goat polyclonal anti cd40l antibody  (R&D Systems)

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Overview of the treatment scheme. i.t: intratumorally; i.v: intravenously; ICOSL: inducible co-stimulator ligand.

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques:

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Viral DNA extraction from CsCl-purified virus particles using proteinase K. The AdV-D24-ICOSL-CD40L sequencing library was made using a Nextera XT kit (Illumina). The size of the library was assessed by high-resolution agarose gel electrophoresis. The presence of the 24-bp deletion in the E1A CR2 domain, the CMV-ICOSL-IRES-CD40L expression cassette inserted in place of the E3 region and the hybrid Ad5/3 fiber were confirmed. Analysis of sequence coverage did not detect the presence of a virus sub-population with a genome characterized by a major rearrangement such as a large insertion or deletion. The figure was generated by SnapGene ® .

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques: DNA Extraction, Purification, Sequencing, Agarose Gel Electrophoresis, Expressing, Generated

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Evaluation of ICOSL and CD40L expression in tested melanoma cells. ( A ) ICOSL concentration was detected on supernatants collected 24–48 h after treatment using ELISA kit. ( B ) CD40L was detected from supernatants collected 24–48 h after treatment using an ELISA kit according to the manufacturer’s instructions. Statistical analysis was carried out with a Mann–Whitney test to compare two groups (ns = not significant, p > 0.05). ( C ) Transgene expression. The expression of ICOSL and CD40L from AdV-D24-ICOSL-CD40L was assessed by infecting A549 cells with the virus, harvesting proteins 48 h after the infection and detecting ICOSL and CD40L by Western blot. 1–4: protein extracts from A549 cells infected with AdV-D24-ICOSL-CD40L clones #1–4. N: protein extract from non-infected A549 cells. M: PageRuler Prestained Protein Ladder (ThermoScientific # 26616). ICOSL MW: 70 kDa (due to heavy protein glycosylation); CD40L MW: 30 kDa.

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Infection, Western Blot, Clone Assay

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Evaluation of cell viability by MTS assay (cell cytotoxicity assay). ( A ) Cell viability was evaluated 72 h post-infection with AdV-24-ICOSL-CD40L and AdV-24 at the concentration of 100 VP/cell and combination with anti-PD1 (pembrolizumab). Data are expressed as the percentage of viable cells according to MTS cell viability assay protocol (CellTiter 96 ® AQueous One Solution Cell Proliferation Assay, Promega). Statistical analysis was carried out with a Mann–Whitney test to compare two groups ( p > 0.05). ( B , C ) Microscopic photographs visualizing the morphology and cytopathic effect (CPE) of the cells representing the investigated groups and assessed by MTS assay at 48 and 72 h post-infection (magnification 10×) (** = p ≤ 0.001).

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques: MTS Assay, Cytotoxicity Assay, Infection, Concentration Assay, Viability Assay, Proliferation Assay, MANN-WHITNEY

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Immunogenic cell death assessment. ( A ) Evaluation of calreticulin (CRT) exposure by melanoma cell lines after treatment with oncolytic adenoviruses AdV-24-ICOSL-CD40L and AdV-D24, and in combination with anti PD-1. CRT exposure was measured 48 h post-treatments with anti-calreticulin antibody staining AlexaFluor ® 488 and subsequent flow cytometry analysis (Beckman-Coulter Cytomics FC500). ( B ) Assessment of ATP release after the treatment. ATP concentration in a supernatant was evaluated 72 h after infection with CellTiter-Glo ® Luminescent Cell Viability Assay ATP detection kit by Promega. ( C ) Evaluation of high-mobility group box 1 (HMGB-1) release after treatment with oncolytic adenoviruses and the combination with anti PD-1. HMGB-1 level was measured from the supernatant collected 72 h after infection with ELISA kit (MBL International, Woburn, MA, USA), according to manufacturer’s dispositions. Statistical analysis was carried out with a Mann–Whitney test to compare two groups (* = p ≤ 0.05; ** = p ≤ 0.001, *** = p ≤ 0.0001).

