081

A novel NFKB2 mutation and NF-κB sequence alignments. (A) Sanger sequencing revealed a heterozygous c.2563 A > T (p.855: Lys > ∗ ) mutation (arrow) in NFKB2 gene of the proband. This variation was not identified in her parents. (B) NF-κB p100 C-terminus amino acid sequence alignments. Lysine 855, highlighted in blue, serves as an acceptor for ubiquitination. Serine 866 and 870, highlighted in yellow, are phosphorylation sites that lead to proteolysis. (C) NFKB2 transcripts in peripheral blood mononuclear cells from proband and a healthy control were quantified by real-time PCR.
Figure Legend Snippet: A novel NFKB2 mutation and NF-κB sequence alignments. (A) Sanger sequencing revealed a heterozygous c.2563 A > T (p.855: Lys > ∗ ) mutation (arrow) in NFKB2 gene of the proband. This variation was not identified in her parents. (B) NF-κB p100 C-terminus amino acid sequence alignments. Lysine 855, highlighted in blue, serves as an acceptor for ubiquitination. Serine 866 and 870, highlighted in yellow, are phosphorylation sites that lead to proteolysis. (C) NFKB2 transcripts in peripheral blood mononuclear cells from proband and a healthy control were quantified by real-time PCR.

Techniques Used: Mutagenesis, Sequencing, Real-time Polymerase Chain Reaction

4) Product Images from "Pregnancy, Microchimerism, and the Maternal Grandmother"

Article Title: Pregnancy, Microchimerism, and the Maternal Grandmother

Journal: PLoS ONE

doi: 10.1371/journal.pone.0024101

Schematic representation of strategy used to identify microchimerism. In this example, HLA genotyping for DRB1 is shown for a proband, her mother (MP), and her fetus. Once a polymorphism unique to the MP is identified (DRB1*01 in this example), polymorphism-specific quantitative PCR can then be used to quantify MP microchimerism in DNA extracted from proband PBMC.
Figure Legend Snippet: Schematic representation of strategy used to identify microchimerism. In this example, HLA genotyping for DRB1 is shown for a proband, her mother (MP), and her fetus. Once a polymorphism unique to the MP is identified (DRB1*01 in this example), polymorphism-specific quantitative PCR can then be used to quantify MP microchimerism in DNA extracted from proband PBMC.

Techniques Used: Real-time Polymerase Chain Reaction

5) Product Images from "Persistence of Viral Reservoirs in Multiple Tissues after Antiretroviral Therapy Suppression in a Macaque RT-SHIV Model"

Article Title: Persistence of Viral Reservoirs in Multiple Tissues after Antiretroviral Therapy Suppression in a Macaque RT-SHIV Model

Journal: PLoS ONE

doi: 10.1371/journal.pone.0084275

The ratio of RT-SHIV gag DNA copies per 10 6 macaque CCR5 DNA copies were measured in PBMC isolated at different time points from each of the untreated and treated macaques.
Figure Legend Snippet: The ratio of RT-SHIV gag DNA copies per 10 6 macaque CCR5 DNA copies were measured in PBMC isolated at different time points from each of the untreated and treated macaques.

Techniques Used: Isolation

6) Product Images from "Phenotype and envelope gene diversity of nef-deleted HIV-1 isolated from long-term survivors infected from a single source"

Article Title: Phenotype and envelope gene diversity of nef-deleted HIV-1 isolated from long-term survivors infected from a single source

Journal: Virology Journal

doi: 10.1186/1743-422X-4-75

V1V2 length polymorphism analysis . The HIV-1 Env V1V2 region incorporating a 6-carboxy-fluorescien fluorophore was amplified by PCR from genomic DNA of HIV-1 infected PBMC and subjected to GeneScan analysis, as described in the Methods and elsewhere [41, 56]. (A) GeneScan sample files generated from amplified products. (B) Fraction of sequences with a given V1V2 nucleotide length, which was calculated from GeneScan sample files. Peaks and bars shown in red represent V1V2 amplimers from early viruses, and peaks and bars shown in blue represent V1V2 amplimers from late viruses. Similar results were obtained in two independent experiments.
Figure Legend Snippet: V1V2 length polymorphism analysis . The HIV-1 Env V1V2 region incorporating a 6-carboxy-fluorescien fluorophore was amplified by PCR from genomic DNA of HIV-1 infected PBMC and subjected to GeneScan analysis, as described in the Methods and elsewhere [41, 56]. (A) GeneScan sample files generated from amplified products. (B) Fraction of sequences with a given V1V2 nucleotide length, which was calculated from GeneScan sample files. Peaks and bars shown in red represent V1V2 amplimers from early viruses, and peaks and bars shown in blue represent V1V2 amplimers from late viruses. Similar results were obtained in two independent experiments.

Techniques Used: Amplification, Polymerase Chain Reaction, Infection, Generated

V1V2 HTA analysis . HIV-1 Env V1V2 regions were amplified by PCR from genomic DNA of HIV-1 infected PBMC and subjected to HTA analysis as described in the Methods. HTA analysis using a [ 32 P]-labelled ADA V1V2 Env probe is shown in the left panel, and HTA analysis using a [ 32 P]-labelled NL4-3 V1V2 Env probe is shown in the right panel. Similar results were obtained in three independent experiments.
Figure Legend Snippet: V1V2 HTA analysis . HIV-1 Env V1V2 regions were amplified by PCR from genomic DNA of HIV-1 infected PBMC and subjected to HTA analysis as described in the Methods. HTA analysis using a [ 32 P]-labelled ADA V1V2 Env probe is shown in the left panel, and HTA analysis using a [ 32 P]-labelled NL4-3 V1V2 Env probe is shown in the right panel. Similar results were obtained in three independent experiments.

Techniques Used: Amplification, Polymerase Chain Reaction, Infection

V3 HTA analysis . The HIV-1 Env V3 region was amplified by PCR from genomic DNA of HIV-1 infected PBMC and subjected to HTA analysis as described in the Methods. HTA analysis using a [ 32 P]-labelled ADA V3 Env probe is shown in the left panel, and HTA analysis using a [ 32 P]-labelled NL4-3 V3 Env probe is shown in the right panel. Similar results were obtained in three independent experiments.
Figure Legend Snippet: V3 HTA analysis . The HIV-1 Env V3 region was amplified by PCR from genomic DNA of HIV-1 infected PBMC and subjected to HTA analysis as described in the Methods. HTA analysis using a [ 32 P]-labelled ADA V3 Env probe is shown in the left panel, and HTA analysis using a [ 32 P]-labelled NL4-3 V3 Env probe is shown in the right panel. Similar results were obtained in three independent experiments.

Techniques Used: Amplification, Polymerase Chain Reaction, Infection

7) Product Images from "A TNF-Regulated Recombinatorial Macrophage Immune Receptor Implicated in Granuloma Formation in Tuberculosis"

Article Title: A TNF-Regulated Recombinatorial Macrophage Immune Receptor Implicated in Granuloma Formation in Tuberculosis

