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    ATCC raw264 7 cells raw264 7 macrophage cells
    Concentration-dependent stimulation of COX-2 mRNA and protein expression in <t>RAW264.7</t> cells. ( A ) Analysis for COX-2 mRNA by real-time PCR: RAW cells were stimulated with increasing concentrations (25 ng to100 ng/ml) of LPS or BLP for 6 hours. Total RNA from treated cells was isolated and real-time PCR was performed as described in ‘Methods’. Results are expressed as 2 −∆∆CT , that is the expression of target gene relative to the housekeeping gene (GAPDH) and normalized to the untreated control. The data shown are mean ± SEM (n = 3), ***p ≤ 0.0001 when compared between LPS and BLP at same doses. ( B ) COX-2 protein expression in response to endotoxins: Lysates from the stated amounts of endotoxin-treated cells were prepared using RIPA buffer and immunoblots were developed using specific primary and appropriate secondary antibodies for COX-2 and GAPDH and visualized as described in ‘Methods’. The results represent data obtained in two independent experiments. One of the best among two full length blot is presented in Supplementary Figure S2 .
    Raw264 7 Cells Raw264 7 Macrophage Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 519 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DS Pharma Biomedical raw264 7
    Dose-Response Curves of Curcumin/Liposome in Inhibition of Cell Proliferation. THP-1 (panel A) or <t>RAW264.7</t> (panel B) cells were cultured for 3 days in the presence of curcumin/liposome (●), curcumin (▲), and control liposomes (○), respectively. After culturing, they were incubated with WST-8 for 3 h to assess viable cells in the culture. Resultant coloring reaction by WST-8 was measured with a plate-reader at the absorbance of 450 nm. In the vertical axis, the absorbance of only the medium was defined as 0%. Symbols in the Figure represent the percentage of absorbance with curucmin or liposomes to that without the test samples. Data represent mean values ± range (bars) in duplicate assays.
    Raw264 7, supplied by DS Pharma Biomedical, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Concentration-dependent stimulation of COX-2 mRNA and protein expression in RAW264.7 cells. ( A ) Analysis for COX-2 mRNA by real-time PCR: RAW cells were stimulated with increasing concentrations (25 ng to100 ng/ml) of LPS or BLP for 6 hours. Total RNA from treated cells was isolated and real-time PCR was performed as described in ‘Methods’. Results are expressed as 2 −∆∆CT , that is the expression of target gene relative to the housekeeping gene (GAPDH) and normalized to the untreated control. The data shown are mean ± SEM (n = 3), ***p ≤ 0.0001 when compared between LPS and BLP at same doses. ( B ) COX-2 protein expression in response to endotoxins: Lysates from the stated amounts of endotoxin-treated cells were prepared using RIPA buffer and immunoblots were developed using specific primary and appropriate secondary antibodies for COX-2 and GAPDH and visualized as described in ‘Methods’. The results represent data obtained in two independent experiments. One of the best among two full length blot is presented in Supplementary Figure S2 .

    Journal: Scientific Reports

    Article Title: Escherichia coli Braun Lipoprotein (BLP) exhibits endotoxemia – like pathology in Swiss albino mice

    doi: 10.1038/srep34666

    Figure Lengend Snippet: Concentration-dependent stimulation of COX-2 mRNA and protein expression in RAW264.7 cells. ( A ) Analysis for COX-2 mRNA by real-time PCR: RAW cells were stimulated with increasing concentrations (25 ng to100 ng/ml) of LPS or BLP for 6 hours. Total RNA from treated cells was isolated and real-time PCR was performed as described in ‘Methods’. Results are expressed as 2 −∆∆CT , that is the expression of target gene relative to the housekeeping gene (GAPDH) and normalized to the untreated control. The data shown are mean ± SEM (n = 3), ***p ≤ 0.0001 when compared between LPS and BLP at same doses. ( B ) COX-2 protein expression in response to endotoxins: Lysates from the stated amounts of endotoxin-treated cells were prepared using RIPA buffer and immunoblots were developed using specific primary and appropriate secondary antibodies for COX-2 and GAPDH and visualized as described in ‘Methods’. The results represent data obtained in two independent experiments. One of the best among two full length blot is presented in Supplementary Figure S2 .

    Article Snippet: Proinflammatory mRNA in RAW264.7 cells RAW264.7 macrophage cells were obtained from ATCC and maintained as described previously .

    Techniques: Concentration Assay, Expressing, Real-time Polymerase Chain Reaction, Isolation, Western Blot

    Induction of proinflammatory genes by BLP in RAW264.7 cells. RAW264.7 cells were cultured and maintained as described in ‘Methods’. 5 × 10 5 cells/ml were treated for 2 hours with vehicle, LPS (100 ng/ml) or BLP (100 ng/ml). All the treatments were carried out in triplicates. After incubation, total RNA was extracted and real-time PCR analysis was carried out for ( A ) TNF-α, ( B ) IL-1β, ( C ) COX-2 and ( D ) COX-1. The results are represented in terms of 2 −∆∆CT with respect to GAPDH and the data shown are mean ± SEM (n = 3). *p ≤ 0.01; **p ≤ 0.001 when compared with the vehicle treated group. The results are representative of two independent experiments.

    Journal: Scientific Reports

    Article Title: Escherichia coli Braun Lipoprotein (BLP) exhibits endotoxemia – like pathology in Swiss albino mice

    doi: 10.1038/srep34666

    Figure Lengend Snippet: Induction of proinflammatory genes by BLP in RAW264.7 cells. RAW264.7 cells were cultured and maintained as described in ‘Methods’. 5 × 10 5 cells/ml were treated for 2 hours with vehicle, LPS (100 ng/ml) or BLP (100 ng/ml). All the treatments were carried out in triplicates. After incubation, total RNA was extracted and real-time PCR analysis was carried out for ( A ) TNF-α, ( B ) IL-1β, ( C ) COX-2 and ( D ) COX-1. The results are represented in terms of 2 −∆∆CT with respect to GAPDH and the data shown are mean ± SEM (n = 3). *p ≤ 0.01; **p ≤ 0.001 when compared with the vehicle treated group. The results are representative of two independent experiments.

    Article Snippet: Proinflammatory mRNA in RAW264.7 cells RAW264.7 macrophage cells were obtained from ATCC and maintained as described previously .

    Techniques: Cell Culture, Incubation, Real-time Polymerase Chain Reaction

    Effect of GLP-1(9–36) on inflammatory gene expression in RAW264.7 macrophages. mRNA expression of a IL-1β, b TNF-α, c Arg1, d Fizz1, e IL-10, f IL-12, g IL-6, and h TGF-β 3 by real-time RT-PCR ( n = 5–12). Mean ± SEM. * P

    Journal: Cardiovascular Diabetology

    Article Title: Metabolically-inactive glucagon-like peptide-1(9–36)amide confers selective protective actions against post-myocardial infarction remodelling

    doi: 10.1186/s12933-016-0386-5

    Figure Lengend Snippet: Effect of GLP-1(9–36) on inflammatory gene expression in RAW264.7 macrophages. mRNA expression of a IL-1β, b TNF-α, c Arg1, d Fizz1, e IL-10, f IL-12, g IL-6, and h TGF-β 3 by real-time RT-PCR ( n = 5–12). Mean ± SEM. * P

    Article Snippet: RAW264.7 macrophage culture RAW264.7 murine macrophages (ATCC® TIB-71™) were maintained in DMEM containing 10 % FCS, 100 U/ml penicillin and 100 µg/ml streptomycin.