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques: Staining, Flow Cytometry, Concentration Assay, Infection, Cell Viability Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Antitumor efficacy of oncolytic vectors and the combination therapy with anti PD-1 antibody in murine melanoma B16V (1 × 10 6 cells/flank, one mouse had 2 tumors/6 tumors per group) xenograft immunocompetent C57BL/6 model. ( A ) Tumor volume (mm 3 ) measured through the study. The treatment was performed once per day on days 1–6. The mice were treated according to the scheme with viruses (i.t.) and anti PD-1 antibody (i.v.) ( B ) At the end of the study, mice were sacrificed and tumors harvested for weight assessment. ( C , D ) Tumor volume measurement on days 6 and 20. ( E ) Body weight measurements throughout the study. ( F ) Survival profile was calculated by Kaplan–Meier test. ( H ) Evaluation of CRT exposure after the treatment with oncolytic adenoviruses AdV-24-ICOSL-CD40L and AdV-D24, and in combination with anti PD-1. CRT exposure was measured in the end of the study (after mice sacrifice) with anti-calreticulin antibody staining and subsequent flow cytometry analysis (Beckman-Coulter Cytomics FC500). ( G ) Assessment of ATP release after the treatment. ATP concentration from the tumors was evaluated in the end of the study (after mice sacrifice) with CellTiter-Glo ® Luminescent Cell Viability Assay ATP detection kit by Promega. Error bars, mean ± SEM, * = p ≤ 0.05, *** = p ≤ 0.0001.

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques: Staining, Flow Cytometry, Concentration Assay, Cell Viability Assay

Journal:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8 + T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

doi: 10.1016/j.vaccine.2008.05.018

Figure Lengend Snippet: Expression of murine CD40L by recombinant canarypox vector. Recombinant canarypox viruses vCPmCD40L and vCPmSP-D-CD40L were generated as described in Materials and Methods. (A) RT-PCR. CEF cells were infected by vCPmCD40L, vCPmSP-D-CD40L or its parental control ALVAC II at 5 MOI for 3 days. Total RNA was isolated from infected CEF cells using Trizol Reagent and subjected to RT-PCR with specific primers amplifying the coding sequence of murine CD40L or SP-D-CD40L. The identity of the amplified product was further verified by DNA sequencing (not shown). (B) Flow cytometry. Human PBMCs were infected by vCPmCD40L, vCPmSP-D-CD40L or ALVAC II at 5 MOI for 24 hrs and stained with PE-labeled anti-mouse CD40L mAb. (C) Western blot. Hela cells were infected by vCPmCD40L, vCPmSP-D-CD40L, or ALVAC II at 10 MOI for 24–48hrs. Cell lysates or magnetic beads incubated with supernatant of infected cells were subjected to Western blot and detected with a goat anti-mouse CD40L or anti-mouse SP-D antibody. As expected, the membrane CD40L was about 35–40kD and soluble multimeric form of CD40L was about 70kD.

Article Snippet: The membrane was then blocked and probed with goat anti-mouse CD40L or goat anti-human CD40L antibody (R&D Systems, Inc., Minneapolis, MN), followed by horseradish peroxidase-conjugated anti-goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Expressing, Recombinant, Plasmid Preparation, Generated, Reverse Transcription Polymerase Chain Reaction, Infection, Control, Isolation, Sequencing, Amplification, DNA Sequencing, Flow Cytometry, Staining, Labeling, Western Blot, Magnetic Beads, Incubation, Membrane

