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R&D Systems elisa procedure
Kinetics of IFN-α and <t>TNF-α</t> responses of porcine AMϕ. (A and C) ZMAC cells were stimulated with poly(I·C) (25 μg/ml) (A) or LPS (100 ng/ml) (C), and the amount of IFN-α (A) or TNF-α (C) present in cell-free culture supernatant at the indicated time after stimulation was determined by <t>ELISA.</t> The results represent the means ± standard deviations of three independent experiments for each agonist. (B) ZMAC cells were either mock treated or exposed to poly(I·C) (25 μg/ml) for 1 h, and their whole-cell lysates were analyzed by Western blotting to detect p-IRF3, total IRF3, and β-actin. (D) ZMAC cells were either mock treated or exposed to LPS (100 ng/ml), and their whole-cell lysates were harvested at the time points indicated and analyzed by Western blotting to detect p-NF-κB, total NF-κB, and β-actin. The results shown are representative of two independent experiments.
Elisa Procedure, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Genotype 2 Strains of Porcine Reproductive and Respiratory Syndrome Virus Dysregulate Alveolar Macrophage Cytokine Production via the Unfolded Protein Response"

Article Title: Genotype 2 Strains of Porcine Reproductive and Respiratory Syndrome Virus Dysregulate Alveolar Macrophage Cytokine Production via the Unfolded Protein Response

Journal: Journal of Virology

doi: 10.1128/JVI.01251-17

Kinetics of IFN-α and TNF-α responses of porcine AMϕ. (A and C) ZMAC cells were stimulated with poly(I·C) (25 μg/ml) (A) or LPS (100 ng/ml) (C), and the amount of IFN-α (A) or TNF-α (C) present in cell-free culture supernatant at the indicated time after stimulation was determined by ELISA. The results represent the means ± standard deviations of three independent experiments for each agonist. (B) ZMAC cells were either mock treated or exposed to poly(I·C) (25 μg/ml) for 1 h, and their whole-cell lysates were analyzed by Western blotting to detect p-IRF3, total IRF3, and β-actin. (D) ZMAC cells were either mock treated or exposed to LPS (100 ng/ml), and their whole-cell lysates were harvested at the time points indicated and analyzed by Western blotting to detect p-NF-κB, total NF-κB, and β-actin. The results shown are representative of two independent experiments.
Figure Legend Snippet: Kinetics of IFN-α and TNF-α responses of porcine AMϕ. (A and C) ZMAC cells were stimulated with poly(I·C) (25 μg/ml) (A) or LPS (100 ng/ml) (C), and the amount of IFN-α (A) or TNF-α (C) present in cell-free culture supernatant at the indicated time after stimulation was determined by ELISA. The results represent the means ± standard deviations of three independent experiments for each agonist. (B) ZMAC cells were either mock treated or exposed to poly(I·C) (25 μg/ml) for 1 h, and their whole-cell lysates were analyzed by Western blotting to detect p-IRF3, total IRF3, and β-actin. (D) ZMAC cells were either mock treated or exposed to LPS (100 ng/ml), and their whole-cell lysates were harvested at the time points indicated and analyzed by Western blotting to detect p-NF-κB, total NF-κB, and β-actin. The results shown are representative of two independent experiments.

Techniques Used: Enzyme-linked Immunosorbent Assay, Western Blot

Infection of AMϕ with PRRSV fails to stimulate IFN-α and inhibits their ability to produce IFN-α in response to stimulation with poly(I·C). (A) ZMAC cells were infected with PRRSV strain FL12 or TGEV strain Purdue, and the frequency of IFN-α-secreting cells after 16 h of culture was determined by ELISPOT assay. (B) Duplicate cultures of ZMAC cells were either mock treated or incubated in the presence of UV light-inactivated or viable PRRSV strain NADC20 for 2 h. Afterward, one member of each pair was mock stimulated or stimulated with poly(I·C) (25 μg/ml) for 8 h, and the amount of IFN-α present in the supernatant was determined by ELISA. The results represent the means ± standard deviations of three independent experiments. The asterisks indicate statistically significant differences (**, P
Figure Legend Snippet: Infection of AMϕ with PRRSV fails to stimulate IFN-α and inhibits their ability to produce IFN-α in response to stimulation with poly(I·C). (A) ZMAC cells were infected with PRRSV strain FL12 or TGEV strain Purdue, and the frequency of IFN-α-secreting cells after 16 h of culture was determined by ELISPOT assay. (B) Duplicate cultures of ZMAC cells were either mock treated or incubated in the presence of UV light-inactivated or viable PRRSV strain NADC20 for 2 h. Afterward, one member of each pair was mock stimulated or stimulated with poly(I·C) (25 μg/ml) for 8 h, and the amount of IFN-α present in the supernatant was determined by ELISA. The results represent the means ± standard deviations of three independent experiments. The asterisks indicate statistically significant differences (**, P