    Techniques: Expressing, Quantitative RT-PCR

    Short anthophyllite and tremolite fibers are engulfed whereas long anthophyllite fibers are attached by the pseudopod of MeT5A and RAW264.7 cells, as observed with time‐lapse light microscopy. A, Long anthophyllite fibers revealed periodical swinging movement after making mechanical contact; however, a small number of short fibers were incorporated into the cytoplasm of the MeT5A cells. Tremolite was internalized from the bottom of the dish into the cytoplasm of MeT5A cells, like a cleaning floor mop. Some MeT5A cells, which were burdened with an abundance of tremolite, lost contact with the dish and disappeared from the camera, suggesting that an overload of tremolite caused MeT5A cell death (Video S1 , anthophyllite; Video S2 , tremolite). B, Anthophyllite that touched the cell was vibrated by the cells that aggregated around the fiber. In contrast to the cells from the epithelial or mesothelial cell lines, the RAW264.7 cells rarely swung the fibers. Tremolite was phagocytosed into Raw 264.7 cells in a manner similar to that of MeT5A cells. At 36 and 48 h, RAW264.7 cells became swollen and extended numerous protrusions explosively (Video S3 , anthophyllite; Video S4 , tremolite)

    Journal: Cancer Science

    Article Title: Frequent homozygous deletion of Cdkn2a/2b in tremolite‐induced malignant mesothelioma in rats, et al. Frequent homozygous deletion of Cdkn2a/2b in tremolite‐induced malignant mesothelioma in rats

    doi: 10.1111/cas.14358

    Figure Lengend Snippet: Short anthophyllite and tremolite fibers are engulfed whereas long anthophyllite fibers are attached by the pseudopod of MeT5A and RAW264.7 cells, as observed with time‐lapse light microscopy. A, Long anthophyllite fibers revealed periodical swinging movement after making mechanical contact; however, a small number of short fibers were incorporated into the cytoplasm of the MeT5A cells. Tremolite was internalized from the bottom of the dish into the cytoplasm of MeT5A cells, like a cleaning floor mop. Some MeT5A cells, which were burdened with an abundance of tremolite, lost contact with the dish and disappeared from the camera, suggesting that an overload of tremolite caused MeT5A cell death (Video S1 , anthophyllite; Video S2 , tremolite). B, Anthophyllite that touched the cell was vibrated by the cells that aggregated around the fiber. In contrast to the cells from the epithelial or mesothelial cell lines, the RAW264.7 cells rarely swung the fibers. Tremolite was phagocytosed into Raw 264.7 cells in a manner similar to that of MeT5A cells. At 36 and 48 h, RAW264.7 cells became swollen and extended numerous protrusions explosively (Video S3 , anthophyllite; Video S4 , tremolite)

    Article Snippet: MeT5A, RAW264.7, and HeLa cell lines were obtained from ATCC and 3 mesothelioma cell lines, named Meso8A, Meso8D, and Meso12A, were provided by Dr Yoshitaka Sekido (Division of Cancer Biology, Aichi Cancer Center) and maintained in RPMI‐1640 with FBS (10%) and an antibiotic‐antimycotic solution (1×).

    Techniques: Light Microscopy

    Short anthophyllite and tremolite fibers are phagocytosed and localized in phagosomes, while long anthophyllite fibers attach to the plasma membrane of MeT5A and RAW264.7 cells, as observed by transmission electron microscopy. A, Large fibers of anthophyllite crushed the diamond knife; thus, only clefts were sometimes observed after ultrathin cutting. Short anthophyllite fibers were phagocytosed, whereas long ones were attached to the pseudopod of plasma membrane. Tremolite was observed in phagosomes more frequently and with fewer artificial clefts than anthophyllite, consistent with the short and thin dimensions of tremolite. B, Short anthophyllite fibers were phagocytosed. Pseudopods extended to catch anthophyllite fibers. Some bundles of tremolite fibers attached to the plasma membrane, and other tremolite fibers were observed in the phagosomes (arrows indicate internalized asbestos fibers)

    Journal: Cancer Science

    Article Title: Frequent homozygous deletion of Cdkn2a/2b in tremolite‐induced malignant mesothelioma in rats, et al. Frequent homozygous deletion of Cdkn2a/2b in tremolite‐induced malignant mesothelioma in rats

    doi: 10.1111/cas.14358

    Figure Lengend Snippet: Short anthophyllite and tremolite fibers are phagocytosed and localized in phagosomes, while long anthophyllite fibers attach to the plasma membrane of MeT5A and RAW264.7 cells, as observed by transmission electron microscopy. A, Large fibers of anthophyllite crushed the diamond knife; thus, only clefts were sometimes observed after ultrathin cutting. Short anthophyllite fibers were phagocytosed, whereas long ones were attached to the pseudopod of plasma membrane. Tremolite was observed in phagosomes more frequently and with fewer artificial clefts than anthophyllite, consistent with the short and thin dimensions of tremolite. B, Short anthophyllite fibers were phagocytosed. Pseudopods extended to catch anthophyllite fibers. Some bundles of tremolite fibers attached to the plasma membrane, and other tremolite fibers were observed in the phagosomes (arrows indicate internalized asbestos fibers)

    Article Snippet: MeT5A, RAW264.7, and HeLa cell lines were obtained from ATCC and 3 mesothelioma cell lines, named Meso8A, Meso8D, and Meso12A, were provided by Dr Yoshitaka Sekido (Division of Cancer Biology, Aichi Cancer Center) and maintained in RPMI‐1640 with FBS (10%) and an antibiotic‐antimycotic solution (1×).

    Techniques: Transmission Assay, Electron Microscopy

    Anthophyllite does not affect proliferation, whereas tremolite suppresses proliferation of MeT5A and RAW264.7 cells. A, Anthophyllite (Ant) at 1, 5, or 10 μg/cm 2 did not suppress MeT5A proliferation. Tremolite (Tre) decreased MeT5A proliferation in a dose‐dependent manner. At 10 μg/cm 2 , tremolite and crocidolite (Cro) induced similar levels of cytotoxicity. B, Ant at 1, 5, or 10 μg/cm 2 did not suppress RAW264.7 proliferation. Tre decreased the proliferation of RAW264.7 cells in a dose‐dependent manner. At 10 μg/cm 2 , Tre and Cro induced similar levels of cytotoxicity. Notably, 1 μg/cm 2 Tre significantly decreased cellular proliferation. C, Ant at 1, 5, or 10 μg/cm 2 did not suppress MeT5A proliferation, whereas Tre decreased MeT5A proliferation in a dose‐dependent manner at days 1, 2, and 3. At 10 μg/cm 2 , Tre and Cro caused similar levels of cytotoxicity. D, Ant did not suppress RAW264.7 proliferation. Significant suppression was observed only on day 3 for 5 and 10 μg/cm 2 Tre and for 10 μg/cm 2 Cro. E, After 24 h, release of intracellular lactate dehydrogenase (LDH) was significantly increased in MeT5A cells treated with 10 μg/cm 2 Tre or Cro. F, Release of intracellular LDH was not significantly elevated in RAW264.7 cells with every asbestos tested after 24 h (* P

    Journal: Cancer Science

    Article Title: Frequent homozygous deletion of Cdkn2a/2b in tremolite‐induced malignant mesothelioma in rats, et al. Frequent homozygous deletion of Cdkn2a/2b in tremolite‐induced malignant mesothelioma in rats

    doi: 10.1111/cas.14358

    Figure Lengend Snippet: Anthophyllite does not affect proliferation, whereas tremolite suppresses proliferation of MeT5A and RAW264.7 cells. A, Anthophyllite (Ant) at 1, 5, or 10 μg/cm 2 did not suppress MeT5A proliferation. Tremolite (Tre) decreased MeT5A proliferation in a dose‐dependent manner. At 10 μg/cm 2 , tremolite and crocidolite (Cro) induced similar levels of cytotoxicity. B, Ant at 1, 5, or 10 μg/cm 2 did not suppress RAW264.7 proliferation. Tre decreased the proliferation of RAW264.7 cells in a dose‐dependent manner. At 10 μg/cm 2 , Tre and Cro induced similar levels of cytotoxicity. Notably, 1 μg/cm 2 Tre significantly decreased cellular proliferation. C, Ant at 1, 5, or 10 μg/cm 2 did not suppress MeT5A proliferation, whereas Tre decreased MeT5A proliferation in a dose‐dependent manner at days 1, 2, and 3. At 10 μg/cm 2 , Tre and Cro caused similar levels of cytotoxicity. D, Ant did not suppress RAW264.7 proliferation. Significant suppression was observed only on day 3 for 5 and 10 μg/cm 2 Tre and for 10 μg/cm 2 Cro. E, After 24 h, release of intracellular lactate dehydrogenase (LDH) was significantly increased in MeT5A cells treated with 10 μg/cm 2 Tre or Cro. F, Release of intracellular LDH was not significantly elevated in RAW264.7 cells with every asbestos tested after 24 h (* P

    Article Snippet: MeT5A, RAW264.7, and HeLa cell lines were obtained from ATCC and 3 mesothelioma cell lines, named Meso8A, Meso8D, and Meso12A, were provided by Dr Yoshitaka Sekido (Division of Cancer Biology, Aichi Cancer Center) and maintained in RPMI‐1640 with FBS (10%) and an antibiotic‐antimycotic solution (1×).