Journal:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8 + T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

doi: 10.1016/j.vaccine.2008.05.018

Figure Lengend Snippet: CD40L expressed by recombinant ALVAC virus boosts the immunogenicity of an HIV-1 canarypox vaccine in mice. Female Balb/c mice of 6–8 wks age were immunized 3 times with an HIV-1 canarypox vaccine, vCP1452, with either of vCPmCD40L or vCPmSP-D-CD40L or ALVAC II. Six weeks after the last immunization, mice were sacrificed and spleens were taken and used for preparation of splenocytes. (A) IFN-γ ELISpot. Splenocytes were stimulated with P815 cells pulsed with an H-2Kd restricted HIV-1 Gag peptide, AMQMLKETI, for 16 hrs. Cells producing IFN-γ were detected and counted as described in Materials and Methods. (B) and (C) MHC-I tetramer analysis. Splenocytes were stained with anti-mouse CD8 mAb and AMQMLKETI/H-2Kd tetramer. (B) shows representative flow cytometry results for each group of mice. Numbers are percentage of tetramer positive cells in CD8+ cells. (C) shows summary of the tetramer data for each group of mice. (D) to (G) Polyfunctional CD8+ T cells analyzed by flow cytometry. Splenocytes were stained with anti-mouse IFN-γ and anti-mouse TNF-α mAbs and co-expressing cells are measured in (D) and (E) or anti-mouse IFN-γ and anti-mouse CD107a mAbs and co-expressing cells are measured in (F) and (G). (D) and (F) show representative flow cytometry results and (E) and (G) show summary of the polyfunctional CD8+ T cell data for each group of mice. (H) CD8+ T cells producing IFN-γ analyzed by flow cytometry. Splenocytes were stained with anti-mouse CD8 and anti-mouse IFN-γ mAbs. (I) and (J) Memory HIV-1-Gag specific CD8+ cells analyzed by flow cytometry. Splenocytes were stained with anti-mouse CD8, anti-mouse CD127 (IL-7Rα) mAbs, and AMQMLKETI/H-2Kd tetramer. In (I) CD8+/Tetramer+ cells are gated and measured by CD127 (y-axis). CD8+ T cells from naïve mice unstained for CD127 was used as control for CD127 gating. (J) shows summary data of CD127 expression in tetramer+ cells. (K) Lymphocyte proliferation. Splenocytes were stimulated with HIV-1 p24 Gag for 6 days and [3H]-thymidine incorporation was measured. (L) Cytokine production. Splenocytes were stimulated with HIV-1 p24 Gag for 5 days. IFN-γ and IL-4 in the supernatant were measured by ELISA. (M) Serum anti-Gag antibody. HIV-1 p24 Gag was used as antigen, and mice serum anti-Gag antibodies were measured by ELISA. Data shown are mean±SEM. n=4–10. *: P<0.05. **: P<0.01.

Article Snippet: The membrane was then blocked and probed with goat anti-mouse CD40L or goat anti-human CD40L antibody (R&D Systems, Inc., Minneapolis, MN), followed by horseradish peroxidase-conjugated anti-goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Recombinant, Virus, Immunopeptidomics, Enzyme-linked Immunospot, Staining, Flow Cytometry, Expressing, Control, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8 + T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

doi: 10.1016/j.vaccine.2008.05.018

Figure Lengend Snippet: CD40L expressed by recombinant ALVAC virus enhances TNF-α and IL-12 production of human MDDCs. Human immature MDDCs were infected with recombinant ALVAC virus expressing human CD40L called vA3131-2, an ALVAC-HIV vaccine called vCP205, or a parental control, ALVAC II at an MOI of 10, or incubated with medium alone, or with CD40L trimer (2 μg/ml) as a positive control at 37°C for 24 h. The production of TNF-α and IL-12 was determined by intracellular staining flow cytometry. (A) Data are taken from the HIV-1-infected participant #1 and are representative of experiments with MDDCs derived from two HIV-1-infected and two HIV-1-uninfected individuals. The numbers in each gate represent the percentage of TNF-α or IL-12-positive MDDCs in total MDDCs. (B) Pooled data from all participants are shown. Data shown are mean±SEM. **: P<0.01.