Techniques Used: Infection, Enzyme-linked Immunospot, Incubation, Enzyme-linked Immunosorbent Assay

The ability of PRRSV to inhibit TNF-α production by AMϕ stimulated with LPS coincides with the time after infection when p-eIF2α is markedly present. (A and B) ZMAC cells were either mock infected or infected with PRRSV strain FL12 or NADC20 (MOI = 5) for 2 (@-2) or 6 (@-6) h and then exposed to LPS (100 ng/ml). Cell-free supernatants and cells from the same cultures were harvested 2 h later. (A) The amounts of TNF-α present in the supernatants were determined by ELISA. (B) The presence of p-eIF2α and total eIF2α in whole-cell lysates was assessed by Western blotting. Additional replicate sets were mock infected and cultured for 2 h before an additional 2- or 1-h incubation in the presence of LPS or DTT, respectively. Shown are the means ± standard deviations (A) and corresponding immunoblots representative of three independent experiments (B). Statistical comparisons in panel A were made between the amounts of cytokine present in the supernatants of virus-infected versus mock-infected cultures stimulated with LPS. The asterisks indicate statistical significance (**, P
Figure Legend Snippet: The ability of PRRSV to inhibit TNF-α production by AMϕ stimulated with LPS coincides with the time after infection when p-eIF2α is markedly present. (A and B) ZMAC cells were either mock infected or infected with PRRSV strain FL12 or NADC20 (MOI = 5) for 2 (@-2) or 6 (@-6) h and then exposed to LPS (100 ng/ml). Cell-free supernatants and cells from the same cultures were harvested 2 h later. (A) The amounts of TNF-α present in the supernatants were determined by ELISA. (B) The presence of p-eIF2α and total eIF2α in whole-cell lysates was assessed by Western blotting. Additional replicate sets were mock infected and cultured for 2 h before an additional 2- or 1-h incubation in the presence of LPS or DTT, respectively. Shown are the means ± standard deviations (A) and corresponding immunoblots representative of three independent experiments (B). Statistical comparisons in panel A were made between the amounts of cytokine present in the supernatants of virus-infected versus mock-infected cultures stimulated with LPS. The asterisks indicate statistical significance (**, P

Techniques Used: Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Cell Culture, Incubation

The repressed ability of AMϕ infected with PRRSV to produce IFN-α in response to poly(I·C) coincides with the timing of eIF2α phosphorylation induced by virus infection. ZMAC cells (A to C) or primary PAMϕ (D to F) were either mock infected or infected with the indicated strain of PRRSV (MOI = 5) for 2 h prior to their stimulation with poly(I·C) (25 μg/ml). (A and D) The amounts of IFN-α present in the culture supernatants at the indicated times after stimulation were determined by ELISA. The data represent the means ± SD of triplicate values obtained in a representative of three (A) or two (D) independent experiments. For virus-infected cultures, the times postinfection at which the cell culture supernatants and cells were harvested for analysis are indicated. The bottom rows indicate the percent inhibition of IFN-α production in virus-infected cultures relative to that detected in identically stimulated mock-infected cultures incubated for a corresponding length of time. This value was calculated using the following formula: 100 − [(IFN-α in virus-infected culture supernatant/IFN-α in mock-infected culture supernatant) × 100]. At each of the time points analyzed, whole-cell lysates were also prepared from the virus-infected (B and E) and mock-infected (C and F) cell cultures and proved by Western blotting for p-eIF2α and total eIF2α. The statistical comparisons in panels A and C were made between the amounts of cytokine present in the supernatants from mock-infected versus virus-infected cultures stimulated with poly(I·C). The asterisks indicate statistical significance (**, P
Figure Legend Snippet: The repressed ability of AMϕ infected with PRRSV to produce IFN-α in response to poly(I·C) coincides with the timing of eIF2α phosphorylation induced by virus infection. ZMAC cells (A to C) or primary PAMϕ (D to F) were either mock infected or infected with the indicated strain of PRRSV (MOI = 5) for 2 h prior to their stimulation with poly(I·C) (25 μg/ml). (A and D) The amounts of IFN-α present in the culture supernatants at the indicated times after stimulation were determined by ELISA. The data represent the means ± SD of triplicate values obtained in a representative of three (A) or two (D) independent experiments. For virus-infected cultures, the times postinfection at which the cell culture supernatants and cells were harvested for analysis are indicated. The bottom rows indicate the percent inhibition of IFN-α production in virus-infected cultures relative to that detected in identically stimulated mock-infected cultures incubated for a corresponding length of time. This value was calculated using the following formula: 100 − [(IFN-α in virus-infected culture supernatant/IFN-α in mock-infected culture supernatant) × 100]. At each of the time points analyzed, whole-cell lysates were also prepared from the virus-infected (B and E) and mock-infected (C and F) cell cultures and proved by Western blotting for p-eIF2α and total eIF2α. The statistical comparisons in panels A and C were made between the amounts of cytokine present in the supernatants from mock-infected versus virus-infected cultures stimulated with poly(I·C). The asterisks indicate statistical significance (**, P

