Pig proximal kidney tubular epithelial cells (LLC‐PK1, ATCC, Manassas) were cultured in Medium‐199 (Gibco BRL) containing 3% FBS 19 alone or cocultured with Lean‐ and MetS‐MSC‐derived EVs (5 μg of EV protein, n = 5 each). Proliferation of confluent PK1 cells was evaluated by calculating the doubling time of cell numbers between the second and third passages using an online formula (http://www.doubling‐time.com/compute.php ), as previously described 28 . Nuclear translocation of the proinflammatory transcription factor nuclear factor (NF)‐kB was evaluated in tubular epithelial cells by immunofluorescent staining (Abcam, 1:200, Cambridge, MA). Nuclear DNA was stained with 4′,6‐diamidino‐2‐phenylindole (DAPI). Double positive (NFkB/DAPI) areas were quantified in 15–20 random fields using a computer‐aided image analysis program (ZEN 2012 blue edition; Carl Zeiss SMT, Oberkochen, Germany), and the results from all fields were averaged. Tubular epithelial cell inflammation was evaluated by immunofluorescent staining with antibodies against tumor necrosis factor (TNF)‐α (Santa Cruz Biotechnology Inc., Santa Cruz, CA, 1:200), monocyte chemoattractant protein (MCP)‐1, interleukin (IL)‐6, and IL‐1β (MyBioSource, San Diego, CA, http://www.mybiosource.com 1:7,500).
Il 1β
Manufactured by MyBioSource
Sourced in United States
Interleukin-1 beta (IL-1β) is a pro-inflammatory cytokine that plays a key role in the immune response and inflammatory processes. It is involved in the regulation of various cellular activities, including cell proliferation, differentiation, and apoptosis.
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Lab products found in correlation
Tnf α, by MyBioSource (8 mentions)
Interleukin 6 (il 6), by MyBioSource (8 mentions)
Il 10, by MyBioSource (5 mentions)
Lipopolysaccharides (lps), by MyBioSource (2 mentions)
Medium 199, by Thermo Fisher Scientific (1 mentions)
Zen 2012 blue edition, by Zeiss (1 mentions)
Tnf α, by Abcam (1 mentions)
Interleukin 6 (il 6), by Huabio (1 mentions)
Anti mouse igg hrp linked secondary antibody, by Cell Signaling Technology (1 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Ifn γ, by MyBioSource (1 mentions)
Il 12, by MyBioSource (1 mentions)
Il 1β, by BD (1 mentions)
Oxldl, by MyBioSource (1 mentions)
Cobas c111 analyzer, by Roche (1 mentions)
Acetylcholinesterase, by Merck Group (1 mentions)
Annexin 5 fitc assay kit, by BD (1 mentions)
Active caspase 3 apoptosis kit, by BD (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
23 protocols using il 1β
1
Renal Tubular Epithelial Cell Inflammation and Proliferation
2
Renal Cortical Protein Extraction and Western Blot Analysis
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Total protein was extracted from renal cortical tissues using the PRO‐PREP Protein Extraction Solution (iNtRON Biotechnology, Gyeonggi‐Do, Republic of Korea), according to the manufacturer's instructions. Western blot analysis was performed using the following antibodies: angiopoietin‐1 (Proteintech, Rosemont, IL, USA; 1:2000), angiopoietin‐2 (Proteintech, Rosemont, IL, USA; 1:2000), TNF‐α (Abcam, Cambridge, UK; 1:500), IL‐6 (Huabio, MA, USA; 1:2000), IL‐1β (MyBioSource, CA, USA; 1:3000), β‐actin (Sigma, St Louis, MO, USA; 1:10000), anti‐rabbit IgG HRP‐linked secondary antibody (Cell Signaling Technology, MA, USA; 1:2000), and anti‐mouse IgG HRP‐linked secondary antibody (Cell Signaling Technology, MA, USA; 1:2000).
3
Quantifying Guinea Pig Cytokine Levels
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Cytokines in the BAL and serum were quantified with enzyme-linked immunosorbent assays (ELISA) following the manufacturer’s instructions of the following kits for the guinea pigs: TNF-α (MBS9303082; MyBioSource, San Diego, CA, USA), IFN-γ (MBS701377; MyBioSource, San Diego, CA, USA), TGF-β1 (CSB-E06773GU; CUSABIO TECHNOLOGY LLC; Houston, TX, USA), IL-1β (MBS765173; MyBioSource, San Diego, CA, USA), 1L-6 (MBS269054; MyBioSource, San Diego, CA, USA) IL-8 (MBS282965; MyBioSource, San Diego, CA, USA), IL-12 (MBS704591; MyBioSource, San Diego, CA, USA).