Article Snippet: The membrane was then blocked and probed with goat anti-mouse CD40L or goat anti-human CD40L antibody (R&D Systems, Inc., Minneapolis, MN), followed by horseradish peroxidase-conjugated anti-goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Recombinant, Virus, Infection, Expressing, Control, Incubation, Positive Control, Staining, Flow Cytometry, Derivative Assay

Journal:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8 + T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

doi: 10.1016/j.vaccine.2008.05.018

Figure Lengend Snippet: CD40L expressed by recombinant ALVAC virus promotes human MDDCs maturation independent of TNF-α secretion. Immature MDDCs were infected with recombinant ALVAC virus expressing human CD40L, vA3131-2; an ALVAC-HIV vaccine, vCP205; or a parental control, ALVAC II at an MOI of 10, or incubated with medium alone at 37°C for 48 h. Expression of surface molecules was analyzed by flow cytometry. (A) Numbers in CD83 row represent percentage of CD83 positive cells in total MDDCs and numbers in CD86 and CD80 rows represent the mean fluorescence intensity of specific staining (histogram with solid line) subtracted from the value of background staining with matched isotype control mouse mAbs (dotted histogram). Data shown are from HIV-1-infected participant #1 and representative of six experiments performed with MDDCs obtained from three HIV-1-uninfected blood donors and three HIV-1-infected individuals. (B) Pooled data from all six participants studied are shown. Data shown are mean±SEM. **: P<0.01. (C) Immature human MDDCs were infected or not (medium) with vA3131-2 (CD40L expressing), vCP205, or ALVAC II at an MOI of 10 and then incubated in the presence of a blocking anti-human TNF-α Ab or the control isotype immunoglobulin (20 μg/mL). After 24 h incubation, MDDCs were collected and stained for CD83 expression to monitor DC maturation. Numbers in each dot plot represent percentage of CD83 positive cells in total MDDCs. Expression of CD83 on MDDCs treated with isotype immunoglobulin is similar to that on MDDCs treated with medium. Increasing the concentration of blocking anti-human TNF-α Ab up to 100 μg/mL did not further reduce the CD83 expression in all conditions (data not shown). The results represent one of five experiments.

Article Snippet: The membrane was then blocked and probed with goat anti-mouse CD40L or goat anti-human CD40L antibody (R&D Systems, Inc., Minneapolis, MN), followed by horseradish peroxidase-conjugated anti-goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Recombinant, Virus, Infection, Expressing, Control, Incubation, Flow Cytometry, Fluorescence, Staining, Blocking Assay, Concentration Assay

Journal:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8 + T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

doi: 10.1016/j.vaccine.2008.05.018

Figure Lengend Snippet: Effects of ALVAC recombinant vA3131-2 expressing human CD40L on apoptosis of DCs. Immature MDDCs were infected or not (medium control) with either parental ALVAC II, ALVAC recombinant vA3131-2 expressing human CD40L, or an HIV canarypox vaccine vCP205 at an MOI of 10 at 37°C for 48 h. Infected cells were harvested and early apoptotic marker caspase-3 was determined by intracellular staining and flow cytometry using FITC- or PE-conjugated mAb against human caspase-3. (A) MDDCs were intracellularly stained for caspase-3 expression to detect early apoptotic MDDCs. The level of caspase-3 expression (x axis) is shown for each of the conditions. Background staining with FITC-conjugated isotype control Ab is shown by a solid gray histogram; medium-treated MDDCs are shown by a histogram with a dotted line, MDDCs infected with vA3131-2 are represented with a histogram with a dark line, and MDDCs infected with parental ALVAC II are represented with a histogram with a light line. The results represent one of five experiments from two HIV-1-uninfected and three HIV-1-infected donors. (B) Pooled data from all participants are shown. Data shown are mean±SEM. **: P<0.01.