Techniques Used: Infection, Enzyme-linked Immunosorbent Assay, Cell Culture, Inhibition, Incubation, Western Blot

Infection of AMϕ with PRRSV does not inhibit the activation of IRF3 induced by transfection with poly(I·C) despite the inhibition of IFN-α production. (A) ZMAC cells were mock infected or infected with PRRSV strain NADC20 or FL12 (MOI = 5). At 2 hpi, the cells were exposed to poly(I·C) either by transfection (400 ng/ml) or in free form (25 μg/ml). At 2 h after poly(I·C) treatment, whole-cell lysates were analyzed by Western blotting for the presence of p-IRF3 and total IRF3. Identical cultures were treated with the transfection reagent without poly(I·C) (mock transfection). (B) Duplicate cultures of ZMAC cells were mock infected or infected with PRRSV strain FL12 or NADC20 (MOI = 5). At 2 hpi, one member of each pair was exposed to either poly(I·C) (400 ng/ml) complexed with transfection reagent or mock transfected [exposed to transfection reagent without poly(I·C)]. After 8 h of culture, the amounts of IFN-α present in cell-free supernatants were determined by ELISA. The data represent the means ± standard deviations of two independent experiments. Statistical comparisons were made between the amounts of the cytokine present in the supernatants of infected versus mock-infected cultures stimulated with poly(I·C). The asterisks indicate statistical significance (**, P
Figure Legend Snippet: Infection of AMϕ with PRRSV does not inhibit the activation of IRF3 induced by transfection with poly(I·C) despite the inhibition of IFN-α production. (A) ZMAC cells were mock infected or infected with PRRSV strain NADC20 or FL12 (MOI = 5). At 2 hpi, the cells were exposed to poly(I·C) either by transfection (400 ng/ml) or in free form (25 μg/ml). At 2 h after poly(I·C) treatment, whole-cell lysates were analyzed by Western blotting for the presence of p-IRF3 and total IRF3. Identical cultures were treated with the transfection reagent without poly(I·C) (mock transfection). (B) Duplicate cultures of ZMAC cells were mock infected or infected with PRRSV strain FL12 or NADC20 (MOI = 5). At 2 hpi, one member of each pair was exposed to either poly(I·C) (400 ng/ml) complexed with transfection reagent or mock transfected [exposed to transfection reagent without poly(I·C)]. After 8 h of culture, the amounts of IFN-α present in cell-free supernatants were determined by ELISA. The data represent the means ± standard deviations of two independent experiments. Statistical comparisons were made between the amounts of the cytokine present in the supernatants of infected versus mock-infected cultures stimulated with poly(I·C). The asterisks indicate statistical significance (**, P

Techniques Used: Infection, Activation Assay, Transfection, Inhibition, Western Blot, Enzyme-linked Immunosorbent Assay