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002375

ΤCRαβ expression by subpopulations of human and murine monocytes/macrophages. ( A ) Fluorescence immunocytochemistry demonstrating that a ∼5% subpopulation of human peripheral blood CD14 + /MHC-II + monocytes expresses the ΤCRαβ. CD14-MACS purified peripheral blood monocytes were isolated from a healthy donor and double-immunostained with Abs against the ΤCRαβ (red) and MHC-II (green). Isotype controls for the anti-ΤCRαβ and anti-MHC-II antibodies are shown (right). Scale bars are indicated. Data shown are representative of n = 12 donors. ( B ) Flow cytometry of peripheral blood mononuclear cells from a healthy individual demonstrates the presence of the TCRβ on the surface of a CD14 + monocyte subpopulation (3.5%, red arrows). Staining for TCRβ and lineage surface markers are shown. CD14 + monocytes are in pink color, CD3 + lymphocytes in blue. ( C ) (Left) Laser scanning cytometry (LSC) of unstimulated (naïve) monocyte-derived macrophages stained for ΤCRαβ (red). Nuclei are counterstained with DAPI (blue). The cytometric analysis shows a subpopulation (5%) with high fluoresence indicative of ΤCRαβ positive naïve macrophages (top right) which is highlighted in the histogram below (arrow). (Bottom) LSC of naïve and IL-4 (10 ng/ml) or IFNγ (1000 U/ml) stimulated monocyte-derived macrophages, respectively, cultured for 6 days. The percentage of ΤCRαβ + cells in each macrophage population is shown for three healthy individuals. ( D ) Detection of the TCR α- and β-chain in CD14 + monocytes and IFNγ or IL-4 polarized macrophages by immunoblot. β-actin, loading control. ( E ) Immunogold electron microscopy demonstrating the presence of the TCR α-subunit on the cell surface of a human IFNγ stimulated macrophage (arrows). (Right) isotype control. ( F ) Immunocytochemical double-staining reveals the presence of the TCRαβ (green) in alveolar macrophages from a 45 year old male with normal BAL cytology. Shown is a ΤCRαβ + alveolar macrophage (top right, arrow) next to a ΤCRαβ + T cell (asterisk). The merged image (bottom right) demonstrates that the majority of the cells express the macrophage marker CD163 (red). Giemsa-staining of the BAL cytospin preparation and isotype controls are shown in the left panel. The results are representative of three individuals. Nuclei (blue), DRAQ5. ( G ) Confocal immunofluorescence microscopy shows ΤCRαβ expression in murine macrophages. Spleen macrophages pooled from three normal C57BL/6 J mice were CD11b-MACS purified and immunofluorescence double-staining was performed using the anti-macrophage antibody F4/80 (red) and an anti-mouse TCRβ antibody that recognizes a common epitope of the murine TCRαβ complex (green). The outlined area is shown at a higher magnification. Nuclei (blue) are counterstained with DRAQ5. Isotype controls are shown. (Bottom) RT-PCR demonstrating expression of the murine TCRα and TCRβ constant chain genes in CD11b-MACS purified spleen macrophages (MΦ) from C57Bl6/J mice. Ly6G + neutrophils are shown as positive control.
Figure Legend Snippet: ΤCRαβ expression by subpopulations of human and murine monocytes/macrophages. ( A ) Fluorescence immunocytochemistry demonstrating that a ∼5% subpopulation of human peripheral blood CD14 + /MHC-II + monocytes expresses the ΤCRαβ. CD14-MACS purified peripheral blood monocytes were isolated from a healthy donor and double-immunostained with Abs against the ΤCRαβ (red) and MHC-II (green). Isotype controls for the anti-ΤCRαβ and anti-MHC-II antibodies are shown (right). Scale bars are indicated. Data shown are representative of n = 12 donors. ( B ) Flow cytometry of peripheral blood mononuclear cells from a healthy individual demonstrates the presence of the TCRβ on the surface of a CD14 + monocyte subpopulation (3.5%, red arrows). Staining for TCRβ and lineage surface markers are shown. CD14 + monocytes are in pink color, CD3 + lymphocytes in blue. ( C ) (Left) Laser scanning cytometry (LSC) of unstimulated (naïve) monocyte-derived macrophages stained for ΤCRαβ (red). Nuclei are counterstained with DAPI (blue). The cytometric analysis shows a subpopulation (5%) with high fluoresence indicative of ΤCRαβ positive naïve macrophages (top right) which is highlighted in the histogram below (arrow). (Bottom) LSC of naïve and IL-4 (10 ng/ml) or IFNγ (1000 U/ml) stimulated monocyte-derived macrophages, respectively, cultured for 6 days. The percentage of ΤCRαβ + cells in each macrophage population is shown for three healthy individuals. ( D ) Detection of the TCR α- and β-chain in CD14 + monocytes and IFNγ or IL-4 polarized macrophages by immunoblot. β-actin, loading control. ( E ) Immunogold electron microscopy demonstrating the presence of the TCR α-subunit on the cell surface of a human IFNγ stimulated macrophage (arrows). (Right) isotype control. ( F ) Immunocytochemical double-staining reveals the presence of the TCRαβ (green) in alveolar macrophages from a 45 year old male with normal BAL cytology. Shown is a ΤCRαβ + alveolar macrophage (top right, arrow) next to a ΤCRαβ + T cell (asterisk). The merged image (bottom right) demonstrates that the majority of the cells express the macrophage marker CD163 (red). Giemsa-staining of the BAL cytospin preparation and isotype controls are shown in the left panel. The results are representative of three individuals. Nuclei (blue), DRAQ5. ( G ) Confocal immunofluorescence microscopy shows ΤCRαβ expression in murine macrophages. Spleen macrophages pooled from three normal C57BL/6 J mice were CD11b-MACS purified and immunofluorescence double-staining was performed using the anti-macrophage antibody F4/80 (red) and an anti-mouse TCRβ antibody that recognizes a common epitope of the murine TCRαβ complex (green). The outlined area is shown at a higher magnification. Nuclei (blue) are counterstained with DRAQ5. Isotype controls are shown. (Bottom) RT-PCR demonstrating expression of the murine TCRα and TCRβ constant chain genes in CD11b-MACS purified spleen macrophages (MΦ) from C57Bl6/J mice. Ly6G + neutrophils are shown as positive control.

Techniques Used: Expressing, Fluorescence, Immunocytochemistry, Magnetic Cell Separation, Purification, Isolation, Flow Cytometry, Cytometry, Staining, Derivative Assay, Cell Culture, Electron Microscopy, Double Staining, Marker, Immunofluorescence, Microscopy, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Positive Control

The monocyte/macrophage TCRαβ is a recombinatorial receptor. ( A ) Detection of D → J (i) and V → DJ (ii) rearrangements in the TCRβ gene locus of human CD14 + monocytes and IFNγ macrophages. Arrows denote the presence of Dβ1→ Jβ and Vβ1→ Jβ rearrangements which were confirmed by sequencing. Genomic organization of the identified rearrangements is schematically drawn. Peripheral blood mononuclear cells (PBMC), positive control. HepG2 cells, Ø control. ( B ) Expression of individual-specific TCR Vβ repertoires by monocytes, IL-4 macrophages (blue) and IFNγ macrophages (red) from three healthy donors (1–3) representatively shown for Vβ13a. A scaled synopsis of the three cell populations is shown at the bottom (∑). ( C ) TCR clonotype analysis by sequencing of the antigen-binding CDR3 loop of Vβ13a representatively shown for individual 2. IL-4 (blue) and IFNγ activated macrophages (red) express completely different Vβ13a clonotypes. The Vβ13a chain is not expressed by the monocytes of this individual (cf. B). Colored letters represent deduced amino acid sequences of the newly identified CDR3 β regions (GenBank Acc. No. JF923737-JF923744). D ) Expression of rearranged TCR Vβ CDR3 clonotypes in granulocyte/macrophage progenitor colonies (CFU-GM) obtained from CD34 + progenitors of two healthy individuals (A and B). Filled boxes indicate positive expression of at least one of the 25 known human TCR Vβ chains (x-axis) in a single colony. Colonies are identified by numbering on the y-axis. The repertoires for each of the expressed Vβ chains were determined by length variant analysis of the antigen-binding CDR3 β region. The detailed Vβ repertoire is representatively shown for colony CFU-GM2 (donor B). The repertoires of additional CFU-GM colonies are summarized in Figure S2B . RT-PCR lineage marker expression profiling documents the monocytic nature of this granulocyte/macrophage progenitor colony. CD2, CD8: T lymphoid markers; MMP25, MPO: granulocyte markers; CD14, CD68, CD163: monocyte markers. ( E ) Direct mass spectrometric identification of multiple TCR Vα- and Vβ-chain variants in human macrophages. Protein lysates from IFNγ macrophages of a healthy donor were immunoprecipitated using an anti-TCRβ antibody and the predicted 58 kD band (boxed) was analyzed by MALDI-TOF mass spectrometry. Peaks 1–6 represent TCR Vα- and Vβ-specific peptide fragments whose amino acid sequence identities with known TCR Vαβ-clonotypes are bolded. In three cases (2, 4 and 6), the identified peptides span V→ J and J→ C junctions (denoted by a gap) indicative of genomic rearrangements in the macrophage TCRα and -β loci. ( F ) Peritoneal macrophages from C57Bl6/J mice (rag1 +/+ ) but not recombination defective rag1 –/– mice express Vα (left) and Vβ repertoires (right) as evidenced by TCR V-chain mRNA expression profiling (top) and CDR3 spectratyping of representative TCR Vα- and Vβ-chains (bottom). Peritoneal macrophages were pooled from three rag1 +/+ mice and an equal number of rag1 –/– mice, respectively.
Figure Legend Snippet: The monocyte/macrophage TCRαβ is a recombinatorial receptor. ( A ) Detection of D → J (i) and V → DJ (ii) rearrangements in the TCRβ gene locus of human CD14 + monocytes and IFNγ macrophages. Arrows denote the presence of Dβ1→ Jβ and Vβ1→ Jβ rearrangements which were confirmed by sequencing. Genomic organization of the identified rearrangements is schematically drawn. Peripheral blood mononuclear cells (PBMC), positive control. HepG2 cells, Ø control. ( B ) Expression of individual-specific TCR Vβ repertoires by monocytes, IL-4 macrophages (blue) and IFNγ macrophages (red) from three healthy donors (1–3) representatively shown for Vβ13a. A scaled synopsis of the three cell populations is shown at the bottom (∑). ( C ) TCR clonotype analysis by sequencing of the antigen-binding CDR3 loop of Vβ13a representatively shown for individual 2. IL-4 (blue) and IFNγ activated macrophages (red) express completely different Vβ13a clonotypes. The Vβ13a chain is not expressed by the monocytes of this individual (cf. B). Colored letters represent deduced amino acid sequences of the newly identified CDR3 β regions (GenBank Acc. No. JF923737-JF923744). D ) Expression of rearranged TCR Vβ CDR3 clonotypes in granulocyte/macrophage progenitor colonies (CFU-GM) obtained from CD34 + progenitors of two healthy individuals (A and B). Filled boxes indicate positive expression of at least one of the 25 known human TCR Vβ chains (x-axis) in a single colony. Colonies are identified by numbering on the y-axis. The repertoires for each of the expressed Vβ chains were determined by length variant analysis of the antigen-binding CDR3 β region. The detailed Vβ repertoire is representatively shown for colony CFU-GM2 (donor B). The repertoires of additional CFU-GM colonies are summarized in Figure S2B . RT-PCR lineage marker expression profiling documents the monocytic nature of this granulocyte/macrophage progenitor colony. CD2, CD8: T lymphoid markers; MMP25, MPO: granulocyte markers; CD14, CD68, CD163: monocyte markers. ( E ) Direct mass spectrometric identification of multiple TCR Vα- and Vβ-chain variants in human macrophages. Protein lysates from IFNγ macrophages of a healthy donor were immunoprecipitated using an anti-TCRβ antibody and the predicted 58 kD band (boxed) was analyzed by MALDI-TOF mass spectrometry. Peaks 1–6 represent TCR Vα- and Vβ-specific peptide fragments whose amino acid sequence identities with known TCR Vαβ-clonotypes are bolded. In three cases (2, 4 and 6), the identified peptides span V→ J and J→ C junctions (denoted by a gap) indicative of genomic rearrangements in the macrophage TCRα and -β loci. ( F ) Peritoneal macrophages from C57Bl6/J mice (rag1 +/+ ) but not recombination defective rag1 –/– mice express Vα (left) and Vβ repertoires (right) as evidenced by TCR V-chain mRNA expression profiling (top) and CDR3 spectratyping of representative TCR Vα- and Vβ-chains (bottom). Peritoneal macrophages were pooled from three rag1 +/+ mice and an equal number of rag1 –/– mice, respectively.

