human il-10 quantikine elisa kit  (Bio-Techne corporation)

Journal: Life Science Alliance

Article Title: Reprogramming of breast tumor–associated macrophages with modulation of arginine metabolism

doi: 10.26508/lsa.202302339

Figure Lengend Snippet: (A) Levels of NO (left), PAs (middle), and NO/PA ratios for THP-1–derived M0-, M1-, and M2-TAMs after being treated with DMSO (vehicle) and SEP (100 μM) for 3 d (n = 5). (B) One-way ANOVA with post hoc Tukey’s test was used for statistical analysis. Error bars: ±SEM. GraphPad Prism version 9.5.1. was used to perform all statistical analyses. (B) Levels of NO (left), PAs (middle), and NO/PA ratios for THP-1–derived Mn-, M0-, M1-, and M2-TAMs after being treated with DMSO (vehicle), NOS2 inhibitor, 1400W (50 μM), or arginase 1 (Arg1) inhibitor, nor-NOHA (50 μM), for 3 d (n = 5). Note the significant decrease of the NO level in 1400W-treated M1-TAMs and the significant decrease of the PA level in nor-NOHA–treated M2-TAMs. (C) Immunofluorescence imaging of THP-1–derived M0-, M1-, and M2-TAMs stained for an M1 marker (green, TNFα) versus M2 marker (red, CD206) and counterstained with DAPI (blue). M1-TAMs were treated with DMSO (control: Ctrl) or NOS2 inhibitor (100 μM 1400W), whereas M2-TAMs were treated with DMSO (Ctrl) or ARG1 inhibitor (50 μM nor-NOHA) for 3 d (n = 3). (D) Levels of type 1 cytokine IL12 (left) and type 2 cytokine IL10 (middle), as well as IL12/IL10 ratios for THP-1–derived TAM subsets measured with ELISA. M1-TAMs were treated with DMSO or NOS inhibitors, 1400W (50 μM) and L-NAME (2.5 mM). M2-TAMs were treated with DMSO, SEP (100 μM), or positive control LPS (5 ng/ml) plus IFNγ (20 ng/ml) for 3 d (n = 6). The cytokine levels were measured using ELISA and normalized against the total protein levels. Error bars: ±SEM. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001; and ns, P > 0.05. (E) Working scheme for the induction of M1 versus M2 polarization by activation of NOS2 versus ARG1/OCD1 pathways and M2-to-M1 reprogramming by SEP.

Article Snippet: The ELISA kits used were as follows: IL12 (Cat. No. D1200; R&D Systems), IL6 (Cat. No. D6050; R&D System), IL1β (Cat. No. DLB50; R&D System), TNFα (Cat. No. DTA00D; R&D System), IL10 (Cat. No. D1000B; R&D Systems), and TGFβ (Cat. No. ab108912; Abcam).

Techniques: Derivative Assay, Immunofluorescence, Imaging, Staining, Marker, Control, Enzyme-linked Immunosorbent Assay, Positive Control, Activation Assay

Journal: Life Science Alliance

Article Title: Reprogramming of breast tumor–associated macrophages with modulation of arginine metabolism

doi: 10.26508/lsa.202302339

Figure Lengend Snippet: (A) Western blot analysis on M1 markers: TLR2, STAT1, and pSTAT1 S727 versus M2 marker: CD206 in THP-1–derived M0-TAMs treated with DMSO (vehicle control), SEP (100 μM), or NO donor [GSNO (100 and 200 μM) and SNAP (10 and 20 μM)] in comparison with M1- and M2-TAMs (n = 4). GAPDH was used as the internal loading control. (B) Western blot analysis on M1 markers: STAT1 and TLR2 versus M2 marker: CD206 in M0-TAMs treated with DMSO (vehicle control), SEP (100 μM), or PAs (5 or 7.5 mM spermine) in comparison with M1- and M2-TAMs (n = 4). (A, B) β-Actin was used as the internal loading control. (For quantification of (A, B), see Appendix .) (C) Ratios of IL12 to IL10 secreted by THP-1–derived M0-TAMs treated with DMSO, SEP, NO donors, and PAs in comparison with M1- and M2-TAMs. (D) Western blot analysis on M1 markers: TLR2, STAT1, and pSTAT1 S727 in M1-TAMs treated with NO scavenger (50 μM cPTIO) with and without NO donor (100 μM GSNO) and M2-TAMs treated with DMSO or SEP (n = 4). GAPDH was used as the internal loading control. (E) Western blot analysis on M1 markers: TLR2, STAT1, and pSTAT1 S727 versus M2 marker: CD206 in THP-1–derived M2-TAMs treated with a PA analog (50 and 100 μM DENSPM) and PAs (5 mM spermine) and M1-TAMs treated with DMSO or SEP (n = 4). (D, E) β-Actin was used as the internal loading control. (For quantification of (D, E), see Appendix .) (D, E, F) Ratios of IL12 to IL10 secreted by THP-1–derived M1- and M2-TAMs with treatment combinations shown in (D, E). Error bars: ± SEM. GraphPad Prism version 9.5.1. was used to perform all statistical analyses. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001; and ns, P > 0.05. (G) Scheme for the induction of M1 versus M2 polarization by NO versus PAs and M2-to-M1 reprogramming by SEP.

Article Snippet: The ELISA kits used were as follows: IL12 (Cat. No. D1200; R&D Systems), IL6 (Cat. No. D6050; R&D System), IL1β (Cat. No. DLB50; R&D System), TNFα (Cat. No. DTA00D; R&D System), IL10 (Cat. No. D1000B; R&D Systems), and TGFβ (Cat. No. ab108912; Abcam).

Techniques: Western Blot, Marker, Derivative Assay, Control, Comparison

Journal: Life Science Alliance

Article Title: Reprogramming of breast tumor–associated macrophages with modulation of arginine metabolism

doi: 10.26508/lsa.202302339

Figure Lengend Snippet: (A) Scheme of the experiment where MMTV-neu (unactivated) mice were allowed to develop palpable mammary tumors (tumor latency of 6–14 mo) and given DMSO or SEP (10 mg/kg) in drinking water ad libitum for 6 wk (n = 7). (B) Tumor growth was measured by caliper, and the volume was determined (V = (W(2) x L)/2). (C) Pictures of exercised tumors. (D) Exercised tumors were analyzed for M1- versus M2-TAM profiles (CD80 versus CD163; IL12 versus IL10; and IFNγ) by FACS. (Top row) DMSO-treated tumors; (bottom row) SEP-treated tumors. (D, E) Quantification of the expression of M1- versus M2-TAM markers as in (D) in exercised tumors (n = 6) in comparison with spleens (n = 6) of the same animals. Error bars: ± SEM. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; and **** P ≤ 0.0001.

Article Snippet: The ELISA kits used were as follows: IL12 (Cat. No. D1200; R&D Systems), IL6 (Cat. No. D6050; R&D System), IL1β (Cat. No. DLB50; R&D System), TNFα (Cat. No. DTA00D; R&D System), IL10 (Cat. No. D1000B; R&D Systems), and TGFβ (Cat. No. ab108912; Abcam).

Techniques: Expressing, Comparison