Infection of AMϕ with PRRSV initially enhances but later suppresses the TNF-α response to LPS. (A) ZMAC cells were either mock infected or infected with PRRSV strain FL12 (MOI = 5) for 2 or 6 h prior to their stimulation with LPS (100 ng/ml). After 2 or 4 h of additional culture, the presence of TNF-α in the supernatants was determined by ELISA. The data shown are the means ± SD of a representative of three independent experiments. Statistically significant differences between the amounts of cytokine present in supernatants from infected versus mock-infected cultures stimulated with LPS are indicated with asterisks (**, P
Figure Legend Snippet: Infection of AMϕ with PRRSV initially enhances but later suppresses the TNF-α response to LPS. (A) ZMAC cells were either mock infected or infected with PRRSV strain FL12 (MOI = 5) for 2 or 6 h prior to their stimulation with LPS (100 ng/ml). After 2 or 4 h of additional culture, the presence of TNF-α in the supernatants was determined by ELISA. The data shown are the means ± SD of a representative of three independent experiments. Statistically significant differences between the amounts of cytokine present in supernatants from infected versus mock-infected cultures stimulated with LPS are indicated with asterisks (**, P

Techniques Used: Infection, Enzyme-linked Immunosorbent Assay

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Article Title: Genotype 2 Strains of Porcine Reproductive and Respiratory Syndrome Virus Dysregulate Alveolar Macrophage Cytokine Production via the Unfolded Protein Response
Article Snippet: .. For TNF-α detection, the same ELISA procedure was followed except that the wells were coated with 50 μl of 4-μg/ml anti-pig TNF-α monoclonal antibody (MAb) (clone103304; R & D Systems, Minneapolis, MN, USA). .. The captured cytokine was detected with 50 μl of 2.5-μg/ml biotin-labeled, anti-pig TNF-α MAb (clone 103302; R & D Systems).

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Cell Culture:

Article Title: Exosomes derived from oxLDL-stimulated macrophages induce neutrophil extracellular traps to drive atherosclerosis
Article Snippet: .. Enzyme-linked immunosorbent assay (ELISA)Quantitative analyses of interleukin (IL)-8, tumor necrosis factor (TNF)-α, IL-6, and IL-1β in cell culture supernatants or mouse sera were performed following the ELISA procedures using a quantitative ELISA kit (R & D Systems, Minneapolis, MN, USA). .. Determination of serum TC, TG and LDLTotal cholesterol (TC), triglycerides (TG) and low-density lipoprotein (LDL) were detected by automatic biochemical analyzer (Hitachi, Tokyo, Japan) using a commercial kit (Beijing North Institute of biological technology, Beijing, China).