Article Snippet: The membrane was then blocked and probed with goat anti-mouse CD40L or goat anti-human CD40L antibody (R&D Systems, Inc., Minneapolis, MN), followed by horseradish peroxidase-conjugated anti-goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Recombinant, Expressing, Infection, Control, Marker, Staining, Flow Cytometry

Journal:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8 + T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

doi: 10.1016/j.vaccine.2008.05.018

Figure Lengend Snippet: CD40L expressed by recombinant ALVAC virus enhances specific antiviral CTL activity. CD4+ T cell-containing or -depleted PBMCs from four HIV-1-infected individuals and three HIV-1-uninfected individuals were cocultured with autologous MDDCs that were either pulsed or not with HLA-restricted epitopes of HIV-1 proteins (for HIV-1 infected individuals) or EBV proteins (for HIV-1-uninfected individuals). MDDCs were previously infected or not (medium) with parental ALVAC II or ALVAC recombinant vA3131-2 expressing human CD40L at an MOI of 10 for 48 h. On day 10, specific CTL activity was assessed by intracellular flow cytometric analysis of IFN-γproducing CD8+ T cells and 51Cr release assay. (A) Representative intracellular IFN-γ flow cytometric data obtained from HIV-1-positive participant #1. (B) Summary data from intracellular IFN-γ flow cytometry of HIV-1-positive participant #2–#4 are graphically depicted. Open bars represent CD4+ T cell-containing condition and dark bar represent CD4+ T cell -depleted conditions. (C) A representative 51Cr release assay result from HIV-1-positive participant #1 is shown. Similar results were obtained with HIV-1-positive participant #2 (data not shown). (D) Summary data from intracellular IFN-γ flow cytometric analysis of EBV-positive participant #5–#7 are graphically depicted. Open bars represent CD4+ T cell -containing condition and dark bars represent CD4+ T cell -depleted conditions. (E) A representative 51Cr release assay result from EBV-positive participant #6 is shown. Similar results were obtained with EBV-positive participant #5 (data not shown). DC, MDDCs not pulsed with peptide; DCp, MDDCs pulsed with peptide; vA3131-2/DCp, vA3131-2-infected MDDCs pulsed with peptide; ALVAC/DCp, parental ALVAC II-infected MDDCs pulsed with peptide.

Article Snippet: The membrane was then blocked and probed with goat anti-mouse CD40L or goat anti-human CD40L antibody (R&D Systems, Inc., Minneapolis, MN), followed by horseradish peroxidase-conjugated anti-goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Recombinant, Virus, Activity Assay, Infection, Expressing, Release Assay, Flow Cytometry

Journal: Clinical and Vaccine Immunology

Article Title: Macaque Multimeric Soluble CD40 Ligand and GITR Ligand Constructs Are Immunostimulatory Molecules In Vitro

doi: 10.1128/cvi.00198-06

Figure Lengend Snippet: FIG. 2. Recombinant DNA constructs for pmacSP-D-CD40L, pmacAcrp30-CD40L, and pmacSP-D-GITRL. On the basis of the in vivo activities of mouse versions of the plasmids encoding SP-D- CD40L, Acrp30-CD40L, and SP-D-GITRL (35), similar constructs were made using the macaque orthologs. The numbers shown corre- spond to the amino acid residues of each component in the construct. For the macaque Acrp30-CD40L recombinant, a linker of lysine-glu- tamine was inserted between the two genes. SP-D-CD40L contained the full extracellular domain of CD40L, including the membrane- proximal stalk, while the Acrp30-CD40L gene encoded only the CD40L head group following the design of Holler et al. (11).

Article Snippet: The membranes were blocked and then probed with goat anti-human CD40L (R&D Systems) or goat anti-human GITRL (R&D Systems), followed by horseradish peroxidase-conjugated anti-goat antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).