Techniques Used: Sequencing, Positive Control, Expressing, Binding Assay, Variant Assay, Reverse Transcription Polymerase Chain Reaction, Marker, Immunoprecipitation, Mass Spectrometry, Mouse Assay

8) Product Images from "A Cell-Based Systems Biology Assessment of Human Blood to Monitor Immune Responses after Influenza Vaccination"

Article Title: A Cell-Based Systems Biology Assessment of Human Blood to Monitor Immune Responses after Influenza Vaccination

Journal: PLoS ONE

doi: 10.1371/journal.pone.0118528

Visualization of differentially expressed RNA transcripts in PBMC and individual immune cell types. Circos plots of differentially expressed RNA transcripts from a vaccinated subject at (a) day 1, (b) day 3, and (c) day 7 post-TIV vaccination (fold change of ≥1.5x and p ≤ 0.05). All RNA transcript classes are represented. For each cell type, the colored bar on the outer circle represents the entire human genome; segments within the bars divide the genome into chromosomes. Red lines indicate DE transcripts that are shared between PBMC and purified immune cell types. Gray lines indicate DE transcripts that are shared between the purified immune cell types.
Figure Legend Snippet: Visualization of differentially expressed RNA transcripts in PBMC and individual immune cell types. Circos plots of differentially expressed RNA transcripts from a vaccinated subject at (a) day 1, (b) day 3, and (c) day 7 post-TIV vaccination (fold change of ≥1.5x and p ≤ 0.05). All RNA transcript classes are represented. For each cell type, the colored bar on the outer circle represents the entire human genome; segments within the bars divide the genome into chromosomes. Red lines indicate DE transcripts that are shared between PBMC and purified immune cell types. Gray lines indicate DE transcripts that are shared between the purified immune cell types.

Techniques Used: Purification

RNA-Seq analysis of purified immune cells after TIV vaccination. (a) Pair-wise comparison of day 0 RNA profiles (all transcript classes represented, filtered to remove zero values; 32,505 transcripts) from a vaccinated subject shows that the transcriptome of each sorted cell type correlates weakly with PBMC and other sorted cell types. (b) PCA of RNA profiles (all transcript classes represented, filtered to remove zero values; 37,606 transcripts) from a TIV-vaccinated subject at four time points shows that the purified immune cell types cluster into distinct groups, although monocytes and mDC cluster closely. (c-h) Semi-supervised hierarchical clustering analysis of RNA expression from a vaccinated individual reveals that purified immune cells have distinct RNA expression profiles compared to PBMC at all time-points. Data (non-zero transcripts with an RPKM of 1 in at least one sample) was centered for normalized signal value across gene and cell type; red = up, black = no change, green = down. (c) All transcript classes (21,438 transcripts). (d) Protein coding transcripts (13,243 transcripts, including Ig and TCR transcripts). (e) Pseudogenes (3,466 transcripts, 2x scale). (f) Anti-sense RNA (1,310 transcripts, 2x scale). (g) lincRNA (1,047 transcripts, 2x scale). (h) New genes (167 transcripts, 5x scale).
Figure Legend Snippet: RNA-Seq analysis of purified immune cells after TIV vaccination. (a) Pair-wise comparison of day 0 RNA profiles (all transcript classes represented, filtered to remove zero values; 32,505 transcripts) from a vaccinated subject shows that the transcriptome of each sorted cell type correlates weakly with PBMC and other sorted cell types. (b) PCA of RNA profiles (all transcript classes represented, filtered to remove zero values; 37,606 transcripts) from a TIV-vaccinated subject at four time points shows that the purified immune cell types cluster into distinct groups, although monocytes and mDC cluster closely. (c-h) Semi-supervised hierarchical clustering analysis of RNA expression from a vaccinated individual reveals that purified immune cells have distinct RNA expression profiles compared to PBMC at all time-points. Data (non-zero transcripts with an RPKM of 1 in at least one sample) was centered for normalized signal value across gene and cell type; red = up, black = no change, green = down. (c) All transcript classes (21,438 transcripts). (d) Protein coding transcripts (13,243 transcripts, including Ig and TCR transcripts). (e) Pseudogenes (3,466 transcripts, 2x scale). (f) Anti-sense RNA (1,310 transcripts, 2x scale). (g) lincRNA (1,047 transcripts, 2x scale). (h) New genes (167 transcripts, 5x scale).