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    R&D Systems ki 67 staining
    Summary of Drug-LGIR assay in monkeys. ( A – F ) The results of Drug-LGIR assay with dexamethasone (Dexa), prednisolone (PSL), betamethasone (Beta), hydrocortisone (Hydro), triamcinolone (TA), and cyclosporine A (CsA) in PBMC from six monkeys: HM-5 ( A ), HM-6 ( B ), HM-7 ( C ), and HM-8 ( D ) that were not transplanted ( n = 4) and HM-1 ( E ) and HM-2 ( F ) that showed RPE-rejection after transplantation ( n = 2). The rate of proliferative immune cells indicated by expression of <t>Ki-67</t> by FACS are shown in bar graphs to evaluate the suppressive effect of each drug. Red dotted-lines indicate the baseline (Control = 1.0, PBMC + iPS-RPE cells without drug). nt: not tested.
    Ki 67 Staining, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems pe labeled anti cxcr3
    Stat4 requirement in T-bet function (A) Wild type, Stat4-deficient ( Stat4 −/−), T-bet-deficient ( Tbx21 −/−) and Stat4-T-bet-double deficient ( Stat4-Tbx21 −/−) CD4+ T cells were cultured under Th1 conditions (IL-12 + anti-IL-4) for five days and total cell extracts were immunoblotted for T-bet, Stat4 and GAPDH as a control. (B) Cells cultured as in (A) were assessed for the expression of Th1 genes before ( Il18rap , Runx3 ) or after ( Lta ) re-stimulation with anti-CD3. (C-G) Wild type, T-bet-deficient ( Tbx21 −/−) and Stat4-T-bet-double deficient ( Stat4 −/− Tbx21 −/−) CD4+ T cells were cultured under Th1 conditions. On day 2 of the culture period, cells were transduced with a bicistronic retrovirus expressing EGFP only (MIEG) or T-bet and EGFP (T-bet). At the end of the culture, cells were sorted for EGFP expression and stimulated for 18 hours with anti-CD3. Supernatants were analyzed for IFNγ levels using ELISA (C). RNA was isolated from each population to determine the expression levels of the indicated genes using qPCR (D). Surface expression of <t>CXCR3</t> was determined using flow cytometry (E). ChIP assay was performed for acetylated-H3 or -H4 at the Hlx1 and Ifng promoters (F) or acetylated-H4 at the Xcl1 promoter (G). Results are the average ± SD of replicate samples and are representative of 2–3 experiments with similar results.
    Pe Labeled Anti Cxcr3, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems anti il 2 polyclonal antibody
    <t>HSV-IL-2</t> replication in tissue culture. Subconfluent RS cell monolayers were infected with 0.01, 1, or 10 PFU of HSV-IL-2, parental dLAT2903, or wild-type McKrae virus per cell with or without incubation with anti-IL-2 <t>polyclonal</t> antibody, as described in Materials and Methods. Total virus was harvested at the indicated times postinfection by two cycles of freeze-thawing. The amount of virus at each time for each virus was determined by standard plaque assays on RS cells.
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    R&D Systems elisa kits
    Effect of eBV on <t>TNF-α</t> and IL-4 in compound 48/80-stimulated RBL-2H3 cells. (A, B) RBL-2H3 cells were treated with compound 48/80 (5 μg/mL) in the presence of eBV, BV, and melittin (0.5, 1, and 2 μg/mL) for 30 min. TNF-α and IL-1β levels were measured using an <t>ELISA</t> kit. (C, D) RBL-2H3 cells were treated with compound 48/80 (5 μg/mL) in the presence of eBV, BV, and melittin (0.5, 1, and 2 μg/mL) for 30 min. Total RNA was isolated and further analyzed by real-time PCR. The results are presented as relative expression levels compared with those in unstimulated cells and were normalized to β-actin expression. Data are presented as mean ± SD (n = 3). ** P
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    Summary of Drug-LGIR assay in monkeys. ( A – F ) The results of Drug-LGIR assay with dexamethasone (Dexa), prednisolone (PSL), betamethasone (Beta), hydrocortisone (Hydro), triamcinolone (TA), and cyclosporine A (CsA) in PBMC from six monkeys: HM-5 ( A ), HM-6 ( B ), HM-7 ( C ), and HM-8 ( D ) that were not transplanted ( n = 4) and HM-1 ( E ) and HM-2 ( F ) that showed RPE-rejection after transplantation ( n = 2). The rate of proliferative immune cells indicated by expression of Ki-67 by FACS are shown in bar graphs to evaluate the suppressive effect of each drug. Red dotted-lines indicate the baseline (Control = 1.0, PBMC + iPS-RPE cells without drug). nt: not tested.

    Journal: International Journal of Molecular Sciences

    Article Title: A Strategy for Personalized Treatment of iPS-Retinal Immune Rejections Assessed in Cynomolgus Monkey Models

    doi: 10.3390/ijms21093077

    Figure Lengend Snippet: Summary of Drug-LGIR assay in monkeys. ( A – F ) The results of Drug-LGIR assay with dexamethasone (Dexa), prednisolone (PSL), betamethasone (Beta), hydrocortisone (Hydro), triamcinolone (TA), and cyclosporine A (CsA) in PBMC from six monkeys: HM-5 ( A ), HM-6 ( B ), HM-7 ( C ), and HM-8 ( D ) that were not transplanted ( n = 4) and HM-1 ( E ) and HM-2 ( F ) that showed RPE-rejection after transplantation ( n = 2). The rate of proliferative immune cells indicated by expression of Ki-67 by FACS are shown in bar graphs to evaluate the suppressive effect of each drug. Red dotted-lines indicate the baseline (Control = 1.0, PBMC + iPS-RPE cells without drug). nt: not tested.

    Article Snippet: Procedure of Ki-67 staining and antibody information were described previously [ , , , ].

    Techniques: Transplantation Assay, Expressing, FACS

    Representative results of LGIR with peripheral blood mononuclear cells (PBMC) from a monkey. PBMCs (2 × 10 6 cells/well) from a healthy monkey HM-1 were cultured with iPSC-RPE cells for 5 days. Before the assay, iPSC-RPE cells were irradiated (20 Gy) and 1 × 10 4 cells were used for a 24-well culture plate. After 5 days of coculture, PBMC were harvested and stained with anti-CD4 (helper T cell-marker), anti-CD8 (cytotoxic T cell-marker), anti-CD11b (monocyte-, macrophage-, NK cell-, and granulocyte-marker), anti-CD20 (B cell-marker), anti-NKG2A (natural killer (NK) group 2 member A; NK cell-marker), and anti-Ki-67 (proliferation marker) antibodies. As a positive control (PC), irradiated allogenic B cells were used. The samples were analyzed by a fluorescence-activated cell sorting (FACS) flow cytometer. Numbers (%) in the scatterplots indicate double-positive cells (e.g., CD4/Ki-67).

