Journal: Life sciences

Article Title: Aging-related hyperphosphatemia triggers the release of TNF-α from macrophages, promoting indicators of sarcopenia through the reduction of IL-15 expression in skeletal muscle.

doi: 10.1016/j.lfs.2025.123507

Figure Lengend Snippet: Fig. 3. Conditioned medium collected from BGP-treated macrophages impairs myogenic differentiation of myoblasts. A,B,C) C2C12 cells were incubated with 2 % HS to promote myogenic differentiation for 72 h in the presence or absence (Control, CT) of 5 % conditioned medium collected from macrophages treated with β-glycerophosphate (BGP) (CM(BGP)) for 6, 8 or 24 h or the vehicle (CM). MHC (A), MyoG (B) and fibronectin (FN) (C) expressions were analysed by western blotting. Representative blots are shown in the left panels. Bar graphs represent the densitometric analysis of the bands and results are expressed as the mean ± SEM from 12 experiments. D,E) C2C12 were incubated in the same conditions as in panels A, B and C in the presence or absence of a TNF-α neutralising antibody (Ab TNF, 2 μg/mL) or IgG antibody as a control (Ab IgG). MHC (D), and FN (E) expressions were analysed by western blotting. Representative blots are shown in the upper panels. Bar graphs represent the densitometric analysis of the bands and results are expressed as the mean ± SEM from 8 experiments. *p < 0.05 vs CT. #p < 0.05 vs CM. $p < 0.05 vs CM(BGP).

Article Snippet: To analyse the role of the cytokine TNF-α in the conditioned medium, certain experiments were performed in the presence of a TNF-α neutralising antibody (2 μg/ mL) (MAB4101, R&D Systems, Minneapolis, MN, USA) or an IgG antibody (sc-2025, Santa Cruz BioTechnology, Sta.

Techniques: Incubation, Control, Western Blot

Journal: Life sciences

Article Title: Aging-related hyperphosphatemia triggers the release of TNF-α from macrophages, promoting indicators of sarcopenia through the reduction of IL-15 expression in skeletal muscle.

doi: 10.1016/j.lfs.2025.123507

Figure Lengend Snippet: Fig. 4. Conditioned medium collected from BGP-treated macrophages induces senescence in myoblasts. A,B) C2C12 cells were incubated in the presence or absence (Control, CT) of 3 % conditioned medium collected from macrophages treated with β-glycerophosphate (BGP) (CM(BGP)) for 6, 8 or 24 h or the vehicle (CM) for 72 h. C,D) C2C12 cells were incubated in the in the same conditions as in panels A and B in the presence or absence of a TNF-α neutralising antibody (Ab TNF, 2 μg/mL) or IgG antibody as a control (Ab IgG). A,C) Senescence-associated β-galactosidase activity was analysed by confocal microscopy using the fluorogenic substrate C12FDG. Representative images are shown in the left panels. Bar graphs represent the densitometric analysis of the green fluorescence intensity and results are expressed as the mean ± SEM from 7 (A) or 5 (B) experiments. B,D) p21 expression was analysed by western blotting. Representative blots are shown in the left panels. Bar graphs represent the densitometric analysis of the bands and results are expressed as the mean ± SEM from 5 experiments. *p < 0.05 vs CT. #p < 0.05 vs CM. $p < 0.05 vs CM(BGP). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: To analyse the role of the cytokine TNF-α in the conditioned medium, certain experiments were performed in the presence of a TNF-α neutralising antibody (2 μg/ mL) (MAB4101, R&D Systems, Minneapolis, MN, USA) or an IgG antibody (sc-2025, Santa Cruz BioTechnology, Sta.