    Techniques:

    Lipopolysaccharide-induced upregulation of Il6, Il1β, Cox2, iNos and Tnfα mRNA expression is blocked by the NE and isolated GA. RAW264.7 were pre-incubated with either NE (left column) GA (right column) or solvent (DMSO) for 24 h (white bars). For LPS-induced experiments, macrophages were co-incubated with 100 ng/ml LPS and either solvent, NE or GA at the doses indicated for another 24 h (grey bars). Samples co-cultured with solvent and LPS were defined as one. Expression levels of the inflammatory response genes (A) Tnfα, (B) Il6, (C) Il1β, (D) Cox2 and (E) iNos were measured using RT-qPCR and normalized to the mRNA expression of the reference gene (F) peptidylprolyl isomerase B (Ppib). Error bars display calculated minimum and maximum of SEM (SEM ± min, max) expression levels of four independent biological experiments, each measured in one or two technical replicates. *, p

    Journal: Redox Biology

    Article Title: The vitamin E derivative garcinoic acid from Garcinia kola nut seeds attenuates the inflammatory response

    doi: 10.1016/j.redox.2019.101166

    Figure Lengend Snippet: Lipopolysaccharide-induced upregulation of Il6, Il1β, Cox2, iNos and Tnfα mRNA expression is blocked by the NE and isolated GA. RAW264.7 were pre-incubated with either NE (left column) GA (right column) or solvent (DMSO) for 24 h (white bars). For LPS-induced experiments, macrophages were co-incubated with 100 ng/ml LPS and either solvent, NE or GA at the doses indicated for another 24 h (grey bars). Samples co-cultured with solvent and LPS were defined as one. Expression levels of the inflammatory response genes (A) Tnfα, (B) Il6, (C) Il1β, (D) Cox2 and (E) iNos were measured using RT-qPCR and normalized to the mRNA expression of the reference gene (F) peptidylprolyl isomerase B (Ppib). Error bars display calculated minimum and maximum of SEM (SEM ± min, max) expression levels of four independent biological experiments, each measured in one or two technical replicates. *, p

    Article Snippet: 2.3 RAW264.7 macrophage culture Murine RAW264.7 macrophages (ATCC, Manassas, VA) were cultivated as described previously [ ].

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

    Lipopolysaccharide-induced expression of iNos and Cox2 protein and secretion of respective signaling molecules are more potently blocked by GA compared to NE. RAW264.7 macrophages were incubated with either solvent (DMSO, white bars), NE or GA, or co-incubated with 100 ng/ml LPS (grey bars). The NE and GA decreased the protein expression of (A) Cox2 and (B) iNos after 24 h pre-incubation with NE or GA followed by 14 h and 24 h co-incubation with LPS, respectively. Samples incubated with LPS were defined as reference and were set as one. Protein levels were normalized to α-tubulin for quantification and representative Western blots are shown (for un-chopped versions see Suppl. Fig. S6 ). (C) Basal NO production, determined using Griess assay, were affected neither by the NE nor GA, whereas LPS-induced the formation of NO was significantly decreased by NE and even more effectively by purified GA. Treatment of RAW264.7 macrophages to measure released (D) TxB 2 and (E) PGs into culture supernatants followed the description in Fig. 2 except for use of 2.5 μM GA. Neither the NE nor GA altered the basal release of prostanoids. Treatment with LPS significantly induced TxB 2 and PG levels in the supernatant of macrophages and was set to 100% or one, respectively. Both, NE and GA decreased the release of TxB 2, PGE 2 and PGD 2 by LPS-activated macrophages Means of three independent biological experiments measured in two technical replicates (A,B) , three (C) , six (D) or four to five (E) independent biological experiments are shown; error bars display SEM. *, p

    Journal: Redox Biology

    Article Title: The vitamin E derivative garcinoic acid from Garcinia kola nut seeds attenuates the inflammatory response

    doi: 10.1016/j.redox.2019.101166

    Figure Lengend Snippet: Lipopolysaccharide-induced expression of iNos and Cox2 protein and secretion of respective signaling molecules are more potently blocked by GA compared to NE. RAW264.7 macrophages were incubated with either solvent (DMSO, white bars), NE or GA, or co-incubated with 100 ng/ml LPS (grey bars). The NE and GA decreased the protein expression of (A) Cox2 and (B) iNos after 24 h pre-incubation with NE or GA followed by 14 h and 24 h co-incubation with LPS, respectively. Samples incubated with LPS were defined as reference and were set as one. Protein levels were normalized to α-tubulin for quantification and representative Western blots are shown (for un-chopped versions see Suppl. Fig. S6 ). (C) Basal NO production, determined using Griess assay, were affected neither by the NE nor GA, whereas LPS-induced the formation of NO was significantly decreased by NE and even more effectively by purified GA. Treatment of RAW264.7 macrophages to measure released (D) TxB 2 and (E) PGs into culture supernatants followed the description in Fig. 2 except for use of 2.5 μM GA. Neither the NE nor GA altered the basal release of prostanoids. Treatment with LPS significantly induced TxB 2 and PG levels in the supernatant of macrophages and was set to 100% or one, respectively. Both, NE and GA decreased the release of TxB 2, PGE 2 and PGD 2 by LPS-activated macrophages Means of three independent biological experiments measured in two technical replicates (A,B) , three (C) , six (D) or four to five (E) independent biological experiments are shown; error bars display SEM. *, p

    Article Snippet: 2.3 RAW264.7 macrophage culture Murine RAW264.7 macrophages (ATCC, Manassas, VA) were cultivated as described previously [ ].

    Techniques: Expressing, Incubation, Western Blot, Griess Assay, Purification

    Catalase inhibition accelerates inflammation in RAW264.7 macrophages. RAW264.7 cells were treated for 6 h with 0, 1, 2, 5, and 10 mM 3-AT. (a) JNK phosphorylation was quantified by western blot (upper panel) and densitometry (lower panel). (b) IL-1 β , (c) IL-6, and (d) TNF- α mRNA levels were measured by real-time PCR. (e) JNK phosphorylation was also determined by western blot (upper panel) and densitometry (lower panel) in cells treated with 0, 1, 2, 5, and 10 mM 3-AT 1 h prior to stimulation with 200 μ M PA for 6 h. (f) IL-1 β , (g) IL-6, and (h) TNF- α mRNA levels were also measured by real-time PCR in these cells. Data are mean ± SE of four experiments. BSA was used as control for the effects of PA. ∗ p

    Journal: Mediators of Inflammation

    Article Title: Novel Role of Endogenous Catalase in Macrophage Polarization in Adipose Tissue

    doi: 10.1155/2016/8675905

    Figure Lengend Snippet: Catalase inhibition accelerates inflammation in RAW264.7 macrophages. RAW264.7 cells were treated for 6 h with 0, 1, 2, 5, and 10 mM 3-AT. (a) JNK phosphorylation was quantified by western blot (upper panel) and densitometry (lower panel). (b) IL-1 β , (c) IL-6, and (d) TNF- α mRNA levels were measured by real-time PCR. (e) JNK phosphorylation was also determined by western blot (upper panel) and densitometry (lower panel) in cells treated with 0, 1, 2, 5, and 10 mM 3-AT 1 h prior to stimulation with 200 μ M PA for 6 h. (f) IL-1 β , (g) IL-6, and (h) TNF- α mRNA levels were also measured by real-time PCR in these cells. Data are mean ± SE of four experiments. BSA was used as control for the effects of PA. ∗ p

    Article Snippet: Culture of RAW264.7 Macrophages RAW264.7 cells were procured from American Type Culture Collection (Manassas, VA, USA) and maintained at 37°C in humidified air with 5% CO2 and in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μ g/mL streptomycin, and 44 mM NaHCO3 .

    Techniques: Inhibition, Western Blot, Real-time Polymerase Chain Reaction

    Pharmacological or genetic inhibition of catalase alters macrophage activation in response to LPS or IL-4. RAW264.7 cells were treated with 0, 1, 2, 5, and 10 mM 3-AT and stimulated with 1 ng/mL LPS for 6 h or 24 h. (a) JNK phosphorylation at 6 h and (b) iNOS levels at 24 h were quantified by western blot (upper panel) and densitometry (lower panel). ∗ p

    Journal: Mediators of Inflammation

    Article Title: Novel Role of Endogenous Catalase in Macrophage Polarization in Adipose Tissue

    doi: 10.1155/2016/8675905

    Figure Lengend Snippet: Pharmacological or genetic inhibition of catalase alters macrophage activation in response to LPS or IL-4. RAW264.7 cells were treated with 0, 1, 2, 5, and 10 mM 3-AT and stimulated with 1 ng/mL LPS for 6 h or 24 h. (a) JNK phosphorylation at 6 h and (b) iNOS levels at 24 h were quantified by western blot (upper panel) and densitometry (lower panel). ∗ p

    Article Snippet: Culture of RAW264.7 Macrophages RAW264.7 cells were procured from American Type Culture Collection (Manassas, VA, USA) and maintained at 37°C in humidified air with 5% CO2 and in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μ g/mL streptomycin, and 44 mM NaHCO3 .