    Journal: International Journal of Molecular Sciences

    Article Title: A Strategy for Personalized Treatment of iPS-Retinal Immune Rejections Assessed in Cynomolgus Monkey Models

    doi: 10.3390/ijms21093077

    Figure Lengend Snippet: Representative results of LGIR with peripheral blood mononuclear cells (PBMC) from a monkey. PBMCs (2 × 10 6 cells/well) from a healthy monkey HM-1 were cultured with iPSC-RPE cells for 5 days. Before the assay, iPSC-RPE cells were irradiated (20 Gy) and 1 × 10 4 cells were used for a 24-well culture plate. After 5 days of coculture, PBMC were harvested and stained with anti-CD4 (helper T cell-marker), anti-CD8 (cytotoxic T cell-marker), anti-CD11b (monocyte-, macrophage-, NK cell-, and granulocyte-marker), anti-CD20 (B cell-marker), anti-NKG2A (natural killer (NK) group 2 member A; NK cell-marker), and anti-Ki-67 (proliferation marker) antibodies. As a positive control (PC), irradiated allogenic B cells were used. The samples were analyzed by a fluorescence-activated cell sorting (FACS) flow cytometer. Numbers (%) in the scatterplots indicate double-positive cells (e.g., CD4/Ki-67).

    Article Snippet: Procedure of Ki-67 staining and antibody information were described previously [ , , , ].

    Techniques: Cell Culture, Irradiation, Staining, Marker, Positive Control, Fluorescence, FACS, Flow Cytometry

    Representative FACS results of Drug-LGIR in PBMC from healthy or transplanted monkeys. In the Drug-LGIR assay, PBMCs were cultured with human iPSC-RPE cells in the presence of indicated drugs for 5 days, and the suppressive effects of the drugs were evaluated by FACS of Ki-67 expressing cells. ( A ) The results of monkey HM-5 before transplantation. Hydrocortisone, prednisolone, and cyclosporine A exhibited significant suppression of the proliferation of the immune cell. By contrast, dexamethasone, betamethasone, and triamcinolone were not suppressive. ( B ) The results of monkey HM-2 that showed immune rejection against human iPSC-RPE cells in vivo. In the results of the in vitro assay, dexamethasone exhibited significant suppression of the immune reaction. Prednisolone and triamcinolone also exhibited suppressive effects. However, other drugs were not suppressive. Dexa: dexamethasone, PSL: prednisolone, Beta: betamethasone, Hydro: hydrocortisone, TA: triamcinolone, and CsA: cyclosporine A.

    Journal: International Journal of Molecular Sciences

    Article Title: A Strategy for Personalized Treatment of iPS-Retinal Immune Rejections Assessed in Cynomolgus Monkey Models

    doi: 10.3390/ijms21093077

    Figure Lengend Snippet: Representative FACS results of Drug-LGIR in PBMC from healthy or transplanted monkeys. In the Drug-LGIR assay, PBMCs were cultured with human iPSC-RPE cells in the presence of indicated drugs for 5 days, and the suppressive effects of the drugs were evaluated by FACS of Ki-67 expressing cells. ( A ) The results of monkey HM-5 before transplantation. Hydrocortisone, prednisolone, and cyclosporine A exhibited significant suppression of the proliferation of the immune cell. By contrast, dexamethasone, betamethasone, and triamcinolone were not suppressive. ( B ) The results of monkey HM-2 that showed immune rejection against human iPSC-RPE cells in vivo. In the results of the in vitro assay, dexamethasone exhibited significant suppression of the immune reaction. Prednisolone and triamcinolone also exhibited suppressive effects. However, other drugs were not suppressive. Dexa: dexamethasone, PSL: prednisolone, Beta: betamethasone, Hydro: hydrocortisone, TA: triamcinolone, and CsA: cyclosporine A.

    Article Snippet: Procedure of Ki-67 staining and antibody information were described previously [ , , , ].