Techniques Used: RNA Sequencing Assay, Purification, RNA Expression

9) Product Images from "A phase II study of AT-101 (gossypol) in chemotherapy-sensitive recurrent extensive stage small cell lung cancer (ES-SCLC)"

Article Title: A phase II study of AT-101 (gossypol) in chemotherapy-sensitive recurrent extensive stage small cell lung cancer (ES-SCLC)

Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

doi: 10.1097/JTO.0b013e31822e2941

Relative caspase activity in peripheral blood mononuclear cells
Figure Legend Snippet: Relative caspase activity in peripheral blood mononuclear cells

Techniques Used: Activity Assay

10) Product Images from "A common mechanism of clinical HIV-1 resistance to the CCR5 antagonist maraviroc despite divergent resistance levels and lack of common gp120 resistance mutations"

Article Title: A common mechanism of clinical HIV-1 resistance to the CCR5 antagonist maraviroc despite divergent resistance levels and lack of common gp120 resistance mutations

Journal: Retrovirology

doi: 10.1186/1742-4690-10-43

Profiles of resistance to MVC and cross-resistance to other CCR5 antagonists. Luciferase reporter viruses pseudotyped with Envs from subject 17 or subject 24 were used to infect NP2-CD4/CCR5 ( A , left panels) or PBMC ( A , right panels) in the presence of increasing concentrations of MVC. The same virus preparations were used to infect NP2-CD4/CCR5 cells in the presence of increasing concentrations of VVC or TAK-779 ( B ). Virus inhibition curves were constructed as described in the Methods. The data points represent the mean and standard error of the mean from quadruplicate wells, and are the results from 5 (MVC) or 3 (VVC and TAK-779) independent experiments. The independent PBMC experiments were performed in cells obtained from different donors. Inhibition curves were constructed using Prism, version 4.0c (GraphPad).
Figure Legend Snippet: Profiles of resistance to MVC and cross-resistance to other CCR5 antagonists. Luciferase reporter viruses pseudotyped with Envs from subject 17 or subject 24 were used to infect NP2-CD4/CCR5 ( A , left panels) or PBMC ( A , right panels) in the presence of increasing concentrations of MVC. The same virus preparations were used to infect NP2-CD4/CCR5 cells in the presence of increasing concentrations of VVC or TAK-779 ( B ). Virus inhibition curves were constructed as described in the Methods. The data points represent the mean and standard error of the mean from quadruplicate wells, and are the results from 5 (MVC) or 3 (VVC and TAK-779) independent experiments. The independent PBMC experiments were performed in cells obtained from different donors. Inhibition curves were constructed using Prism, version 4.0c (GraphPad).

Techniques Used: Luciferase, Inhibition, Construct

11) Product Images from "Evaluation of antigen specific recognition and cell mediated cytotoxicity by a modified lysispot assay in a rat colon carcinoma model"

Article Title: Evaluation of antigen specific recognition and cell mediated cytotoxicity by a modified lysispot assay in a rat colon carcinoma model

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/1756-9966-31-9

IFN-γ release . IFN-γ-ELISpot results from three different experiments, expressed as number of spots per well (mean ± SD), showed the immune-response of DHD-K12-inoculated rats (dark grey) against CSH-275 peptide. No effect was produced on PBMC from control rats (light grey). Increasing concentration of peptide yielded an increasing numbers of IFN-γ producing PBMC. Under each histogram there is the corresponding image illustrative of blue spots. As negative contros we showed the non stimulated PBMC (W/O).
Figure Legend Snippet: IFN-γ release . IFN-γ-ELISpot results from three different experiments, expressed as number of spots per well (mean ± SD), showed the immune-response of DHD-K12-inoculated rats (dark grey) against CSH-275 peptide. No effect was produced on PBMC from control rats (light grey). Increasing concentration of peptide yielded an increasing numbers of IFN-γ producing PBMC. Under each histogram there is the corresponding image illustrative of blue spots. As negative contros we showed the non stimulated PBMC (W/O).

Techniques Used: Enzyme-linked Immunospot, Cell Surface Hydrophobicity, Produced, Concentration Assay

Panel A - LysiSpot assay . LysiSpot assay results, expressed as net number of spots per well (spots from wells containing only target cells were subtracted), from four different experiments (mean ± SD). Increasing numbers of target cells were plated in short term cultures with effector cells (2 × 10 5 /well PBMC). Spots were the imprint of β-gal, released by the transfected DHD-K12 target cells after lysis. Cytotoxic activity of PBMC from DHD-K12-inoculated rats or control rats are represented by dark and light grey respectively. Panel B - LDH-Cytotoxicity assay. Cytotoxic activity expressed as percent of specific lysis (mean ± SD) of DHD-K12 target cells from PBMC of intact (control) or DHD-K12-inoculated rats (Primed) evaluated by Promega CytoTox 96 kit. Concentration ratio of effector and target cells was 10:1 (light grey), 5:1 (dark grey), 2.5:1 (white), 1.25:1 (black) and corresponding respectively to 2 × 10 4 ,1 × 10 4 , 5 × 10 3 , 2.5 × 10 3 of DHD-K12 target cells.
Figure Legend Snippet: Panel A - LysiSpot assay . LysiSpot assay results, expressed as net number of spots per well (spots from wells containing only target cells were subtracted), from four different experiments (mean ± SD). Increasing numbers of target cells were plated in short term cultures with effector cells (2 × 10 5 /well PBMC). Spots were the imprint of β-gal, released by the transfected DHD-K12 target cells after lysis. Cytotoxic activity of PBMC from DHD-K12-inoculated rats or control rats are represented by dark and light grey respectively. Panel B - LDH-Cytotoxicity assay. Cytotoxic activity expressed as percent of specific lysis (mean ± SD) of DHD-K12 target cells from PBMC of intact (control) or DHD-K12-inoculated rats (Primed) evaluated by Promega CytoTox 96 kit. Concentration ratio of effector and target cells was 10:1 (light grey), 5:1 (dark grey), 2.5:1 (white), 1.25:1 (black) and corresponding respectively to 2 × 10 4 ,1 × 10 4 , 5 × 10 3 , 2.5 × 10 3 of DHD-K12 target cells.

Techniques Used: Transfection, Lysis, Activity Assay, LDH Cytotoxicity Assay, Concentration Assay

Dual-colour LysiSpot assay . Dual-colour LysiSpot assay results from three different experiments (mean ± SD), expressed as net number of spots per well (spots from control wells containing only target cells or PBMC cultured alone were subtracted from the spots counted in the cocultures. DHD-K12 transfected cells (2 × 10 4 /well) were cocultured with 2 × 10 5 /well PBMC. The panel shows the image of the different spots left on the wells at the end of assay: 1) pure red spots indicate cell lysis by IFN-γ non-producing cells; 2) pure blue spots indicate IFN-γ secreting cells; 3) violet spots indicate cell lysis by IFN-γ producing cells. Dark and light grey bars represent number of spots from DHD-K12-inoculated rats or from control rats respectively.
Figure Legend Snippet: Dual-colour LysiSpot assay . Dual-colour LysiSpot assay results from three different experiments (mean ± SD), expressed as net number of spots per well (spots from control wells containing only target cells or PBMC cultured alone were subtracted from the spots counted in the cocultures. DHD-K12 transfected cells (2 × 10 4 /well) were cocultured with 2 × 10 5 /well PBMC. The panel shows the image of the different spots left on the wells at the end of assay: 1) pure red spots indicate cell lysis by IFN-γ non-producing cells; 2) pure blue spots indicate IFN-γ secreting cells; 3) violet spots indicate cell lysis by IFN-γ producing cells. Dark and light grey bars represent number of spots from DHD-K12-inoculated rats or from control rats respectively.

Techniques Used: Cell Culture, Transfection, Lysis

12) Product Images from "Chimeric Matrix Proteins Encoded by Defective Proviruses with Large Internal Deletions in Human T-Cell Leukemia Virus Type 1-Infected Humans"

Article Title: Chimeric Matrix Proteins Encoded by Defective Proviruses with Large Internal Deletions in Human T-Cell Leukemia Virus Type 1-Infected Humans

Journal: Journal of Virology

doi:

Detection of HTLV-1 proviruses with large internal deletions by nested PCR-Southern blotting in the DNA from PBMCs of the ATLL and TSP/HAM patients. PCR was performed with the A1-B1 and A2-B2 pairs of primers. Lanes: 1, DNA from MT-2 cells; 2, DNA from noninfected CEM cells; 3 to 8, DNAs from ATLL patients; 9 to 12, DNAs from TSP/HAM patients; 13, water control. Position of the p28 provirus from MT-2 cells is marked by an arrow.
Figure Legend Snippet: Detection of HTLV-1 proviruses with large internal deletions by nested PCR-Southern blotting in the DNA from PBMCs of the ATLL and TSP/HAM patients. PCR was performed with the A1-B1 and A2-B2 pairs of primers. Lanes: 1, DNA from MT-2 cells; 2, DNA from noninfected CEM cells; 3 to 8, DNAs from ATLL patients; 9 to 12, DNAs from TSP/HAM patients; 13, water control. Position of the p28 provirus from MT-2 cells is marked by an arrow.