    Techniques: Inhibition, Activation Assay, Western Blot

    Analyte and transcription factor response to WTC-PM and LPA in RAW264.7 Supernatants were assayed after 24 h of WTC-PM and/or LPA exposure, n = 3. ( A ) IL-1α, ( B ) IL-10, ( C ) TNF-α, ( D ) NF-κB. All values reported as mean ± SD of fold change over PBS. Independent LPA exposure is denoted as the left red point. ( A – D ) Synergistic inflammatory expression of ( B ) IL-10 observed after WTC-PM/LPA co-exposure. ( D ) PM-induced NF-κB elaboration greater than that of PM/LPA co-exposure. ( E ) Immunoblots display p -Akt, p -STAT3, STAT5b, and GAPDH expression: Lane 1 Media Alone, Lane 2 WTC-PM, Lane 3 LPA, Lane 4 WTC-PM/LPA. ( F ) Immunoblots display RAGE and GAPDH expression: Lane 1 WTC-PM, Lane 2 LPA, Lane 3 WTC-PM/LPA. ( G ) Densitometry analyses of immunoblots; fold change over media alone (MA). * denotes p

    Journal: International Journal of Environmental Research and Public Health

    Article Title: Synergistic Effect of WTC-Particulate Matter and Lysophosphatidic Acid Exposure and the Role of RAGE: In-Vitro and Translational Assessment

    doi: 10.3390/ijerph17124318

    Figure Lengend Snippet: Analyte and transcription factor response to WTC-PM and LPA in RAW264.7 Supernatants were assayed after 24 h of WTC-PM and/or LPA exposure, n = 3. ( A ) IL-1α, ( B ) IL-10, ( C ) TNF-α, ( D ) NF-κB. All values reported as mean ± SD of fold change over PBS. Independent LPA exposure is denoted as the left red point. ( A – D ) Synergistic inflammatory expression of ( B ) IL-10 observed after WTC-PM/LPA co-exposure. ( D ) PM-induced NF-κB elaboration greater than that of PM/LPA co-exposure. ( E ) Immunoblots display p -Akt, p -STAT3, STAT5b, and GAPDH expression: Lane 1 Media Alone, Lane 2 WTC-PM, Lane 3 LPA, Lane 4 WTC-PM/LPA. ( F ) Immunoblots display RAGE and GAPDH expression: Lane 1 WTC-PM, Lane 2 LPA, Lane 3 WTC-PM/LPA. ( G ) Densitometry analyses of immunoblots; fold change over media alone (MA). * denotes p

    Article Snippet: THP-1-derived macrophages were phorbol-12-myristate-13-acetate (PMA)-differentiated and cultured in RPMI1640 (Gibco) and murine RAW264.7 (ATCC) (murine) macrophage-like cells were cultured in DMEM (ATCC).

    Techniques: Expressing, Western Blot

    Response of RAW264.7 macrophage cells to different laminarins in the presence or absence of a Dectin-1 agonist A. RAW cells were treated with increasing concentrations of the indicated laminarin for 18-20 h followed by analysis of mouse TNFα via ELISA. For ease of interpretation, dose-response graphs on left show the mean response of four individual experiments, while the graph at right represents the mean TNFα response induced with 12.5 μg/ml of the indicated laminarin ± SEM. B. Dose-response curves were log(x) transformed, normalized and analyzed using nonlinear regression as described in methods to determine each compound's EC50. Data were analyzed using one-way ANOVA and all groups were determined to be significantly (p ≤ 0.0001) different from one another. C. RAW cells were treated and analyzed as in A , but 1 h post-laminarin treatment 10 μg/ml of purified C. albicans β-glucan was added for the remaining incubation. Dashed line indicates the average TNFα level induced by β-glucan alone. D. Data were analyzed and are represented as in B . All groups were determined to be significantly different from one another (p ≤ 0.01) expect those denoted.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Immunoregulatory activity of the natural product laminarin varies widely as a result lt of its physical properties

    doi: 10.4049/jimmunol.1701258

    Figure Lengend Snippet: Response of RAW264.7 macrophage cells to different laminarins in the presence or absence of a Dectin-1 agonist A. RAW cells were treated with increasing concentrations of the indicated laminarin for 18-20 h followed by analysis of mouse TNFα via ELISA. For ease of interpretation, dose-response graphs on left show the mean response of four individual experiments, while the graph at right represents the mean TNFα response induced with 12.5 μg/ml of the indicated laminarin ± SEM. B. Dose-response curves were log(x) transformed, normalized and analyzed using nonlinear regression as described in methods to determine each compound's EC50. Data were analyzed using one-way ANOVA and all groups were determined to be significantly (p ≤ 0.0001) different from one another. C. RAW cells were treated and analyzed as in A , but 1 h post-laminarin treatment 10 μg/ml of purified C. albicans β-glucan was added for the remaining incubation. Dashed line indicates the average TNFα level induced by β-glucan alone. D. Data were analyzed and are represented as in B . All groups were determined to be significantly different from one another (p ≤ 0.01) expect those denoted.

    Article Snippet: Raw264.7 (RAW) and THP-1 cells were obtained from ATCC (Manassas, Virginia) and maintained in complete DMEM or RPMI media, respectively, that was supplemented with 10% fetal bovine serum and penicillin/streptomycin/glutamine (Fisher Scientific).

    Techniques: Enzyme-linked Immunosorbent Assay, Transformation Assay, Purification, Incubation

    Effect of stable overexpression of VEGF on macrophages in tubular networks formation After 24 h of culture in Matrigel, hVEGF 165 -ZsGreen1-RAW264.7 formed tubular structures. But no similar structure was detected in untransfected RAW264.7, ZsGreen1-RAW264.7, or exogenous VEGF-treated RAW264.7. Scale bars =100 μm. All images are representatives of three independent experiments.

    Journal: Bioscience Reports

    Article Title: Vascular endothelial growth factor modified macrophages transdifferentiate into endothelial-like cells and decrease foam cell formation

    doi: 10.1042/BSR20170002

    Figure Lengend Snippet: Effect of stable overexpression of VEGF on macrophages in tubular networks formation After 24 h of culture in Matrigel, hVEGF 165 -ZsGreen1-RAW264.7 formed tubular structures. But no similar structure was detected in untransfected RAW264.7, ZsGreen1-RAW264.7, or exogenous VEGF-treated RAW264.7. Scale bars =100 μm. All images are representatives of three independent experiments.

    Article Snippet: It is approximately three-fold higher than untransfected RAW264.7 and ZsGreen1-RAW264.7 (both P < 0.01) ( B).

    Techniques: Over Expression

    Effect of stable overexpression of VEGF on macrophages in phenotypic characteristics ( A ) Quantitative analysis of mRNA expression of endothelial markers by real-time PCR. mRNA expression of FLK-1, vWF, eNOS, VE-cadherin, and Tie-2 were dramatically increased in hVEGF 165 -ZsGreen1-RAW264.7 cells compared with untransfected RAW264.7, ZsGreen1-RAW264.7, and exogenous VEGF-treated RAW264.7. Data are normalized to β-actin and presented as fold difference relative to RAW267.4 control. ( B ) Western blot analysis of endothelial markers expression. Protein expression of FLK-1, vWF, and eNOS were dramatically increased in hVEGF 165 -ZsGreen1-RAW264.7 cells compared with untransfected RAW264.7, ZsGreen1-RAW264.7, and exogenous VEGF-treated RAW264.7. All images are representatives of three independent experiments each performed in triplicate, and graphs depict the value of mean and S.D . * indicates P

    Journal: Bioscience Reports

    Article Title: Vascular endothelial growth factor modified macrophages transdifferentiate into endothelial-like cells and decrease foam cell formation

    doi: 10.1042/BSR20170002

    Figure Lengend Snippet: Effect of stable overexpression of VEGF on macrophages in phenotypic characteristics ( A ) Quantitative analysis of mRNA expression of endothelial markers by real-time PCR. mRNA expression of FLK-1, vWF, eNOS, VE-cadherin, and Tie-2 were dramatically increased in hVEGF 165 -ZsGreen1-RAW264.7 cells compared with untransfected RAW264.7, ZsGreen1-RAW264.7, and exogenous VEGF-treated RAW264.7. Data are normalized to β-actin and presented as fold difference relative to RAW267.4 control. ( B ) Western blot analysis of endothelial markers expression. Protein expression of FLK-1, vWF, and eNOS were dramatically increased in hVEGF 165 -ZsGreen1-RAW264.7 cells compared with untransfected RAW264.7, ZsGreen1-RAW264.7, and exogenous VEGF-treated RAW264.7. All images are representatives of three independent experiments each performed in triplicate, and graphs depict the value of mean and S.D . * indicates P

    Article Snippet: It is approximately three-fold higher than untransfected RAW264.7 and ZsGreen1-RAW264.7 (both P < 0.01) ( B).