    Techniques: FACS, Cell Culture, Expressing, Transplantation Assay, In Vivo, In Vitro

    Stat4 requirement in T-bet function (A) Wild type, Stat4-deficient ( Stat4 −/−), T-bet-deficient ( Tbx21 −/−) and Stat4-T-bet-double deficient ( Stat4-Tbx21 −/−) CD4+ T cells were cultured under Th1 conditions (IL-12 + anti-IL-4) for five days and total cell extracts were immunoblotted for T-bet, Stat4 and GAPDH as a control. (B) Cells cultured as in (A) were assessed for the expression of Th1 genes before ( Il18rap , Runx3 ) or after ( Lta ) re-stimulation with anti-CD3. (C-G) Wild type, T-bet-deficient ( Tbx21 −/−) and Stat4-T-bet-double deficient ( Stat4 −/− Tbx21 −/−) CD4+ T cells were cultured under Th1 conditions. On day 2 of the culture period, cells were transduced with a bicistronic retrovirus expressing EGFP only (MIEG) or T-bet and EGFP (T-bet). At the end of the culture, cells were sorted for EGFP expression and stimulated for 18 hours with anti-CD3. Supernatants were analyzed for IFNγ levels using ELISA (C). RNA was isolated from each population to determine the expression levels of the indicated genes using qPCR (D). Surface expression of CXCR3 was determined using flow cytometry (E). ChIP assay was performed for acetylated-H3 or -H4 at the Hlx1 and Ifng promoters (F) or acetylated-H4 at the Xcl1 promoter (G). Results are the average ± SD of replicate samples and are representative of 2–3 experiments with similar results.

    Journal: Immunity

    Article Title: Stat4 is required for T-bet to promote IL-12-dependent Th1 fate determination

    doi: 10.1016/j.immuni.2008.08.017

    Figure Lengend Snippet: Stat4 requirement in T-bet function (A) Wild type, Stat4-deficient ( Stat4 −/−), T-bet-deficient ( Tbx21 −/−) and Stat4-T-bet-double deficient ( Stat4-Tbx21 −/−) CD4+ T cells were cultured under Th1 conditions (IL-12 + anti-IL-4) for five days and total cell extracts were immunoblotted for T-bet, Stat4 and GAPDH as a control. (B) Cells cultured as in (A) were assessed for the expression of Th1 genes before ( Il18rap , Runx3 ) or after ( Lta ) re-stimulation with anti-CD3. (C-G) Wild type, T-bet-deficient ( Tbx21 −/−) and Stat4-T-bet-double deficient ( Stat4 −/− Tbx21 −/−) CD4+ T cells were cultured under Th1 conditions. On day 2 of the culture period, cells were transduced with a bicistronic retrovirus expressing EGFP only (MIEG) or T-bet and EGFP (T-bet). At the end of the culture, cells were sorted for EGFP expression and stimulated for 18 hours with anti-CD3. Supernatants were analyzed for IFNγ levels using ELISA (C). RNA was isolated from each population to determine the expression levels of the indicated genes using qPCR (D). Surface expression of CXCR3 was determined using flow cytometry (E). ChIP assay was performed for acetylated-H3 or -H4 at the Hlx1 and Ifng promoters (F) or acetylated-H4 at the Xcl1 promoter (G). Results are the average ± SD of replicate samples and are representative of 2–3 experiments with similar results.

    Article Snippet: Flow cytometric analysis was performed using standard methods with a PE-labeled anti-CXCR3 (R & D Systems, Minneapolis, MN)( ).

    Techniques: Cell Culture, Expressing, Transduction, Enzyme-linked Immunosorbent Assay, Isolation, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Chromatin Immunoprecipitation

    Contribution of Stat4 and T-bet to expression of genes in Th1 cells Wild type, Stat4-deficient ( Stat4 −/−) and T-bet-deficient ( Tbx21 −/−) CD4+ T cells were cultured under Th1 conditions (IL−12 + anti-IL-4) for five days. RNA was isolated from cells either before ( Ccr5 , Il18r1 , Etv5 , Cxcr3 ) or six hours after ( Ifng , Hlx1 , Xcl1 , Egr2 , Egr3 , Furin ) re-stimulation of cells with anti-CD3. Quantitative PCR using TaqMan primers specific for each gene was performed and results were normalized to expression of beta2-microglobulin. Results are the average ± SD of replicate samples and are representative of four experiments with similar results.