Techniques Used: Nested PCR, Southern Blot, Polymerase Chain Reaction

13) Product Images from "Efficacy of HIV antiviral polyanionic carbosilane dendrimer G2-S16 in the presence of semen"

Article Title: Efficacy of HIV antiviral polyanionic carbosilane dendrimer G2-S16 in the presence of semen

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S104292

Effect of SEVI on the antiviral activity of G2-S16 polyanionic carbosilane dendrimer to block HIV-1 infection of TZM.bl cells and PBMC. Notes: Cells were pretreated with G2-S16 at a concentration range of 0.01–20 µM. After 1 hour, the cells were infected with R5-HIV-1 NL(AD8) , pCH058.c, and pTHRO.c in the absence and presence of SE at a concentration of 20 ng/10 6 cells. RLU values were measured 72 hours after infection by quantification of luciferase expression. Data represent the mean ± SEM (n=3). Abbreviations: HIV, human immunodeficiency virus; PBMC, peripheral blood mononuclear cells; SE, semen; SEM, standard error of mean; SEVI, semen-derived enhancer of viral infection; RLU, relative light unit.
Figure Legend Snippet: Effect of SEVI on the antiviral activity of G2-S16 polyanionic carbosilane dendrimer to block HIV-1 infection of TZM.bl cells and PBMC. Notes: Cells were pretreated with G2-S16 at a concentration range of 0.01–20 µM. After 1 hour, the cells were infected with R5-HIV-1 NL(AD8) , pCH058.c, and pTHRO.c in the absence and presence of SE at a concentration of 20 ng/10 6 cells. RLU values were measured 72 hours after infection by quantification of luciferase expression. Data represent the mean ± SEM (n=3). Abbreviations: HIV, human immunodeficiency virus; PBMC, peripheral blood mononuclear cells; SE, semen; SEM, standard error of mean; SEVI, semen-derived enhancer of viral infection; RLU, relative light unit.

Techniques Used: Activity Assay, Blocking Assay, Infection, Concentration Assay, Luciferase, Expressing, Derivative Assay

Effect of SEVI on the antiviral activity of G2-S16 polyanionic carbosilane dendrimer to block HIV-1 infection of TZM.bl cells and PBMC. Notes: Cells were pretreated with G2-S16 at a concentration range of 0.01–20 µM. After 1 hour, the cells were infected with R5-HIV-1 NL(AD8) , pCH058.c, and pTHRO.c in the absence and presence of SE at a concentration of 20 ng/10 6 cells. Infection rates were measured 72 hours after infection by quantification of luciferase expression. Data represent the mean ± SEM (n=3). Abbreviations: HIV, human immunodeficiency virus; PBMC, peripheral blood mononuclear cells; SE, semen; SEM, standard error of mean; SEVI, semen-derived enhancer of viral infection; NT, nontreated.
Figure Legend Snippet: Effect of SEVI on the antiviral activity of G2-S16 polyanionic carbosilane dendrimer to block HIV-1 infection of TZM.bl cells and PBMC. Notes: Cells were pretreated with G2-S16 at a concentration range of 0.01–20 µM. After 1 hour, the cells were infected with R5-HIV-1 NL(AD8) , pCH058.c, and pTHRO.c in the absence and presence of SE at a concentration of 20 ng/10 6 cells. Infection rates were measured 72 hours after infection by quantification of luciferase expression. Data represent the mean ± SEM (n=3). Abbreviations: HIV, human immunodeficiency virus; PBMC, peripheral blood mononuclear cells; SE, semen; SEM, standard error of mean; SEVI, semen-derived enhancer of viral infection; NT, nontreated.

Techniques Used: Activity Assay, Blocking Assay, Infection, Concentration Assay, Luciferase, Expressing, Derivative Assay

14) Product Images from "Evaluation of antigen specific recognition and cell mediated cytotoxicity by a modified lysispot assay in a rat colon carcinoma model"

Article Title: Evaluation of antigen specific recognition and cell mediated cytotoxicity by a modified lysispot assay in a rat colon carcinoma model

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/1756-9966-31-9

IFN-γ release . IFN-γ-ELISpot results from three different experiments, expressed as number of spots per well (mean ± SD), showed the immune-response of DHD-K12-inoculated rats (dark grey) against CSH-275 peptide. No effect was produced on PBMC from control rats (light grey). Increasing concentration of peptide yielded an increasing numbers of IFN-γ producing PBMC. Under each histogram there is the corresponding image illustrative of blue spots. As negative contros we showed the non stimulated PBMC (W/O).
Figure Legend Snippet: IFN-γ release . IFN-γ-ELISpot results from three different experiments, expressed as number of spots per well (mean ± SD), showed the immune-response of DHD-K12-inoculated rats (dark grey) against CSH-275 peptide. No effect was produced on PBMC from control rats (light grey). Increasing concentration of peptide yielded an increasing numbers of IFN-γ producing PBMC. Under each histogram there is the corresponding image illustrative of blue spots. As negative contros we showed the non stimulated PBMC (W/O).

Techniques Used: Enzyme-linked Immunospot, Cell Surface Hydrophobicity, Produced, Concentration Assay

Panel A - LysiSpot assay . LysiSpot assay results, expressed as net number of spots per well (spots from wells containing only target cells were subtracted), from four different experiments (mean ± SD). Increasing numbers of target cells were plated in short term cultures with effector cells (2 × 10 5 /well PBMC). Spots were the imprint of β-gal, released by the transfected DHD-K12 target cells after lysis. Cytotoxic activity of PBMC from DHD-K12-inoculated rats or control rats are represented by dark and light grey respectively. Panel B - LDH-Cytotoxicity assay. Cytotoxic activity expressed as percent of specific lysis (mean ± SD) of DHD-K12 target cells from PBMC of intact (control) or DHD-K12-inoculated rats (Primed) evaluated by Promega CytoTox 96 kit. Concentration ratio of effector and target cells was 10:1 (light grey), 5:1 (dark grey), 2.5:1 (white), 1.25:1 (black) and corresponding respectively to 2 × 10 4 ,1 × 10 4 , 5 × 10 3 , 2.5 × 10 3 of DHD-K12 target cells.
Figure Legend Snippet: Panel A - LysiSpot assay . LysiSpot assay results, expressed as net number of spots per well (spots from wells containing only target cells were subtracted), from four different experiments (mean ± SD). Increasing numbers of target cells were plated in short term cultures with effector cells (2 × 10 5 /well PBMC). Spots were the imprint of β-gal, released by the transfected DHD-K12 target cells after lysis. Cytotoxic activity of PBMC from DHD-K12-inoculated rats or control rats are represented by dark and light grey respectively. Panel B - LDH-Cytotoxicity assay. Cytotoxic activity expressed as percent of specific lysis (mean ± SD) of DHD-K12 target cells from PBMC of intact (control) or DHD-K12-inoculated rats (Primed) evaluated by Promega CytoTox 96 kit. Concentration ratio of effector and target cells was 10:1 (light grey), 5:1 (dark grey), 2.5:1 (white), 1.25:1 (black) and corresponding respectively to 2 × 10 4 ,1 × 10 4 , 5 × 10 3 , 2.5 × 10 3 of DHD-K12 target cells.

Techniques Used: Transfection, Lysis, Activity Assay, LDH Cytotoxicity Assay, Concentration Assay

Dual-colour LysiSpot assay . Dual-colour LysiSpot assay results from three different experiments (mean ± SD), expressed as net number of spots per well (spots from control wells containing only target cells or PBMC cultured alone were subtracted from the spots counted in the cocultures. DHD-K12 transfected cells (2 × 10 4 /well) were cocultured with 2 × 10 5 /well PBMC. The panel shows the image of the different spots left on the wells at the end of assay: 1) pure red spots indicate cell lysis by IFN-γ non-producing cells; 2) pure blue spots indicate IFN-γ secreting cells; 3) violet spots indicate cell lysis by IFN-γ producing cells. Dark and light grey bars represent number of spots from DHD-K12-inoculated rats or from control rats respectively.
Figure Legend Snippet: Dual-colour LysiSpot assay . Dual-colour LysiSpot assay results from three different experiments (mean ± SD), expressed as net number of spots per well (spots from control wells containing only target cells or PBMC cultured alone were subtracted from the spots counted in the cocultures. DHD-K12 transfected cells (2 × 10 4 /well) were cocultured with 2 × 10 5 /well PBMC. The panel shows the image of the different spots left on the wells at the end of assay: 1) pure red spots indicate cell lysis by IFN-γ non-producing cells; 2) pure blue spots indicate IFN-γ secreting cells; 3) violet spots indicate cell lysis by IFN-γ producing cells. Dark and light grey bars represent number of spots from DHD-K12-inoculated rats or from control rats respectively.