Techniques: Recombinant, Construct, In Vivo, Membrane

Journal: Clinical and Vaccine Immunology

Article Title: Macaque Multimeric Soluble CD40 Ligand and GITR Ligand Constructs Are Immunostimulatory Molecules In Vitro

doi: 10.1128/cvi.00198-06

Figure Lengend Snippet: FIG. 3. Expression and secretion of macaque SP-D-CD40L, Acrp30-CD40L, and SP-D-GITRL from plasmid-transfected 293T cells. The plasmids described in the legend to Fig. 2 were transfected into 293T cells. The empty vector pVAX1 was used as a control transfection. A human CD40L-specific or human GITRL-specific monoclonal antibody was used to pull down macSP-D-CD40L and macAcrp30-CD40L or macSP-D-GITRL from their respective trans- fection supernatants. The immunoprecipitated proteins were sepa- rated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted with polyclonal anti-human CD40L or anti-human GITRL antibodies. Molecular mass standards (in kilodaltons) were run for each Western blot and are combined into this montage image where lanes 1 to 3 were immunoblotted with antibody for human CD40L and lanes 4 and 5 were immunoblotted with antibody for human GITRL. Lanes 1 and 4 contain negative-control supernatants from pVAX1-transfected 293T cells. Lane 2 contains pmacSP-D-CD40L transfection supernatant, lane 3 contains pmacAcrp30-CD40L transfection supernatant, and lane 5 con- tains pmacSP-D-GITRL transfection supernatant.

Article Snippet: The membranes were blocked and then probed with goat anti-human CD40L (R&D Systems) or goat anti-human GITRL (R&D Systems), followed by horseradish peroxidase-conjugated anti-goat antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).

Techniques: Expressing, Plasmid Preparation, Transfection, Control, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Western Blot, Negative Control

Journal: Clinical and Vaccine Immunology

Article Title: Macaque Multimeric Soluble CD40 Ligand and GITR Ligand Constructs Are Immunostimulatory Molecules In Vitro

doi: 10.1128/cvi.00198-06

Figure Lengend Snippet: FIG. 4. Macaque SP-D-CD40L and Acrp30-CD40L induced hu- man B-cell proliferation in vitro. CD19 B cells were isolated from human PBMC and incubated for 5 days with supernatant from trans- fected 293T cells with () or without () 10 ng/ml human IL-4. B cells were incubated with 10 g/ml immunoglobulin M (IgM) as a positive control. Mock-transfected 293T cell supernatant was used as a negative control. For selected samples, 2 l of anti-human CD40L antibody ( CD40L mAb) was added to 100 l of supernatant and incubated for 20 min at room temperature prior to use. Other supernatant samples were boiled for 10 min and cooled on ice for 5 min before being added. The level of proliferation was determined by [3H]thymidine incorpo- ration, as measured by scintillation counting of cells harvested onto glass-fiber filters. Data are plotted as the means (plus SEMs [error bars]) of triplicate wells. huSP-D-CD40L, human SP-D-CD40L.

Article Snippet: The membranes were blocked and then probed with goat anti-human CD40L (R&D Systems) or goat anti-human GITRL (R&D Systems), followed by horseradish peroxidase-conjugated anti-goat antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).

Techniques: In Vitro, Isolation, Incubation, Positive Control, Transfection, Negative Control

Journal: Clinical and Vaccine Immunology

Article Title: Macaque Multimeric Soluble CD40 Ligand and GITR Ligand Constructs Are Immunostimulatory Molecules In Vitro

doi: 10.1128/cvi.00198-06

Figure Lengend Snippet: FIG. 5. Macaque SP-D-CD40L induced macaque B-cell proliferation in vitro. Macaque CFSE-labeled PBMC were cultured for 72 h in supernatants prepared from 293T cells that had either been mock transfected (mock) or transfected with empty vector (vector control) or pmacSP-D-CD40L. Pokeweed mitogen (PWM) was used as a positive control. Cells were stained for CD4, CD8, and CD20, and the CD4 CD8 CD20 population was scored as B cells. Numbers on the graphs represent the frequencies of CFSE-negative cells as a percentage of total CD20 cells. FSC, forward scatter.

Article Snippet: The membranes were blocked and then probed with goat anti-human CD40L (R&D Systems) or goat anti-human GITRL (R&D Systems), followed by horseradish peroxidase-conjugated anti-goat antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).

Techniques: In Vitro, Labeling, Cell Culture, Transfection, Plasmid Preparation, Control, Positive Control, Staining