    Journal: Immunity

    Article Title: Stat4 is required for T-bet to promote IL-12-dependent Th1 fate determination

    doi: 10.1016/j.immuni.2008.08.017

    Figure Lengend Snippet: Contribution of Stat4 and T-bet to expression of genes in Th1 cells Wild type, Stat4-deficient ( Stat4 −/−) and T-bet-deficient ( Tbx21 −/−) CD4+ T cells were cultured under Th1 conditions (IL−12 + anti-IL-4) for five days. RNA was isolated from cells either before ( Ccr5 , Il18r1 , Etv5 , Cxcr3 ) or six hours after ( Ifng , Hlx1 , Xcl1 , Egr2 , Egr3 , Furin ) re-stimulation of cells with anti-CD3. Quantitative PCR using TaqMan primers specific for each gene was performed and results were normalized to expression of beta2-microglobulin. Results are the average ± SD of replicate samples and are representative of four experiments with similar results.

    Article Snippet: Flow cytometric analysis was performed using standard methods with a PE-labeled anti-CXCR3 (R & D Systems, Minneapolis, MN)( ).

    Techniques: Expressing, Cell Culture, Isolation, Real-time Polymerase Chain Reaction

    HSV-IL-2 replication in tissue culture. Subconfluent RS cell monolayers were infected with 0.01, 1, or 10 PFU of HSV-IL-2, parental dLAT2903, or wild-type McKrae virus per cell with or without incubation with anti-IL-2 polyclonal antibody, as described in Materials and Methods. Total virus was harvested at the indicated times postinfection by two cycles of freeze-thawing. The amount of virus at each time for each virus was determined by standard plaque assays on RS cells.

    Journal: Journal of Virology

    Article Title: Overexpression of Interleukin-2 by a Recombinant Herpes Simplex Virus Type 1 Attenuates Pathogenicity and Enhances Antiviral Immunity

    doi: 10.1128/JVI.76.18.9069-9078.2002

    Figure Lengend Snippet: HSV-IL-2 replication in tissue culture. Subconfluent RS cell monolayers were infected with 0.01, 1, or 10 PFU of HSV-IL-2, parental dLAT2903, or wild-type McKrae virus per cell with or without incubation with anti-IL-2 polyclonal antibody, as described in Materials and Methods. Total virus was harvested at the indicated times postinfection by two cycles of freeze-thawing. The amount of virus at each time for each virus was determined by standard plaque assays on RS cells.

    Article Snippet: As described in Materials and Methods, the virus was mixed with 5 μg of anti-IL-2 polyclonal antibody, and then cells were infected at an MOI of 1 with the virus and antibody (Fig. ).

    Techniques: Infection, Incubation

    Effect of eBV on TNF-α and IL-4 in compound 48/80-stimulated RBL-2H3 cells. (A, B) RBL-2H3 cells were treated with compound 48/80 (5 μg/mL) in the presence of eBV, BV, and melittin (0.5, 1, and 2 μg/mL) for 30 min. TNF-α and IL-1β levels were measured using an ELISA kit. (C, D) RBL-2H3 cells were treated with compound 48/80 (5 μg/mL) in the presence of eBV, BV, and melittin (0.5, 1, and 2 μg/mL) for 30 min. Total RNA was isolated and further analyzed by real-time PCR. The results are presented as relative expression levels compared with those in unstimulated cells and were normalized to β-actin expression. Data are presented as mean ± SD (n = 3). ** P

    Journal: PLoS ONE

    Article Title: In Vitro and In Vivo Anti-Allergic and Anti-Inflammatory Effects of eBV, a Newly Developed Derivative of Bee Venom, through Modulation of IRF3 Signaling Pathway in a Carrageenan-Induced Edema Model

    doi: 10.1371/journal.pone.0168120

    Figure Lengend Snippet: Effect of eBV on TNF-α and IL-4 in compound 48/80-stimulated RBL-2H3 cells. (A, B) RBL-2H3 cells were treated with compound 48/80 (5 μg/mL) in the presence of eBV, BV, and melittin (0.5, 1, and 2 μg/mL) for 30 min. TNF-α and IL-1β levels were measured using an ELISA kit. (C, D) RBL-2H3 cells were treated with compound 48/80 (5 μg/mL) in the presence of eBV, BV, and melittin (0.5, 1, and 2 μg/mL) for 30 min. Total RNA was isolated and further analyzed by real-time PCR. The results are presented as relative expression levels compared with those in unstimulated cells and were normalized to β-actin expression. Data are presented as mean ± SD (n = 3). ** P

    Article Snippet: The levels of prostaglandin E2 (PGE2 ), TNF-α and IL-1β were measured through immunosorbent assay method using ELISA kits (R & D Systems, Minneapolis, MN, USA).

    Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Real-time Polymerase Chain Reaction, Expressing