Techniques Used: Cell Culture, Transfection, Lysis

Related Articles

RNA Extraction:

Article Title: Neonatal Fc receptor is involved in the protection of fibrinogen after its intake in peripheral blood mononuclear cells
Article Snippet: .. RNA extraction, reverse transcription and PCR Total RNA was extracted from SH-SY5Y, T-cells and PBMCs of the same control subject and from Jurkat and HepG2 cells using the ReliaPrep™ RNA cell miniprep system (Z6011, Promega, Milano, Italy) following the manufacturer’s instructions. .. Two micrograms of DNA-free total RNA were reverse transcribed into first-strand cDNA with random primers in a 20 μl final volume using the GoScript™ reverse transcription system (Promega, A5000).

DNA Purification:

Article Title: Anti-Bovine Programmed Death-1 Rat–Bovine Chimeric Antibody for Immunotherapy of Bovine Leukemia Virus Infection in Cattle
Article Snippet: .. Briefly, genomic DNA was extracted from 2 × 106 PBMCs with a Wizard Genomic DNA Purification Kit (Promega). .. Amplification of the BLV tax gene was performed in a reaction mixture containing 5 µl of Cycleave PCR Reaction Mix (Takara Bio, Otsu, Japan), 0.5 µl of Probe/Primer Mix for BLV (Takara Bio), 1 µl of a DNA template, and 3.5 µl of RNase-Free Distilled Water (Takara Bio) with a LightCycler 480 system II (Roche Diagnostics, Mannheim, Germany).

CtB Assay:

Article Title: Analysis of DNA Double-Strand Breaks and Cytotoxicity after 7 Tesla Magnetic Resonance Imaging of Isolated Human Lymphocytes
Article Snippet: .. Viability assay To monitor the metabolic activity of unstimulated PBMCs, CellTiter-Blue assay (Promega, Madison, WI, USA) was performed according to the manufacturer’s instructions. ..

Incubation:

Article Title: Autophagy Plays a Critical Role in ChLym-1-Induced Cytotoxicity of Non-Hodgkin's Lymphoma Cells
Article Snippet: .. Then cells were incubated with 10 µg/ml of chLym-1 in the presence of PBMC at a ratio of 1∶20, 1∶40 or 1∶60 for 5 h. The release of LDH was measured with cytotoxity 96 kit (Promega BioSciences, LLC., CA, USA). .. The percent-specific lysis was determined by the following equation: [(E-T)/(H-T)]×100, where E is the mean LDH released in the test samples with effector cells, H is the mean LDH released in the presence of lysis buffer, and T is the mean LDH released by target cells incubated with medium alone .

Quantitative RT-PCR:

Article Title: Expression of inhibitory regulators of innate immunity in patients with active tuberculosis
Article Snippet: .. Evaluation of mRNA levels by quantitative RT-PCR Total RNA obtained from PBMCs and AMs was reverse transcribed using oligo(dT) primer and moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA) according to recommendations of the suppliers. .. RT-PCRs were performed using FastStart DNA Master SYBR Green I in a Light Cycler apparatus (Roche Applied Science, Penzberg, Germany).

Affinity Magnetic Separation:

Article Title: Expression of inhibitory regulators of innate immunity in patients with active tuberculosis
Article Snippet: .. Evaluation of mRNA levels by quantitative RT-PCR Total RNA obtained from PBMCs and AMs was reverse transcribed using oligo(dT) primer and moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA) according to recommendations of the suppliers. .. RT-PCRs were performed using FastStart DNA Master SYBR Green I in a Light Cycler apparatus (Roche Applied Science, Penzberg, Germany).

other:

Article Title: Immunomodulatory and Anti-IBDV Activities of the Polysaccharide AEX from Coccomyxa gloeobotrydiformis
Article Snippet: Nitric Oxide (NO) Production Assay After a 24 h treatment with various concentrations of AEX, the culture supernatants of the PBMCs were collected, and the nitrite contents were determined by the Griess reaction using the Griess Reagent System (Promega, Madison, WI, USA) according to the manufacturer’s protocol.

Activity Assay:

Article Title: Analysis of DNA Double-Strand Breaks and Cytotoxicity after 7 Tesla Magnetic Resonance Imaging of Isolated Human Lymphocytes
Article Snippet: .. Viability assay To monitor the metabolic activity of unstimulated PBMCs, CellTiter-Blue assay (Promega, Madison, WI, USA) was performed according to the manufacturer’s instructions. ..

Spectrophotometry:

Article Title: Characterization of the Phospholipid Platelet-Activating Factor As a Mediator of Inflammation in Chickens
Article Snippet: .. Nitrite levels were determined in supernatants of BMDM, PBMC, or HD11 cell cultures by spectrophotometry using the Griess reagent system (Promega, UK). ..

Expressing:

Article Title: Combined modality radiation therapy promotes tolerogenic myeloid cell populations and STAT3-related gene expression in head and neck cancer patients
Article Snippet: .. For gene expression analysis, mRNA was isolated from PBMCs from baseline and the second week draw during CMT from 4 HPV+ patients and 2 HPV– patients (number 7, 8, 11, 12, 13, and 14) (Table ) using mRNA isolation kit on Maxwell Rapid Sample Concentrator instrument (Promega). .. RNA quality was verified using the Bioanalyzer-2100 (Agilent).

Isolation:

Article Title: Combined modality radiation therapy promotes tolerogenic myeloid cell populations and STAT3-related gene expression in head and neck cancer patients
Article Snippet: .. For gene expression analysis, mRNA was isolated from PBMCs from baseline and the second week draw during CMT from 4 HPV+ patients and 2 HPV– patients (number 7, 8, 11, 12, 13, and 14) (Table ) using mRNA isolation kit on Maxwell Rapid Sample Concentrator instrument (Promega). .. RNA quality was verified using the Bioanalyzer-2100 (Agilent).

Polymerase Chain Reaction:

Article Title: Neonatal Fc receptor is involved in the protection of fibrinogen after its intake in peripheral blood mononuclear cells
Article Snippet: .. RNA extraction, reverse transcription and PCR Total RNA was extracted from SH-SY5Y, T-cells and PBMCs of the same control subject and from Jurkat and HepG2 cells using the ReliaPrep™ RNA cell miniprep system (Z6011, Promega, Milano, Italy) following the manufacturer’s instructions. .. Two micrograms of DNA-free total RNA were reverse transcribed into first-strand cDNA with random primers in a 20 μl final volume using the GoScript™ reverse transcription system (Promega, A5000).

Viability Assay:

Article Title: Analysis of DNA Double-Strand Breaks and Cytotoxicity after 7 Tesla Magnetic Resonance Imaging of Isolated Human Lymphocytes
Article Snippet: .. Viability assay To monitor the metabolic activity of unstimulated PBMCs, CellTiter-Blue assay (Promega, Madison, WI, USA) was performed according to the manufacturer’s instructions. ..

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    Promega human pbmc cd8
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    Promega pbmcs
    Multiple cytokines and iNOS expression were upregulated and NO production were increased by <t>AEX</t> in <t>PBMCs.</t> ( A – G ) PBMCs were isolated and cultured with AEX (0–100 μg/mL) or LPS (100 ng/mL) for 24 h. Then total RNA was extracted and analyzed by qRT-PCR for IFN-β, IL-1β, IL-6, TNF-α, IL-10, IL-12p40, and iNOS; ( H ) PBMCs were isolated and cultured with AEX (0–100 μg/mL) or LPS (100 ng/mL) for 24 h and 48 h. Then, the culture supernatants were collected and nitrite contents were determined by Griess reaction. Data represent means ± SEM from three wells per group. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Results are representative of two independent experiments.
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    Promega car bearing pbmcs
    Combination of <t>CAR-bearing</t> <t>PBMCs</t> and scFv-IL2 enhances the antitumor effect on MKN-45 cells. Notes: ( A ) Exchange of the mouse scFv gene for its human equivalent in the CAR gene construct. The mouse scFv of mCR-2, which was the most effective among 4 CAR constructs, was exchanged for the 45κHscFv, a human scFv antibody, and this fully human CAR gene was designated hCR-2. ( B ) Expression of hCR-2 inserted into different expression vectors, pcDNA3.1(−) or pIRES, in Jurkat cells was detected by flow cytometry using APC-BSA and APC-CEA. Although hCR-2 was expressed at slightly higher levels in Jurkat cells than mCR-2, no other difference could be detected between the 2 expression vectors. ( C ) hCR-2 in PBMCs was detected by flow cytometry, by using EGFP expression. Approximately 60% of PBMCs expressed hCR-2 after transfection of the CAR gene within a pIRES vector using NEPA21. ( D ) PBMCs expressing hCR-2 in combination with scFv-IL2 demonstrated a higher antitumor activity on MKN-45 cells than those expressing IL-2 or PBMCs alone. Cell viability was determined by measuring the light products using a luciferase assay system. Data represent the mean ± standard error of the mean from at least 3 independent experiments. * P
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    Correlation between progesterone or IFN-γ production with IFN-γ gene methylation in human CD8 Tm cells. CD8 Tm cells were purified from PBMCs of 10 subjects at before, weeks 14 and 28 of pregnancy and around 1 year after delivery. Average percentages of DNA methylation at 6 CpG sites of IFN-γ gene promoter region are shown in ( A ). ( B ) Correlation between serum progesterone levels and IFN-γ gene methylation levels of all samples as shown in (A). Frequencies of IFN-γ-producing PBMC CD8 Tm cells after ex vivo stimulation with PMA and Ionomycin are shown in ( C ). And Correlation between IFN-γ gene methylation levels and frequencies of IFN-γ-producing CD8 Tm cells is shown in ( D ). Horizontal lines in (A) and (C) represent median values. One-way ANOVA and Tukey’s multiple comparisons test was used to compare between multiple groups. Pearson correlation analysis was used to determine the potential correlation between two parameters. * P