    Techniques: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Western Blot

    VEGF 165 secretion level in supernatant after 72 h serum-free culture by ELISA ( A ) hVEGF 165 production increased dramatically in hVEGF 165 -ZsGreen1-RAW264.7, but could not be detected in untransfected RAW264.7 and control transfected ZsGreen1-RAW264.7 ( P

    Journal: Bioscience Reports

    Article Title: Vascular endothelial growth factor modified macrophages transdifferentiate into endothelial-like cells and decrease foam cell formation

    doi: 10.1042/BSR20170002

    Figure Lengend Snippet: VEGF 165 secretion level in supernatant after 72 h serum-free culture by ELISA ( A ) hVEGF 165 production increased dramatically in hVEGF 165 -ZsGreen1-RAW264.7, but could not be detected in untransfected RAW264.7 and control transfected ZsGreen1-RAW264.7 ( P

    Article Snippet: It is approximately three-fold higher than untransfected RAW264.7 and ZsGreen1-RAW264.7 (both P < 0.01) ( B).

    Techniques: Enzyme-linked Immunosorbent Assay, Transfection

    Foam cell formation assay ( A ) Representative images of foam cell formation assay. Untransfected RAW264.7, ZsGreen1-RAW264.7, and hVEGF 165 -ZsGreen1-RAW264.7 were treated with 100 μg/ml ox-LDL for 24 h. After fixation and staining with Oil Red O, the cells were observed by light microscopy. Cytoplasmic lipid droplets accumulated in RAW264.7 or ZsGreen1-RAW264.7, but hVEGF165-ZsGreen1-RAW264.7 cells showed significantly decreased lipid accumulation. Scale bars =50 μm. ( B ) Quantitative analysis of lipid accumulation in cells. The cells stained with Oil Red O were treated with 1 ml 60% isopropanol for 1 h to redissolve the oil red O and absorbance was detected at 518 nm through a spectrophotometer. The absorbance of oil red O was significantly decreased in hVEGF 165 -ZsGreen1-RAW264.7 compared with RAW264.7 or ZsGreen1-RAW264.7 cells. Data represent mean ± S.D. of three independent experiments each performed in triplicate. * indicates P

    Journal: Bioscience Reports

    Article Title: Vascular endothelial growth factor modified macrophages transdifferentiate into endothelial-like cells and decrease foam cell formation

    doi: 10.1042/BSR20170002

    Figure Lengend Snippet: Foam cell formation assay ( A ) Representative images of foam cell formation assay. Untransfected RAW264.7, ZsGreen1-RAW264.7, and hVEGF 165 -ZsGreen1-RAW264.7 were treated with 100 μg/ml ox-LDL for 24 h. After fixation and staining with Oil Red O, the cells were observed by light microscopy. Cytoplasmic lipid droplets accumulated in RAW264.7 or ZsGreen1-RAW264.7, but hVEGF165-ZsGreen1-RAW264.7 cells showed significantly decreased lipid accumulation. Scale bars =50 μm. ( B ) Quantitative analysis of lipid accumulation in cells. The cells stained with Oil Red O were treated with 1 ml 60% isopropanol for 1 h to redissolve the oil red O and absorbance was detected at 518 nm through a spectrophotometer. The absorbance of oil red O was significantly decreased in hVEGF 165 -ZsGreen1-RAW264.7 compared with RAW264.7 or ZsGreen1-RAW264.7 cells. Data represent mean ± S.D. of three independent experiments each performed in triplicate. * indicates P

    Article Snippet: It is approximately three-fold higher than untransfected RAW264.7 and ZsGreen1-RAW264.7 (both P < 0.01) ( B).

    Techniques: Tube Formation Assay, Staining, Light Microscopy, Spectrophotometry

    RA increases proliferation of the osteoclast progenitors. RAW264.7 cells (A) were incubated in a 96-well plate in the presence or absence of RANKL (RL) and 4 nM of RA for 6 days. Human CD14 + cells were incubated in a 96-well plate (B) or 12-well plate (C) in the absence or presence of M-CSF (M), RANKL (RL), and 4 nM of RA for 14 days. Cell proliferation was determined by an MTT based cell growth determination kit or cell number counting with NucleoCounter. Each data point represents the average ± SD of triplicate wells. Similar results were obtained in more than three independent experiments. NS denotes non-significant difference. * P

    Journal: PLoS ONE

    Article Title: Retinoic Acid Increases Proliferation of Human Osteoclast Progenitors and Inhibits RANKL-Stimulated Osteoclast Differentiation by Suppressing RANK

    doi: 10.1371/journal.pone.0013305

    Figure Lengend Snippet: RA increases proliferation of the osteoclast progenitors. RAW264.7 cells (A) were incubated in a 96-well plate in the presence or absence of RANKL (RL) and 4 nM of RA for 6 days. Human CD14 + cells were incubated in a 96-well plate (B) or 12-well plate (C) in the absence or presence of M-CSF (M), RANKL (RL), and 4 nM of RA for 14 days. Cell proliferation was determined by an MTT based cell growth determination kit or cell number counting with NucleoCounter. Each data point represents the average ± SD of triplicate wells. Similar results were obtained in more than three independent experiments. NS denotes non-significant difference. * P

    Article Snippet: Osteoclast formation in RAW264.7 cell culture RAW264.7 cells (from ATCC) were cultured as described previously .

    Techniques: Incubation, MTT Assay

    RA does not inhibit mature osteoclast function. RAW264.7 cells were incubated with RANKL on bone slices or plastic for 5 days followed by incubation in 400 nM of RA for another 2 days (A–B). Human CD14 + cells were incubated on bone slices or plastic with M-CSF (M) and RANKL (RL) for 14 days or 10 days. The cells were then incubated in 400 nM of RA for another 2 days (D–E), or 12 days (D, F) on bone slices or on plastic for 2 days (C, D) or 8 days (G). The CTX measurements (E), secretion of TRAP activity (A, C, F, G) and the TRAP staining (B, D) were carried out as explained in Figure 1 .

    Journal: PLoS ONE

    Article Title: Retinoic Acid Increases Proliferation of Human Osteoclast Progenitors and Inhibits RANKL-Stimulated Osteoclast Differentiation by Suppressing RANK

    doi: 10.1371/journal.pone.0013305

    Figure Lengend Snippet: RA does not inhibit mature osteoclast function. RAW264.7 cells were incubated with RANKL on bone slices or plastic for 5 days followed by incubation in 400 nM of RA for another 2 days (A–B). Human CD14 + cells were incubated on bone slices or plastic with M-CSF (M) and RANKL (RL) for 14 days or 10 days. The cells were then incubated in 400 nM of RA for another 2 days (D–E), or 12 days (D, F) on bone slices or on plastic for 2 days (C, D) or 8 days (G). The CTX measurements (E), secretion of TRAP activity (A, C, F, G) and the TRAP staining (B, D) were carried out as explained in Figure 1 .

    Article Snippet: Osteoclast formation in RAW264.7 cell culture RAW264.7 cells (from ATCC) were cultured as described previously .

    Techniques: Incubation, Activity Assay, Staining

    RA inhibits differentiation of osteoclast progenitors. TRAP activity in medium in RAW264.7 cells incubated with RANKL (RL, 100 ng/ml) with various concentrations of RA (A), or in the presence or absence of 4 nM of RA or 8 nM of RAR pan-antagonist (AGN) (B, C) on bone slices or plastic for 7 days was measured as described in materials and methods . Human CD14 + blood monocytes were incubated with M-CSF (M, 25 ng/ml), RANKL (RL, 25 ng/ml) and various concentrations of RA (D), or in the presence or absence of 4 nM RA or 8 nM AGN on bone slices for 14 days or on plastic for 10 days (E–G). Release of TRAP activity in the culture medium was determined using an adapted Sigma protocol (A, B, D, E). The TRAP staining was carried out using the Acid Phosphatase Leukocyte (TRAP) kit (C, F). CTX was determined by CrossLaps ELISA kit (G). Each data point represents the average ± SD of triplicate wells. Similar results were obtained in more than three independent experiments. NS means non-significant difference. A, D, compared with RL group. * P

    Journal: PLoS ONE

    Article Title: Retinoic Acid Increases Proliferation of Human Osteoclast Progenitors and Inhibits RANKL-Stimulated Osteoclast Differentiation by Suppressing RANK

    doi: 10.1371/journal.pone.0013305

    Figure Lengend Snippet: RA inhibits differentiation of osteoclast progenitors. TRAP activity in medium in RAW264.7 cells incubated with RANKL (RL, 100 ng/ml) with various concentrations of RA (A), or in the presence or absence of 4 nM of RA or 8 nM of RAR pan-antagonist (AGN) (B, C) on bone slices or plastic for 7 days was measured as described in materials and methods . Human CD14 + blood monocytes were incubated with M-CSF (M, 25 ng/ml), RANKL (RL, 25 ng/ml) and various concentrations of RA (D), or in the presence or absence of 4 nM RA or 8 nM AGN on bone slices for 14 days or on plastic for 10 days (E–G). Release of TRAP activity in the culture medium was determined using an adapted Sigma protocol (A, B, D, E). The TRAP staining was carried out using the Acid Phosphatase Leukocyte (TRAP) kit (C, F). CTX was determined by CrossLaps ELISA kit (G). Each data point represents the average ± SD of triplicate wells. Similar results were obtained in more than three independent experiments. NS means non-significant difference. A, D, compared with RL group. * P

    Article Snippet: Osteoclast formation in RAW264.7 cell culture RAW264.7 cells (from ATCC) were cultured as described previously .