Techniques: Incubation, Control, Activity Assay, Confocal Microscopy, Fluorescence, Expressing, Western Blot

Journal: Life sciences

Article Title: Aging-related hyperphosphatemia triggers the release of TNF-α from macrophages, promoting indicators of sarcopenia through the reduction of IL-15 expression in skeletal muscle.

doi: 10.1016/j.lfs.2025.123507

Figure Lengend Snippet: Fig. 6. IL-15 prevents the signs of sarcopenia in myoblasts promoted by the conditioned medium collected from BGP-treated macrophages. A) C2C12 cells were treated with 10 mM BGP for 6, 8 or 24 h. B) C2C12 cells were incubated in the presence or absence (Control, CT) of 5 % conditioned medium collected from macrophages treated with β-glycerophosphate (BGP) (24 h) (CM(BGP)) or the vehicle (CM) for 24 h. C) C2C12 cells were incubated in the same conditions as in panel B in the presence or absence of a TNF-α neutralising antibody (Ab TNF-α, 2 μg/mL) or IgG antibody as a control (Ab IgG). D) C2C12 cells were treated with different doses of TNF-α for 24 h. A,B,C,D) IL-15 expression was measured by RT-qPCR using GAPDH as endogenous control. Bar graphs represent the mean ± SEM from 5 (A), 5 (B), 5 (C) or 6 (D) experiments. E,F) C2C12 cells were cultured with 2 % HS to promote myogenic differentiation and incubated with 5 % CM or CM(BGP) for 72 h in the presence or absence of different doses of recombinant IL-15 (100–1000 ng/mL). MHC (E) and fibronectin (FN) (F) expressions were analysed by western blotting. Representative blots are shown in the left panels. Bar graphs represent the densitometric analysis of the bands and results are expressed as the mean ± SEM from 5 experiments. G) C2C12 cells were incubated with 3 % CM or CM(BGP) for 72 h in the presence or absence of different doses of recombinant IL-15. Senescence- associated β-galactosidase activity was analysed by confocal microscopy using the fluorogenic substrate C12FDG. Representative images are shown in the left panels. Bar graphs represent the densitometric analysis of the green fluorescence intensity and results are expressed as the mean ± SEM from 4 experiments. *p < 0.05 vs CT. #p < 0.05 vs CM. $p < 0.05 vs CM(BGP). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: To analyse the role of the cytokine TNF-α in the conditioned medium, certain experiments were performed in the presence of a TNF-α neutralising antibody (2 μg/ mL) (MAB4101, R&D Systems, Minneapolis, MN, USA) or an IgG antibody (sc-2025, Santa Cruz BioTechnology, Sta.

Techniques: Incubation, Control, Expressing, Quantitative RT-PCR, Cell Culture, Recombinant, Western Blot, Activity Assay, Confocal Microscopy, Fluorescence

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 2. Measuring T cell functions by flow cytometry vs. ELISPOT. Left panel: For the detection of secretory products by ICS, the cells need to be “poisoned” first with Golgi inhibitors to prevent secretion, then permeabilized and fixed/“mummified.” The subsequent standard flow cytometric analysis does not make the distinction whether the analyte is indeed bound for secretion and thus is biologically active, or is retained in/on the cell. Right panel: In contrast, ELISPOT measures the actual secretory activity of pharmacologically untreated, living cells. The cells survive ELISPOT assays unharmed, and can be retested, phenotyped, expanded, cloned, or cryopreserved. Graphic artist: Gabor Pesthy.

Article Snippet: Commercially available ready-to-use ELISPOT assay kits (R&D Systems, Inc.) to study secretion of mouse TNF(EL410).

Techniques: Cytometry, Enzyme-linked Immunospot, Activity Assay, Clone Assay

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 4. The different functional avidities of antigen-reactive T cells. PBMC of an HLA-A2 positive subject were plated with different concentrations of individual A-2 restricted CEF peptides, as specified by the different symbols. A standard 24 h IFN-G ELISPOT assay was performed. Note how far apart the maximum stimulatory concentrations of the different peptides are.

Article Snippet: Commercially available ready-to-use ELISPOT assay kits (R&D Systems, Inc.) to study secretion of mouse TNF(EL410).

Techniques: Functional Assay, Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 1. Typical ELISPOT images of secreted cytokines from human PBMCs. Note the prominent inhibitory effect of oxidative stress on secretion of all cytokines, except IL-4 and IL5.