    Journal: PLoS Pathogens

    Article Title: Progesterone impairs antigen-non-specific immune protection by CD8 T memory cells via interferon-γ gene hypermethylation

    doi: 10.1371/journal.ppat.1006736

    Figure Lengend Snippet: Correlation between progesterone or IFN-γ production with IFN-γ gene methylation in human CD8 Tm cells. CD8 Tm cells were purified from PBMCs of 10 subjects at before, weeks 14 and 28 of pregnancy and around 1 year after delivery. Average percentages of DNA methylation at 6 CpG sites of IFN-γ gene promoter region are shown in ( A ). ( B ) Correlation between serum progesterone levels and IFN-γ gene methylation levels of all samples as shown in (A). Frequencies of IFN-γ-producing PBMC CD8 Tm cells after ex vivo stimulation with PMA and Ionomycin are shown in ( C ). And Correlation between IFN-γ gene methylation levels and frequencies of IFN-γ-producing CD8 Tm cells is shown in ( D ). Horizontal lines in (A) and (C) represent median values. One-way ANOVA and Tukey’s multiple comparisons test was used to compare between multiple groups. Pearson correlation analysis was used to determine the potential correlation between two parameters. * P

    Article Snippet: Bisulfite sequencing Genomic DNA was prepared from purified human PBMC CD8 Tm cells(hCD3+ hCD8+ hCD45RO+ ), or murine splenic CD8 Tm cells (CD3+ CD8+ CD44+ H2Kb -OVA257-264 tetramer+ ) at 40 days post immunization, by using the Wizard Genomic DNA Purification Kit (Promega; Madison, WI, USA).

    Techniques: Methylation, Purification, DNA Methylation Assay, Ex Vivo

    Impact of demethylation treatment on IFN-γ production by CD8 Tm cells from pregnant women. ( A ) Representative distribution of methylation at 6 CpG sites of IFN-γ gene promoter region before and after demethylation treatment (decitabine, Dec) is shown. Numbers refer to position relative to transcription start site. Filled circles represents methylated CpG and open circles represent demethylated CpG. ( B ) Average percentages of DNA methylation at 6 CpG sites of IFN-γ gene promoter region before and after demethylation treatment in PBMC CD8 Tm cells from five women at week 28 of pregnancy. ( C ) Representative graphs of triplicate IFN-γ ELISpot wells of CD8 Tm cells stimulated ex vivo with either PHA or T2 cells pulsed with CMV pp65 peptide, with or without demethylation treatment. ( D ) Statistics of IFN-γ spot forming CD8 Tm cells from five pregnant women, following ex vivo stimulation with PHA or T2 cells pulsed with CMV pp65 peptide, with or without demethylation treatment. Two-tailed paired Student’s t -test was used for statistical comparison between control and decitabine treatment groups. * P

    Journal: PLoS Pathogens

    Article Title: Progesterone impairs antigen-non-specific immune protection by CD8 T memory cells via interferon-γ gene hypermethylation

    doi: 10.1371/journal.ppat.1006736

    Figure Lengend Snippet: Impact of demethylation treatment on IFN-γ production by CD8 Tm cells from pregnant women. ( A ) Representative distribution of methylation at 6 CpG sites of IFN-γ gene promoter region before and after demethylation treatment (decitabine, Dec) is shown. Numbers refer to position relative to transcription start site. Filled circles represents methylated CpG and open circles represent demethylated CpG. ( B ) Average percentages of DNA methylation at 6 CpG sites of IFN-γ gene promoter region before and after demethylation treatment in PBMC CD8 Tm cells from five women at week 28 of pregnancy. ( C ) Representative graphs of triplicate IFN-γ ELISpot wells of CD8 Tm cells stimulated ex vivo with either PHA or T2 cells pulsed with CMV pp65 peptide, with or without demethylation treatment. ( D ) Statistics of IFN-γ spot forming CD8 Tm cells from five pregnant women, following ex vivo stimulation with PHA or T2 cells pulsed with CMV pp65 peptide, with or without demethylation treatment. Two-tailed paired Student’s t -test was used for statistical comparison between control and decitabine treatment groups. * P

    Article Snippet: Bisulfite sequencing Genomic DNA was prepared from purified human PBMC CD8 Tm cells(hCD3+ hCD8+ hCD45RO+ ), or murine splenic CD8 Tm cells (CD3+ CD8+ CD44+ H2Kb -OVA257-264 tetramer+ ) at 40 days post immunization, by using the Wizard Genomic DNA Purification Kit (Promega; Madison, WI, USA).

    Techniques: Methylation, DNA Methylation Assay, Enzyme-linked Immunospot, Ex Vivo, Two Tailed Test

    Multiple cytokines and iNOS expression were upregulated and NO production were increased by AEX in PBMCs. ( A – G ) PBMCs were isolated and cultured with AEX (0–100 μg/mL) or LPS (100 ng/mL) for 24 h. Then total RNA was extracted and analyzed by qRT-PCR for IFN-β, IL-1β, IL-6, TNF-α, IL-10, IL-12p40, and iNOS; ( H ) PBMCs were isolated and cultured with AEX (0–100 μg/mL) or LPS (100 ng/mL) for 24 h and 48 h. Then, the culture supernatants were collected and nitrite contents were determined by Griess reaction. Data represent means ± SEM from three wells per group. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Results are representative of two independent experiments.

    Journal: Marine Drugs

    Article Title: Immunomodulatory and Anti-IBDV Activities of the Polysaccharide AEX from Coccomyxa gloeobotrydiformis

    doi: 10.3390/md15020036

    Figure Lengend Snippet: Multiple cytokines and iNOS expression were upregulated and NO production were increased by AEX in PBMCs. ( A – G ) PBMCs were isolated and cultured with AEX (0–100 μg/mL) or LPS (100 ng/mL) for 24 h. Then total RNA was extracted and analyzed by qRT-PCR for IFN-β, IL-1β, IL-6, TNF-α, IL-10, IL-12p40, and iNOS; ( H ) PBMCs were isolated and cultured with AEX (0–100 μg/mL) or LPS (100 ng/mL) for 24 h and 48 h. Then, the culture supernatants were collected and nitrite contents were determined by Griess reaction. Data represent means ± SEM from three wells per group. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Results are representative of two independent experiments.

    Article Snippet: Nitric Oxide (NO) Production Assay After a 24 h treatment with various concentrations of AEX, the culture supernatants of the PBMCs were collected, and the nitrite contents were determined by the Griess reaction using the Griess Reagent System (Promega, Madison, WI, USA) according to the manufacturer’s protocol.