    Techniques: Activity Assay, Incubation, Staining, Enzyme-linked Immunosorbent Assay

    a Proliferation activity and b mRNA expression of cytokines secretion of NO, IL-6 and TNF-α of RAW264.7 macrophage cells after treatment with polysaccharide from L. barbarum (NX1, QH1, XJ1). The values are presented as mean ± SD (n = 3). Significant differences with control cells were designated as * p

    Journal: Chinese Medicine

    Article Title: Quality evaluation of Lycium barbarum (wolfberry) from different regions in China based on polysaccharide structure, yield and bioactivities

    doi: 10.1186/s13020-019-0273-6

    Figure Lengend Snippet: a Proliferation activity and b mRNA expression of cytokines secretion of NO, IL-6 and TNF-α of RAW264.7 macrophage cells after treatment with polysaccharide from L. barbarum (NX1, QH1, XJ1). The values are presented as mean ± SD (n = 3). Significant differences with control cells were designated as * p

    Article Snippet: RAW264.7 macrophage proliferation RAW264.7 macrophage cells in an RPMI-1640 medium containing 10% FBS were plated in a 96-well microplate (1 × 104 cells/well, 100-µL volume; ATCC).

    Techniques: Activity Assay, Expressing

    Effect of silencing Numb on activation of MAPK and NF-κB pathways in activated macrophages and the interaction of Numb and Itch. ( A ) After stimulation with LPS for indicated times, phosphorylation levels of NF-κB p65, p38, ERK1/2, and JNK MAPK from GFP + macrophages were analyzed. Data are representative of two independent experiments. ( B , C ) GFP + macrophages containing control (open bars) or sh Numb (closed bars) vectors were stimulated with LPS for 1 hr prior to treatment with DMSO or with actinomycin D to inhibit mRNA synthesis. The relative amount of remaining Tnfα mRNA was measured by qPCR. Half-life of Tnfα mRNA was calculated using linear regression line equations and shown in ( C ). Data are the mean ± SEM from representative of two independent experiments performed in triplicates. ( D ) RAW264.7 cell line was transiently transfected with the control plasmid or pCIneoHA-Numb and stimulated with LPS as indicated. p38 MAPK and NF-κB p65 were detected by immunoblotting. ( E ) RAW264.7 cell line transfected with the control plasmid or pCIneoHA-Numb were stimulated with LPS for 1 hr. The amount of TNFα was measured by ELISA. ( F ) Co-immunoprecipitation of endogenous Numb from unstimulated or LPS-stimulated macrophages was analyzed by immunoblotting. ( G ) Expression of Itch in macrophages containing control or sh Numb vector was detected by immunoblotting following LPS stimulation. ( H ) RAW264.7 cell line transfected with the control plasmid or pCIneoHA-Numb were stimulated with LPS for the times indicated and the level of Itch was detected by immunoblotting.

    Journal: Scientific Reports

    Article Title: A Novel Role of Numb as A Regulator of Pro-inflammatory Cytokine Production in Macrophages in Response to Toll-like Receptor 4

    doi: 10.1038/srep12784

    Figure Lengend Snippet: Effect of silencing Numb on activation of MAPK and NF-κB pathways in activated macrophages and the interaction of Numb and Itch. ( A ) After stimulation with LPS for indicated times, phosphorylation levels of NF-κB p65, p38, ERK1/2, and JNK MAPK from GFP + macrophages were analyzed. Data are representative of two independent experiments. ( B , C ) GFP + macrophages containing control (open bars) or sh Numb (closed bars) vectors were stimulated with LPS for 1 hr prior to treatment with DMSO or with actinomycin D to inhibit mRNA synthesis. The relative amount of remaining Tnfα mRNA was measured by qPCR. Half-life of Tnfα mRNA was calculated using linear regression line equations and shown in ( C ). Data are the mean ± SEM from representative of two independent experiments performed in triplicates. ( D ) RAW264.7 cell line was transiently transfected with the control plasmid or pCIneoHA-Numb and stimulated with LPS as indicated. p38 MAPK and NF-κB p65 were detected by immunoblotting. ( E ) RAW264.7 cell line transfected with the control plasmid or pCIneoHA-Numb were stimulated with LPS for 1 hr. The amount of TNFα was measured by ELISA. ( F ) Co-immunoprecipitation of endogenous Numb from unstimulated or LPS-stimulated macrophages was analyzed by immunoblotting. ( G ) Expression of Itch in macrophages containing control or sh Numb vector was detected by immunoblotting following LPS stimulation. ( H ) RAW264.7 cell line transfected with the control plasmid or pCIneoHA-Numb were stimulated with LPS for the times indicated and the level of Itch was detected by immunoblotting.

    Article Snippet: Overexpression of Numb in macrophage-like cell line RAW264.7 RAW264.7 cells (ATCC TIB-71) were transfected with either pCI-OVA , as a control, or pCIneoHA mouse Numb (Addgene plasmid #37012) using FuGENE HD transfection reagent (Promega, USA).

    Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Expressing

    MicroRNA-183/96/182 modulates phagocytosis and intracellular killing capacities of Mϕ and PMNs. (A–D ) In vitro overexpression ( A , B ) and knockdown ( C , D ) of miR-183/96/182 in Mϕ cell line, Raw264.7. ( E , F ) Ex vivo phagocytosis and intracellular killing assays in PMN from miR-183/96/182 ko and wt mice. n = 4 for each group. * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Inactivation of the miR-183/96/182 Cluster Decreases the Severity of Pseudomonas aeruginosa-Induced Keratitis

    doi: 10.1167/iovs.16-19134

    Figure Lengend Snippet: MicroRNA-183/96/182 modulates phagocytosis and intracellular killing capacities of Mϕ and PMNs. (A–D ) In vitro overexpression ( A , B ) and knockdown ( C , D ) of miR-183/96/182 in Mϕ cell line, Raw264.7. ( E , F ) Ex vivo phagocytosis and intracellular killing assays in PMN from miR-183/96/182 ko and wt mice. n = 4 for each group. * P

    Article Snippet: In Vitro Transfection and Phagocytosis and Intracellular Killing Assays A total of 4 × 105 RAW264.7 Mϕ (a cell line derived from the mouse; ATCC) were plated in a 48-well plate and transfected with anti-miR-183/anti-miR-96/anti-miR-182 (10 nM for each) or negative control anti-miR oligonucleotides with scrambled sequences (30 nM; Exiqon, Vedbaek, Denmark) and miRNA mimics of miR-183, miR-96, and miR-182 (10 nM for each) or negative control mimics (30 nM; ThermoFisher) using RNAiMax Lipofectamine (ThermoFisher) as described before.

    Techniques: In Vitro, Over Expression, Ex Vivo, Mouse Assay

    Stable overexpression of VEGF down-regulates expression of CD36 in macrophages ( A ) Quantitative analysis of mRNA expression of CD36 by qRT-PCR. After treating with 100 μg/ml ox-LDL for 24 h, mRNA expression of CD36 were significantly decreased in hVEGF 165 -ZsGreen1-RAW264.7 cells compared with that in RAW264.7 and ZsGreen1-RAW264.7. ( B ) Western blot analysis for endothelial cell markers expression. Protein expression of CD36 was significantly decreased in hVEGF 165 -ZsGreen1-RAW264.7 cells compared with RAW264.7 and ZsGreen1-RAW264.7. Data represent mean ± S.D. of three independent experiments each performed in triplicate. # indicates P

    Journal: Bioscience Reports

    Article Title: Vascular endothelial growth factor modified macrophages transdifferentiate into endothelial-like cells and decrease foam cell formation

    doi: 10.1042/BSR20170002

    Figure Lengend Snippet: Stable overexpression of VEGF down-regulates expression of CD36 in macrophages ( A ) Quantitative analysis of mRNA expression of CD36 by qRT-PCR. After treating with 100 μg/ml ox-LDL for 24 h, mRNA expression of CD36 were significantly decreased in hVEGF 165 -ZsGreen1-RAW264.7 cells compared with that in RAW264.7 and ZsGreen1-RAW264.7. ( B ) Western blot analysis for endothelial cell markers expression. Protein expression of CD36 was significantly decreased in hVEGF 165 -ZsGreen1-RAW264.7 cells compared with RAW264.7 and ZsGreen1-RAW264.7. Data represent mean ± S.D. of three independent experiments each performed in triplicate. # indicates P

    Article Snippet: RAW 264.7 macrophages only transfected with ZsGreen1 were named as ZsGreen1-RAW264.7 and untransfected RAW264.7 (ATCC, Manassas, VA, U.S.A.) were both used as controls.