4
Rat Cytokine Profiling ELISA
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5
Plasma Biomarker ELISA Protocol
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Plasma ZO-1 (Catalog #: MBS706368, MyBioSource, San Diego, CA), LPS (Catalog #: MBS266722, MyBioSource, San Diego, CA), sCD14 (Catalog #: RK01060, Abyntek Biopharma, Vizcaya, Spain), and IL-1β (Catalog #: MBS2510385, MyBioSource, San Diego, CA) were measured using commercially available ELISA kits according to the manufacturers’ instructions. Circulating human anti-β-lactoglobulin antibody levels (IgG isotypes) were assayed using a validated home-made ELISA protocol as detailed below. All the plasma samples were measured in blind duplicates for each biomarker. For all protocols, absorbance at 450 nm with 570 nm correction was measured in a microplate reader, and corrected absorbance was interpolated in each standard curve to determine the concentrations (Sigma Plot).
6
Inflammatory and Oxidative Biomarkers
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Changes in interleukin-1β (IL-1β; BD Biosciences, Franklin Lakes, NJ, USA), thiobarbituric acid reactive substances (TBARS; R&D Systems, Minneapolis, MN, USA), and oxidized low-density lipoprotein (oxLDL; MyBioSource, San Diego, CA, USA) at months 0, 3, and 6 were measured in heparin plasma (for IL-1β) and EDTA plasma (for TBARS and oxLDL) using commercially available ELISA kits. High-sensitivity C-reactive protein (hs-CRP) changes were measured at months 0, 3, and 6 in serum using a Cobas c111 analyzer (Roche Diagnostics, Indianapolis, IN, USA).
7
Apoptosis and Inflammation Assays
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Acetylcholinesterase and phorbol 12-myristate 13-acetate were purchased from Sigma (St. Louis, MO, United States); FITC Annexin V assay kit, active Caspase-3 apoptosis kit and JC-1 were purchased from BD Biosciences (San Jose, CA, United States); IL-1β, IL-6, and TNF-α were purchased from MyBioSource (San Diego, CA, United States); IL-8 and P-selectin ELISA kits were purchased from ThermoFisher (Waltham, MA, United States); IMDM, RPMI1640, FBS, and BSA were purchased from Gibco (Waltham, MA, United States); tanshinone IIA was purchased from Shanghai Pharmaceuticals (Shanghai, China); TPO was purchased from PeproTech (Rocky Hill, NJ, United States).
8
Cytokine and Immunoglobulin Assays in Plasma
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Thiamine and LPS in plasma were determined using similar methods as described above for rumen fluid and a lower measurement limit of LPS as 0.1 endotoxin units/ml. The LBP concentrations were performed using an ELISA kit test (Chinese Horseshoe Crab Reagent Manufactory, Xiamen, China) with a detection range of 15.6–1,000 ng/ml. The determination of IgA in serum was via an immunoturbidimetry assay kit (Bethyl Lab., Inc., Montgomery, USA). The cytokine in plasma was determined using the following commercial kits according to the manufacturer's instructions: TNF-α (Bio Source/Med Probe, Camarillo, CA), IL-1β (MyBioSource Inc., San Diego, CA), IL6 (MyBioSource Inc., San Diego, CA), and IL10 (Bethyl Lab., Inc., Montgomery, USA). The absorbance of all samples was read at 450 nm using a microplate reader (BioTek Instruments, Winooski, VT). The concentrations of TNF-α, IL-1β, IL6, and IL-10 had a detection range of 15.6–500 ng/ml, 31.25–2,000 pg/ml, 15.6–500 ng/ml, and 5–1,000 pg/ml, respectively, and the variation coefficients for inter- and intra-assay were no more than 10%.
9
Cytokine Levels in Isolated Hippocampi
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Isolated hippocampi were homogenized, and aliquots were used for the following determinations using the ELISA technique: IL-10, TNF-α, and IL-1β, levels (MyBioSource, USA). Each sample’s protein content was measured using a colorimetric technique, and all parameters were represented as pg/mg protein.
10
Neurochemical Biomarkers in Neuroprotection
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The following chemicals were employed in the study: 3-NPA, malondialdehyde (MDA), succinate dehydrogenase (SDH), catalase (CAT), glutathione-S-transferase (GST), reduced glutathione (GSH), brain-derived natriuretic factor (BDNF), interleukins (IL-1β), tumor necrosis factor (TNF-α) and cyclooxygenase (COX) (MyBioSource, San Diego, CA, USA), measurement kit ATP estimate kit (Sigma-Aldrich, Inc., St. Louis, MO, USA), and fustin (>98%, stability ≥ 4 years, SKL, Mumbai, Maharashtra, India).
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