    Techniques: Expressing, Isolation, Cell Culture, Quantitative RT-PCR

    Intracellular negative regulators and mediators of TLR signaling. mRNA levels in PBMCs of TB patients (open dots) and healthy controls (closed dots) and TB patient AMs from the diseased (TB-positive) lung segment (open squares) and from the matching contralateral lung (closed squares). PBMC mRNA expression of IRAK-M (A) , MKP-1 (C) , SOCS-3 (E) , TOLLIP (F) and A20 (I) . AM mRNA expression of IRAK-M (B) , MKP-1 (D) , TOLLIP (G) and A20 (J) . TOLLIP (H) expression levels in CD4 or CD8 positive lymphocytes and CD14 positive monocytes, as determined by flow cytometry (mean channel fluorescence intensity, MFI). mRNA expression is normalized to β2-microglobulin. Depicted are dot plots with medians; open dots: TB patients, closed dots: healthy donors. * P

    Journal: BMC Infectious Diseases

    Article Title: Expression of inhibitory regulators of innate immunity in patients with active tuberculosis

    doi: 10.1186/s12879-015-0833-z

    Figure Lengend Snippet: Intracellular negative regulators and mediators of TLR signaling. mRNA levels in PBMCs of TB patients (open dots) and healthy controls (closed dots) and TB patient AMs from the diseased (TB-positive) lung segment (open squares) and from the matching contralateral lung (closed squares). PBMC mRNA expression of IRAK-M (A) , MKP-1 (C) , SOCS-3 (E) , TOLLIP (F) and A20 (I) . AM mRNA expression of IRAK-M (B) , MKP-1 (D) , TOLLIP (G) and A20 (J) . TOLLIP (H) expression levels in CD4 or CD8 positive lymphocytes and CD14 positive monocytes, as determined by flow cytometry (mean channel fluorescence intensity, MFI). mRNA expression is normalized to β2-microglobulin. Depicted are dot plots with medians; open dots: TB patients, closed dots: healthy donors. * P

    Article Snippet: Evaluation of mRNA levels by quantitative RT-PCR Total RNA obtained from PBMCs and AMs was reverse transcribed using oligo(dT) primer and moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA) according to recommendations of the suppliers.

    Techniques: Affinity Magnetic Separation, Expressing, Flow Cytometry, Cytometry, Fluorescence

    ST2 during active pulmonary tuberculosis. (A) ST2 mRNA in PBMCs; (B) ST2 surface expression measured on CD4 and CD8 positive lymphocytes and CD14 positive monocytes (representative histograms). FMO, fluorescence minus one. (C) Idem for individual subjects (geomean channel fluorescence intensity (MFI)). (D) Plasma sST2 concentrations. (E) ST2 mRNA in alveolar macrophages (AM) of TB patients originating from the TB infected lung (open squares) or contralateral lung (closed squares). Depicted mRNA levels are normalized to the house keeping gene β2-microglobulin. Depicted are dot plots with medians; open dots: TB patients, closed dots: healthy donors. *** P

    Journal: BMC Infectious Diseases

    Article Title: Expression of inhibitory regulators of innate immunity in patients with active tuberculosis

    doi: 10.1186/s12879-015-0833-z

    Figure Lengend Snippet: ST2 during active pulmonary tuberculosis. (A) ST2 mRNA in PBMCs; (B) ST2 surface expression measured on CD4 and CD8 positive lymphocytes and CD14 positive monocytes (representative histograms). FMO, fluorescence minus one. (C) Idem for individual subjects (geomean channel fluorescence intensity (MFI)). (D) Plasma sST2 concentrations. (E) ST2 mRNA in alveolar macrophages (AM) of TB patients originating from the TB infected lung (open squares) or contralateral lung (closed squares). Depicted mRNA levels are normalized to the house keeping gene β2-microglobulin. Depicted are dot plots with medians; open dots: TB patients, closed dots: healthy donors. *** P

    Article Snippet: Evaluation of mRNA levels by quantitative RT-PCR Total RNA obtained from PBMCs and AMs was reverse transcribed using oligo(dT) primer and moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA) according to recommendations of the suppliers.

    Techniques: Expressing, Fluorescence, Infection

    SIGIRR during active pulmonary tuberculosis. (A) SIGIRR mRNA in PBMCs. (B) SIGIRR surface expression measured on CD4 and CD8 positive lymphocytes and CD14 positive monocytes (representative histograms). FMO, fluorescence minus one. (C) Idem for individual subjects (mean channel fluorescence intensity, MFI). (D) SIGIRR mRNA in alveolar macrophages (AM) of TB patients originating from the TB infected lung (open squares) or contralateral lung (closed squares). Depicted mRNAs are normalized to the house keeping gene β2-microglobulin. Depicted are dot plots with medians; open dots: TB patients, closed dots: healthy donors.

    Journal: BMC Infectious Diseases

    Article Title: Expression of inhibitory regulators of innate immunity in patients with active tuberculosis

    doi: 10.1186/s12879-015-0833-z

    Figure Lengend Snippet: SIGIRR during active pulmonary tuberculosis. (A) SIGIRR mRNA in PBMCs. (B) SIGIRR surface expression measured on CD4 and CD8 positive lymphocytes and CD14 positive monocytes (representative histograms). FMO, fluorescence minus one. (C) Idem for individual subjects (mean channel fluorescence intensity, MFI). (D) SIGIRR mRNA in alveolar macrophages (AM) of TB patients originating from the TB infected lung (open squares) or contralateral lung (closed squares). Depicted mRNAs are normalized to the house keeping gene β2-microglobulin. Depicted are dot plots with medians; open dots: TB patients, closed dots: healthy donors.

    Article Snippet: Evaluation of mRNA levels by quantitative RT-PCR Total RNA obtained from PBMCs and AMs was reverse transcribed using oligo(dT) primer and moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA) according to recommendations of the suppliers.

    Techniques: Expressing, Fluorescence, Infection

    Combination of CAR-bearing PBMCs and scFv-IL2 enhances the antitumor effect on MKN-45 cells. Notes: ( A ) Exchange of the mouse scFv gene for its human equivalent in the CAR gene construct. The mouse scFv of mCR-2, which was the most effective among 4 CAR constructs, was exchanged for the 45κHscFv, a human scFv antibody, and this fully human CAR gene was designated hCR-2. ( B ) Expression of hCR-2 inserted into different expression vectors, pcDNA3.1(−) or pIRES, in Jurkat cells was detected by flow cytometry using APC-BSA and APC-CEA. Although hCR-2 was expressed at slightly higher levels in Jurkat cells than mCR-2, no other difference could be detected between the 2 expression vectors. ( C ) hCR-2 in PBMCs was detected by flow cytometry, by using EGFP expression. Approximately 60% of PBMCs expressed hCR-2 after transfection of the CAR gene within a pIRES vector using NEPA21. ( D ) PBMCs expressing hCR-2 in combination with scFv-IL2 demonstrated a higher antitumor activity on MKN-45 cells than those expressing IL-2 or PBMCs alone. Cell viability was determined by measuring the light products using a luciferase assay system. Data represent the mean ± standard error of the mean from at least 3 independent experiments. * P

    Journal: OncoTargets and therapy

    Article Title: Enhancement of antitumor activity by using a fully human gene encoding a single-chain fragmented antibody specific for carcinoembryonic antigen

    doi: 10.2147/OTT.S140174

    Figure Lengend Snippet: Combination of CAR-bearing PBMCs and scFv-IL2 enhances the antitumor effect on MKN-45 cells. Notes: ( A ) Exchange of the mouse scFv gene for its human equivalent in the CAR gene construct. The mouse scFv of mCR-2, which was the most effective among 4 CAR constructs, was exchanged for the 45κHscFv, a human scFv antibody, and this fully human CAR gene was designated hCR-2. ( B ) Expression of hCR-2 inserted into different expression vectors, pcDNA3.1(−) or pIRES, in Jurkat cells was detected by flow cytometry using APC-BSA and APC-CEA. Although hCR-2 was expressed at slightly higher levels in Jurkat cells than mCR-2, no other difference could be detected between the 2 expression vectors. ( C ) hCR-2 in PBMCs was detected by flow cytometry, by using EGFP expression. Approximately 60% of PBMCs expressed hCR-2 after transfection of the CAR gene within a pIRES vector using NEPA21. ( D ) PBMCs expressing hCR-2 in combination with scFv-IL2 demonstrated a higher antitumor activity on MKN-45 cells than those expressing IL-2 or PBMCs alone. Cell viability was determined by measuring the light products using a luciferase assay system. Data represent the mean ± standard error of the mean from at least 3 independent experiments. * P

    Article Snippet: In addition, the antitumor effect of the CAR-bearing PBMCs in combination with scFv-IL2 was evaluated by measuring the intensity of light produced by MKN-45 cells expressing luciferase, using a luciferase assay system (Promega, Madison, WI, USA) according to the manufacturer’s protocol.

    Techniques: Construct, Expressing, Flow Cytometry, Cytometry, Transfection, Plasmid Preparation, Activity Assay, Luciferase