    Techniques: Over Expression, Expressing, Quantitative RT-PCR, Western Blot

    Effects of Chlorella 11-peptide on lipopolysaccharide (LPS)-induced monocyte chemoattractant protein (MCP-1) production. RAW264.7 cells ( n = 8) were treated with LPS (1 µg/mL) with and without Chlorella 11-peptide (9 and 38 µM) or indomethacin (0.25 mM) for 6 h prior to MCP-1 concentration being measured. Statistics are shown for LPS-treated cells ### p

    Journal: Marine Drugs

    Article Title: Chlorella 11-Peptide Inhibits the Production of Macrophage-Induced Adhesion Molecules and Reduces Endothelin-1 Expression and Endothelial Permeability

    doi: 10.3390/md11103861

    Figure Lengend Snippet: Effects of Chlorella 11-peptide on lipopolysaccharide (LPS)-induced monocyte chemoattractant protein (MCP-1) production. RAW264.7 cells ( n = 8) were treated with LPS (1 µg/mL) with and without Chlorella 11-peptide (9 and 38 µM) or indomethacin (0.25 mM) for 6 h prior to MCP-1 concentration being measured. Statistics are shown for LPS-treated cells ### p

    Article Snippet: RAW264.7 Macrophage Culture Macrophage RAW264.7 (ATCC number: TIB-71) cells were obtained from Bioresource Collection & Research Center (Taiwan) and cultured in DMEM supplemented with 10% Fetal Bovine Serum (Hyclone), 2 mM glutamine, 1% non-essential amino acid, 25 mM HEPES, 1 mM sodium pyruvate, 100 U/mL penicillin and 100 µg/mL streptomycin.

    Techniques: Concentration Assay

    Effect of TIPE2 Overexpression on LPS-induced iNOS expression. RAW264.7 cells were stably transfected with TIPE2 plasmid or vector control. TIPE2 expression levels were determined by quantitative RT-PCR ( A ) and Western blot ( B ), respectively. For quantitative PCR, the results were presented as folds expression of TIPE2 RNA to that of β-actin. TIPE2 overexpression RAW264.7 cells or control cells were treated with 100 ng/mL LPS for 24 h, and iNOS mRNA ( C ) and protein ( D ) levels were detected by quantitative PCR and Western blot, respectively. Data are shown as mean ±SE of one representative experiment. ** P

    Journal: PLoS ONE

    Article Title: TIPE2 Negatively Regulates Inflammation by Switching Arginine Metabolism from Nitric Oxide Synthase to Arginase

    doi: 10.1371/journal.pone.0096508

    Figure Lengend Snippet: Effect of TIPE2 Overexpression on LPS-induced iNOS expression. RAW264.7 cells were stably transfected with TIPE2 plasmid or vector control. TIPE2 expression levels were determined by quantitative RT-PCR ( A ) and Western blot ( B ), respectively. For quantitative PCR, the results were presented as folds expression of TIPE2 RNA to that of β-actin. TIPE2 overexpression RAW264.7 cells or control cells were treated with 100 ng/mL LPS for 24 h, and iNOS mRNA ( C ) and protein ( D ) levels were detected by quantitative PCR and Western blot, respectively. Data are shown as mean ±SE of one representative experiment. ** P

    Article Snippet: RAW264.7 culture Murine macrophage cell line Raw264.7 was obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM (GIBCO-BRL, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco-BRL, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere containing 5% CO2 .

    Techniques: Over Expression, Expressing, Stable Transfection, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction

    Effect of TIPE2 overexpression on LPS-induced arginases mRNA expression, NO and urea production in macrophages. A and B, RAW264.7 cells overexpressing TIPE2 or control cells were treated with 100/mL LPS for 4 h, and expression levels of arginaseI and arginase II mRNA were determined by quantitative PCR. Culture media were collected for NO and urea measurement (C and D). Data are shown as means ±SE of triplicates from an experiment that was repeated a total of three times with similar results. * P

    Journal: PLoS ONE

    Article Title: TIPE2 Negatively Regulates Inflammation by Switching Arginine Metabolism from Nitric Oxide Synthase to Arginase

    doi: 10.1371/journal.pone.0096508

    Figure Lengend Snippet: Effect of TIPE2 overexpression on LPS-induced arginases mRNA expression, NO and urea production in macrophages. A and B, RAW264.7 cells overexpressing TIPE2 or control cells were treated with 100/mL LPS for 4 h, and expression levels of arginaseI and arginase II mRNA were determined by quantitative PCR. Culture media were collected for NO and urea measurement (C and D). Data are shown as means ±SE of triplicates from an experiment that was repeated a total of three times with similar results. * P

    Article Snippet: RAW264.7 culture Murine macrophage cell line Raw264.7 was obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM (GIBCO-BRL, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco-BRL, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere containing 5% CO2 .

    Techniques: Over Expression, Expressing, Real-time Polymerase Chain Reaction

    Sphk1 activity and S1P analysis in palmitate and LPS treated RAW264.7 cells. A. Sphk1 activity and B. S1P analysis normalized to respective controls. C. Fold change (log2) values of (G)SL analyzed in Raw264.7 challenged with BSA vs 500 μM BSA coupled to Palmitate (BSA-PA). The red color represents high-normalized levels and the blue color represents low levels of the lipids. (n = 3). Significance is evaluated by a student's t-test and indicated as * p

    Journal: PLoS ONE

    Article Title: Induction of Sphk1 activity in obese adipose tissue macrophages promotes survival

    doi: 10.1371/journal.pone.0182075

    Figure Lengend Snippet: Sphk1 activity and S1P analysis in palmitate and LPS treated RAW264.7 cells. A. Sphk1 activity and B. S1P analysis normalized to respective controls. C. Fold change (log2) values of (G)SL analyzed in Raw264.7 challenged with BSA vs 500 μM BSA coupled to Palmitate (BSA-PA). The red color represents high-normalized levels and the blue color represents low levels of the lipids. (n = 3). Significance is evaluated by a student's t-test and indicated as * p

    Article Snippet: Cell culture experiments and reagents RAW264.7 obtained from the American Type Culture Collection were cultured in DMEM/10% FCS supplemented with penicillin-streptomycin.

    Techniques: Activity Assay

    Sphk1 activity contributes to cell survival upon challenge of RAW264.7 cells with palmitate and chloroquine. The CHOP/Sphk1 ratio is reduced in FACS sorted ATM A. HFD ATMs (both F4/80-Mgl1 + [M2] and F4/80-Mgl1 - [M1]) and B. ob/ob ATMs (both F4/80-Mgl1 + [M2] and F4/80-Mgl1 - [M1]). C. The CHOP/Sphk1 ratio is also reduced in CD11b + enriched cells from SVF D. S1P analysis showed that SK1-I inhibits Sphk1 activity. Analysis of the role of Sphk1 in promoting cell viability using E. WST-1 assay E. PARP cleavage analyzed by western blot in RAW264.7 cells challenged with 250 μM BSA coupled palmitate in the presence or absence of the Sphk1 inhibitor SK1-I for 24h (WST-1), or 8 h (PARP cleavage). Data in the graphs are depicted as mean +/- S.D. (n = 3) ** p

    Journal: PLoS ONE

    Article Title: Induction of Sphk1 activity in obese adipose tissue macrophages promotes survival

    doi: 10.1371/journal.pone.0182075

    Figure Lengend Snippet: Sphk1 activity contributes to cell survival upon challenge of RAW264.7 cells with palmitate and chloroquine. The CHOP/Sphk1 ratio is reduced in FACS sorted ATM A. HFD ATMs (both F4/80-Mgl1 + [M2] and F4/80-Mgl1 - [M1]) and B. ob/ob ATMs (both F4/80-Mgl1 + [M2] and F4/80-Mgl1 - [M1]). C. The CHOP/Sphk1 ratio is also reduced in CD11b + enriched cells from SVF D. S1P analysis showed that SK1-I inhibits Sphk1 activity. Analysis of the role of Sphk1 in promoting cell viability using E. WST-1 assay E. PARP cleavage analyzed by western blot in RAW264.7 cells challenged with 250 μM BSA coupled palmitate in the presence or absence of the Sphk1 inhibitor SK1-I for 24h (WST-1), or 8 h (PARP cleavage). Data in the graphs are depicted as mean +/- S.D. (n = 3) ** p

    Article Snippet: Cell culture experiments and reagents RAW264.7 obtained from the American Type Culture Collection were cultured in DMEM/10% FCS supplemented with penicillin-streptomycin.

