Mouse TNF‑ alpha Antibody

Detection of Mouse TNF-alpha by Western Blot

Parameters of inflammation/nitrosative stress are unaltered in adipose tissue after chronic administration of compound 21. (A) and (B) Levels of TNF‐ alpha determined by WB and IHC. (C) and (D) levels of nitrotyrosine determined by WB and IHC. Quantification of specific bands was performed with Gel‐Pro Analyzer software. (C) For nitrotyrosine quantification, the intensity of the most predominant bands in the WB (of unknown identity), with MWs of 53 and 65 kDa, respectively, were quantified in each sample. Bar graphs are the means ± SE. (B) and (D) Positive TNF‐ alpha and nitrotyrosine staining as detected by immunohistochemistry was quantified using Image‐Pro Plus software. Data were analyzed by unpaired two‐tailed Student's t test. C21, n = 9 per group, control, n = 12 per group for TNF‐ alpha quantification. For nitrotyrosine quantification by Western blotting: C21, n = 6 per group; control, n = 6 per group and by immunohistochemistry: C21, n = 9 per group; control, n = 9 per group. WB, western blotting. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/30156060), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse TNF-alpha by Flow Cytometry

IAV- and HA-induced inflammatory programmed cell death is dependent on TNF. Macrophage cultures were pretreated with 1 ng/mL anti-TNF-alpha antibody and then infected with IAV (MOI of 0.25) or exposed to 10 ng/mL viral HA for 24 h. Cell death analysis was performed by flow cytometry analysis of annexin V/PI-positive cells (A, B). Assessment of cell viability through the measurement of LDH release in the supernatant of BMDMs (C). (D) The expression of p-RIPK1 was detected in BMDMs by Western blotting. beta -actin levels were used as a control for protein loading. (E) Graphs of band densitometry obtained after loading normalization and expressed as fold change over mock untreated control. (F) Levels of nitrite were measured by the Griess method in the supernatant of BMDM cultures after 24 h of stimulus. The levels of (G) IL-6 and (H) IL-10 were measured by ELISA in the supernatant of BMDM cultures after 24 h of stimulation. Data are presented as the mean ± SEM of 5 independent experiments *P < 0.05 versus untreated control group (MOCK); #P < 0.05 versus respective untreated infected/stimulated group. (I) Model of A/HA-triggered necroptosis. IAV/HA triggered necroptosis by a loop of events involving TLR4, TNF-alpha, and RIPK1, leading to exacerbation of inflammation. Images were created with BioRender.com. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36875528), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse TNF-alpha by Flow Cytometry

IAV- and HA-induced inflammatory programmed cell death is dependent on TNF. Macrophage cultures were pretreated with 1 ng/mL anti-TNF-alpha antibody and then infected with IAV (MOI of 0.25) or exposed to 10 ng/mL viral HA for 24 h. Cell death analysis was performed by flow cytometry analysis of annexin V/PI-positive cells (A, B). Assessment of cell viability through the measurement of LDH release in the supernatant of BMDMs (C). (D) The expression of p-RIPK1 was detected in BMDMs by Western blotting. beta -actin levels were used as a control for protein loading. (E) Graphs of band densitometry obtained after loading normalization and expressed as fold change over mock untreated control. (F) Levels of nitrite were measured by the Griess method in the supernatant of BMDM cultures after 24 h of stimulus. The levels of (G) IL-6 and (H) IL-10 were measured by ELISA in the supernatant of BMDM cultures after 24 h of stimulation. Data are presented as the mean ± SEM of 5 independent experiments *P < 0.05 versus untreated control group (MOCK); #P < 0.05 versus respective untreated infected/stimulated group. (I) Model of A/HA-triggered necroptosis. IAV/HA triggered necroptosis by a loop of events involving TLR4, TNF-alpha, and RIPK1, leading to exacerbation of inflammation. Images were created with BioRender.com. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36875528), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse TNF-alpha by Western Blot

IAV- and HA-induced inflammatory programmed cell death is dependent on TNF. Macrophage cultures were pretreated with 1 ng/mL anti-TNF-alpha antibody and then infected with IAV (MOI of 0.25) or exposed to 10 ng/mL viral HA for 24 h. Cell death analysis was performed by flow cytometry analysis of annexin V/PI-positive cells (A, B). Assessment of cell viability through the measurement of LDH release in the supernatant of BMDMs (C). (D) The expression of p-RIPK1 was detected in BMDMs by Western blotting. beta -actin levels were used as a control for protein loading. (E) Graphs of band densitometry obtained after loading normalization and expressed as fold change over mock untreated control. (F) Levels of nitrite were measured by the Griess method in the supernatant of BMDM cultures after 24 h of stimulus. The levels of (G) IL-6 and (H) IL-10 were measured by ELISA in the supernatant of BMDM cultures after 24 h of stimulation. Data are presented as the mean ± SEM of 5 independent experiments *P < 0.05 versus untreated control group (MOCK); #P < 0.05 versus respective untreated infected/stimulated group. (I) Model of A/HA-triggered necroptosis. IAV/HA triggered necroptosis by a loop of events involving TLR4, TNF-alpha, and RIPK1, leading to exacerbation of inflammation. Images were created with BioRender.com. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36875528), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse TNF-alpha by Western Blot

IAV- and HA-induced inflammatory programmed cell death is dependent on TNF. Macrophage cultures were pretreated with 1 ng/mL anti-TNF-alpha antibody and then infected with IAV (MOI of 0.25) or exposed to 10 ng/mL viral HA for 24 h. Cell death analysis was performed by flow cytometry analysis of annexin V/PI-positive cells (A, B). Assessment of cell viability through the measurement of LDH release in the supernatant of BMDMs (C). (D) The expression of p-RIPK1 was detected in BMDMs by Western blotting. beta -actin levels were used as a control for protein loading. (E) Graphs of band densitometry obtained after loading normalization and expressed as fold change over mock untreated control. (F) Levels of nitrite were measured by the Griess method in the supernatant of BMDM cultures after 24 h of stimulus. The levels of (G) IL-6 and (H) IL-10 were measured by ELISA in the supernatant of BMDM cultures after 24 h of stimulation. Data are presented as the mean ± SEM of 5 independent experiments *P < 0.05 versus untreated control group (MOCK); #P < 0.05 versus respective untreated infected/stimulated group. (I) Model of A/HA-triggered necroptosis. IAV/HA triggered necroptosis by a loop of events involving TLR4, TNF-alpha, and RIPK1, leading to exacerbation of inflammation. Images were created with BioRender.com. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36875528), licensed under a CC-BY license. Not internally tested by R&D Systems.