Article Snippet: Commercially available ready-to-use ELISPOT assay kits (R&D Systems, Inc.) to study secretion of mouse TNF(EL410).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 2. Analysis of a dual-analyte ELISPOT assay. QuantiHub 4.1 is capable of recognizing and detecting spots that have different colors as well as different shades of the same color within a large dynamic range.

Article Snippet: Commercially available ready-to-use ELISPOT assay kits (R&D Systems, Inc.) to study secretion of mouse TNF(EL410).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 1. Measured versus theoretical distribution of ELISPOT counts. IFN-G-transfected CHO cells were plated into an IFN-G ELISPOT assay at 15, 30, 60, and 120 cells per well, with 192 replicate wells per cell number, developed and counted with ImmonoSpot® Software. Distributions for spot counts are depicted for a Poisson distribution (dotted lines ) and a normal distribution (the solid line) with the means and a standard deviation corresponding to the square root of the means.

Article Snippet: Commercially available ready-to-use ELISPOT assay kits (R&D Systems, Inc.) to study secretion of mouse TNF(EL410).

Techniques: Enzyme-linked Immunospot, Transfection, Software, Standard Deviation

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 2. Flow chart of ex vivo and cultured ELISPOT assay.

Article Snippet: Commercially available ready-to-use ELISPOT assay kits (R&D Systems, Inc.) to study secretion of mouse TNF(EL410).

Techniques: Ex Vivo, Cell Culture, Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 3. Example of cultured ELISPOT application: Correlation of memory responses with protection. Memory cell responses were determined by cultured IFN-G ELISPOT assay. Results of ELISPOT analysis are expressed as mean spot-forming cells (SFCs)/million cells. Cultured ELISPOT assays were performed before Mycobacterium bovis infection 14 weeks post-BCG vaccination and compared to outcome of infection with M. bovis at week 28 post-vaccination (animals were challenged with M. bovis at week 14 post- vaccination). Protection has been determined by bacterial load (log CFU/g tissue). Shown is the correlation of mean cultured ELISPOT responses and mean bacterial loads using data from unvaccinated cattle as well as cattle vaccinated with three different vaccines that induced various degrees of protection (from ref. 2).

Article Snippet: Commercially available ready-to-use ELISPOT assay kits (R&D Systems, Inc.) to study secretion of mouse TNF(EL410).

Techniques: Cell Culture, Enzyme-linked Immunospot, Infection, Vaccines

Journal: Theranostics

Article Title: Salmonella typhimurium Suppresses Tumor Growth via the Pro-Inflammatory Cytokine Interleukin-1β

doi: 10.7150/thno.11432

Figure Lengend Snippet: Role of IL-1β in SLΔppGpp-mediated cancer therapy. (A) Protocols for co-treatment with an IL-1β blocking antibody (left) or recombinant IL-1β (right), plus PBS or SLΔppGpp. (B) CT26 cells were transplanted into mice. Mice then received an intravenous (i.v.) injection of PBS (black rectangles) or SLΔppGpp (black circles). IL-1β depletion: mice received an i.v. injection of anti-IL-1β-specific antibody (IL-1β Ab) 1 day before SLΔppGpp treatment (Day 1). The antibody was then injected twice a week for 2 weeks (open rectangles). Control mice received isotype (IgG) according to the same schedule (black triangles). Treatment with recombinant IL-1β (rIL-1β): mice received an intratumoral (i.t.) injection of rIL-1β on 5 dpi (open triangles), followed by another i.t injection every other day up until 11 dpi. Another group of tumor-bearing mice received four i.t injections of rIL-1β alone (open circles). Data represent the mean ± SD. Results from at least three individual experiments are shown. * P < 0.05, ** P < 0.005, and *** P < 0.001 vs. control at Day 12 and vs. SLΔppGpp at Day 18. (C) Photographs of representative animals in each group were taken before (0 dpi) and after treatment (5 and 16 dpi).

Article Snippet: The Salmonellae and TNF-α combination therapy groups received an intratumoral injection of recombinant TNF-α (410-MT/CF; 0.25 μg in PBS; R&D Systems) every 2 days starting at 5 dpi and continuing until 11 dpi.

Techniques: Blocking Assay, Recombinant, Injection, Control