    Techniques: Activity Assay, FACS, WST-1 Assay, Western Blot

    Pu-erh tea extract inhibited osteoclast differentiation in the RAW264.7 cells. RAW264.7 cells were cultured for 6 days with RANKL (50 ng/mL) in the presence of Xian-Ling-Gu-Bao (XLGB) (10 μg/mL) or the indicated concentrations of PTE and then stained for TRAP (A) . TRAP-positive multinucleated cells with more than five nuclei were considered mature osteoclasts, as observed under a light microscope (B) . The effect of PTE on the viability of RAW264.7 cells as determined by the MTT assay (C) . Representative images were acquired under a light microscope (magnification ×200). Values are shown as the mean ± SEM of three independent experiments. ΔΔΔ P

    Journal: Frontiers in Pharmacology

    Article Title: Pu-erh Tea Extract Ameliorates Ovariectomy-Induced Osteoporosis in Rats and Suppresses Osteoclastogenesis In Vitro

    doi: 10.3389/fphar.2017.00324

    Figure Lengend Snippet: Pu-erh tea extract inhibited osteoclast differentiation in the RAW264.7 cells. RAW264.7 cells were cultured for 6 days with RANKL (50 ng/mL) in the presence of Xian-Ling-Gu-Bao (XLGB) (10 μg/mL) or the indicated concentrations of PTE and then stained for TRAP (A) . TRAP-positive multinucleated cells with more than five nuclei were considered mature osteoclasts, as observed under a light microscope (B) . The effect of PTE on the viability of RAW264.7 cells as determined by the MTT assay (C) . Representative images were acquired under a light microscope (magnification ×200). Values are shown as the mean ± SEM of three independent experiments. ΔΔΔ P

    Article Snippet: Cell Culture and Maintenance RAW264.7 murine macrophages (ATCC, Manassas, VA, United States) were used in this study.

    Techniques: Cell Culture, Staining, Light Microscopy, MTT Assay

    DOPG inhibited Pam 3 CSK 4 -induced NF κ B activation in the macrophage cell line RAW264.7. (A) RAW264.7 cells were treated with or without 2.5 µg/ml Pam 3 CSK 4 (Pam) in the presence and absence of 100 µg/ml DOPG for 10 minutes. Cells were harvested and the phosphorylation (activation) status of NF κ B determined using an antibody recognizing phosphoserine 536 in comparison with total NF κ B levels. (A) shows a representative Western blot, whereas (B) presents the cumulative results from three separate experiments (means ± S.D.); *** P

    Journal: Molecular Pharmacology

    Article Title: Pathogen-Associated Molecular Pattern-Induced TLR2 and TLR4 Activation Increases Keratinocyte Production of Inflammatory Mediators and is Inhibited by Phosphatidylglycerol

    doi: 10.1124/mol.119.118166

    Figure Lengend Snippet: DOPG inhibited Pam 3 CSK 4 -induced NF κ B activation in the macrophage cell line RAW264.7. (A) RAW264.7 cells were treated with or without 2.5 µg/ml Pam 3 CSK 4 (Pam) in the presence and absence of 100 µg/ml DOPG for 10 minutes. Cells were harvested and the phosphorylation (activation) status of NF κ B determined using an antibody recognizing phosphoserine 536 in comparison with total NF κ B levels. (A) shows a representative Western blot, whereas (B) presents the cumulative results from three separate experiments (means ± S.D.); *** P

    Article Snippet: RAW264.7 cells, purchased from American Type Culture Collection (Manassas, VA), were kindly provided by Drs. Qing Zhong and Carlos Isales (Augusta University) and were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 1% penicillin and streptomycin.

    Techniques: Activation Assay, Western Blot

    DOPG had no effect on R848-induced NF κ B activation in the macrophage cell line RAW264.7. (A) RAW264.7 cells were treated with or without 1 µg/ml R848 (Resiquimod) in the presence and absence of 100 µg/ml DOPG for 10 minutes. Cells were harvested and the phosphorylation (activation) status of NF κ B determined using an antibody recognizing phosphoserine 536 in comparison with total NF κ B levels. (A) shows a representative Western blot, whereas (B) presents the cumulative results from three separate experiments (means ± S.D.); * P

    Journal: Molecular Pharmacology

    Article Title: Pathogen-Associated Molecular Pattern-Induced TLR2 and TLR4 Activation Increases Keratinocyte Production of Inflammatory Mediators and is Inhibited by Phosphatidylglycerol

    doi: 10.1124/mol.119.118166

    Figure Lengend Snippet: DOPG had no effect on R848-induced NF κ B activation in the macrophage cell line RAW264.7. (A) RAW264.7 cells were treated with or without 1 µg/ml R848 (Resiquimod) in the presence and absence of 100 µg/ml DOPG for 10 minutes. Cells were harvested and the phosphorylation (activation) status of NF κ B determined using an antibody recognizing phosphoserine 536 in comparison with total NF κ B levels. (A) shows a representative Western blot, whereas (B) presents the cumulative results from three separate experiments (means ± S.D.); * P

    Article Snippet: RAW264.7 cells, purchased from American Type Culture Collection (Manassas, VA), were kindly provided by Drs. Qing Zhong and Carlos Isales (Augusta University) and were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 1% penicillin and streptomycin.

    Techniques: Activation Assay, Western Blot

    DOPG inhibited Pam 3 CSK 4 -induced inflammatory mediator expression in the macrophage cell line RAW264.7. RAW264.7 cells were treated with 0, 1, and 2.5 µM Pam 3 CSK 4 (Pam) in the presence or absence of 50 or 100 µg/ml DOPG for 2 hours. RNA was then isolated, and the expression of the inflammatory mediators (A) IL1 α , (B) IL1 β , (C) IL6, and (D) TNF α was monitored by quantitative RT-PCR with GAPDH used as the housekeeping gene. Results represent the means ± S.D. of four separate experiments; * P

    Journal: Molecular Pharmacology

    Article Title: Pathogen-Associated Molecular Pattern-Induced TLR2 and TLR4 Activation Increases Keratinocyte Production of Inflammatory Mediators and is Inhibited by Phosphatidylglycerol

    doi: 10.1124/mol.119.118166

    Figure Lengend Snippet: DOPG inhibited Pam 3 CSK 4 -induced inflammatory mediator expression in the macrophage cell line RAW264.7. RAW264.7 cells were treated with 0, 1, and 2.5 µM Pam 3 CSK 4 (Pam) in the presence or absence of 50 or 100 µg/ml DOPG for 2 hours. RNA was then isolated, and the expression of the inflammatory mediators (A) IL1 α , (B) IL1 β , (C) IL6, and (D) TNF α was monitored by quantitative RT-PCR with GAPDH used as the housekeeping gene. Results represent the means ± S.D. of four separate experiments; * P

    Article Snippet: RAW264.7 cells, purchased from American Type Culture Collection (Manassas, VA), were kindly provided by Drs. Qing Zhong and Carlos Isales (Augusta University) and were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 1% penicillin and streptomycin.

    Techniques: Expressing, Isolation, Quantitative RT-PCR

    Dose-Response Curves of Curcumin/Liposome in Inhibition of Cell Proliferation. THP-1 (panel A) or RAW264.7 (panel B) cells were cultured for 3 days in the presence of curcumin/liposome (●), curcumin (▲), and control liposomes (○), respectively. After culturing, they were incubated with WST-8 for 3 h to assess viable cells in the culture. Resultant coloring reaction by WST-8 was measured with a plate-reader at the absorbance of 450 nm. In the vertical axis, the absorbance of only the medium was defined as 0%. Symbols in the Figure represent the percentage of absorbance with curucmin or liposomes to that without the test samples. Data represent mean values ± range (bars) in duplicate assays.

    Journal: PLoS ONE

    Article Title: Nanoparticles Containing Curcumin Useful for Suppressing Macrophages In Vivo in Mice

    doi: 10.1371/journal.pone.0137207

    Figure Lengend Snippet: Dose-Response Curves of Curcumin/Liposome in Inhibition of Cell Proliferation. THP-1 (panel A) or RAW264.7 (panel B) cells were cultured for 3 days in the presence of curcumin/liposome (●), curcumin (▲), and control liposomes (○), respectively. After culturing, they were incubated with WST-8 for 3 h to assess viable cells in the culture. Resultant coloring reaction by WST-8 was measured with a plate-reader at the absorbance of 450 nm. In the vertical axis, the absorbance of only the medium was defined as 0%. Symbols in the Figure represent the percentage of absorbance with curucmin or liposomes to that without the test samples. Data represent mean values ± range (bars) in duplicate assays.

    Article Snippet: RAW264.7 was purchased from DS Pharma Biomedical Co., Ltd.

    Techniques: Inhibition, Cell Culture, Incubation