Skin samples were placed in 0.25% dispase II (Roche, Switzerland) solution at 4 °C for 16 h. The epidermis and dermis were separated by forceps. The epidermis was cut and put into 0.25% trypsin and 0.02% EDTA digestion (Beyotime), and the cells were cultured in the defined keratinocyte-SFM (Gibco, New York, USA) for 5 days to get primary keratinocytes. The dermis was cut into small pieces and cultured in Dulbecco’s modified eagle medium (Gibco) containing 10% fetal bovine serum (Sciencell), 1% penicillin/streptomycin (Beyotime) for 7 days to get primary fibroblast. Cells were cultured with recombinant human TNF-α protein (Abcam) for 12 h, cells cultured with medium provided a control group. Culture supernatants were collected to measure the levels of cytokines secreted by keratinocytes and fibroblast. The levels of CXCL8 (Raybiotech, Georgia, USA), CCL20 (Abcam), IL-6 (Invitrogen), IL-1B (Abcam), and IL-17B (Novus Biologicals, Colorado, USA) were determined by ELISA according to the manufacturer’s instructions.
Il 1b
Manufactured by Abcam
Sourced in United States
IL-1b is a laboratory product that functions as a cytokine. It is involved in the regulation of immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Tnf α, by Abcam (4 mentions)
Interleukin 6 (il 6), by Abcam (2 mentions)
Keratinocyte sfm, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Ccl20, by Abcam (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Recombinant human tnf α protein, by Abcam (1 mentions)
Penicillin streptomycin, by Beyotime (1 mentions)
Dispase 2, by Roche (1 mentions)
Trypsin, by Beyotime (1 mentions)
Fetal bovine serum (fbs), by ScienCell (1 mentions)
Il 18, by Abcam (1 mentions)
Sc 2314, by Santa Cruz Biotechnology (1 mentions)
Interleukin 4 (il 4), by Abcam (1 mentions)
Hmgb1, by Abcam (1 mentions)
Sc 2004, by Santa Cruz Biotechnology (1 mentions)
Ecl plu, by Cytiva (1 mentions)
Gapdh, by Abcam (1 mentions)
Gasdermin d gsdmd, by Abcam (1 mentions)
Il 18, by Solarbio (1 mentions)
8 protocols using il 1b
1
Skin Cytokine Profiling in Inflammation
2
Western Blot Analysis of Neuroinflammatory Markers
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Tissue homogenates from the hippocampus and PFC were subjected to Western Blot (WB) analysis for the determination of protein levels. The extraction of protein and WB was performed as previously described [9 (link)–12 (link)]. The primary antibodies IL1b, IL 4, IL-18, TLR4, HMGB 1 and GAPDH were commercially obtained from Abcam, Cambridge, MA. Secondary antibodies were obtained: anti-mouse, 1:5000 dilution and anti-rabbit, 1:5000 dilution (SC-2314 and SC-2004 respectively, Santa Cruz Biotechnology, Santa Cruz, CA). The Immunoreactive bands were visualized using enhanced chemiluminescence (ECL Plus, Amersham), band intensities were quantified using ImageJ imaging software (NIH), and were normalized with GAPDH.
3
Hippocampal Protein Analysis after HSR
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At 12 h after HSR, total protein was extracted from the hippocampal tissues (n=6 per group) as described in the IL-1β and IL-18 assay. Samples containing 30 µg protein quantified by bicinchoninic acid (BCA) assay were separated by 10% SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked with 5% skimmed milk at 37°C for 2 h, and primary antibodies rabbit anti-rat polyclonal cleaved caspase-1 (1:1,000; cat. no. ab1872; Abcam), monoclonal NLRP3 (1:1,000; cat. no. ab210491; Abcam), polyclonal Gasdermin D (GSDMD; 1:1,000; cat. no. ab219800; Abcam), polyclonal IL-1b (1:1,000; cat. no. K107559P; Beijing Solarbio Science & Technology Co., Ltd.) and polyclonal IL-18 (1:1,000; cat. no. K002143P; Beijing Solarbio Science & Technology Co., Ltd.) were applied at 4°C overnight, followed by incubation with a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:2,000; cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) at 25°C for 1 h. Protein bands in each treatment group were detected using an enhanced chemiluminescence western blot detection system ImageJ 1.48u software (National Institutes of Health). β-actin (1:2,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc.) was used as an internal reference (20 (link)).
4
Protein Expression Analysis in Graft Tissues
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Protein samples were extracted from graft tissues. Equal masses of protein samples were separated by SDS-PAGE, and transferred to PVDF membranes that were blocked and reacted with primary antibodies according to the manufacturer's recommendations. The antibodies used include: CDKN2A (1:600, Abcam), TNFAIP6 (1:800, Abcam), ESR2 (1:400, Abcam), UCN (1:400, LifeSpan BioSciences), IL1B (1:1000, Abcam), VIP (1:1000, Abcam), NFKBIA (1:1000, Abcam), GAL (1:600, Santa), CSF1 (1:400, Santa), JUN (1:1000, Abcam), ARG1 (1:800, Abcam), FGF2 (1:1000, Abcam), ATF3 (1:1000, Abcam), ZFP36 (1:400, Millipore), CCL2 (1:1000, Millipore), GFI1 (1:400, Abcam), CCK (1:1000, Santa), RUNX2 (1:600, Abcam), TNFα (1:600, Abcam), and β-actin (1:4,000, Proteintech). The specific binding of primary antibody was detected by HRP-conjugated species-specific secondary antibody (Beyotime, Shanghai, China) and enhanced chemiluminescence (ECL) assay.
5
Serum Biomarkers in Rats
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5 mL of arterial blood was extracted from the femoral artery of rats, and the serum was separated and stored at −20°C. Pepsinogen I (PG I), pepsinogen II (PG II), and gastrin-17 (G-17) ELISA kits were purchased from Shanghai Yuanye Bio-engineering Co., Ltd. (Shanghai, China). Moreover, p-JAK 2, p-STAT 3, cyclin D1, Bcl-2, TNF-α, IFNG, IL1B, IL6, and IL10 ELISA kits were purchased from Abcam (China). The levels of these factors were measured according to the operation instructions of the kits.
6
Multifunctional Wound Dressing Fabrication
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Chitosan (degree of deacetylation≈95%, 100–200 mps), dextran (Mw: ~70 kDa), 3, 4-dihydroxy hydrocinnamic acid, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), lead-oxide (ZnO) were purchased from Aladdin. Sodium periodate (NaIO4) was purchased from Acros. 4-(Methylamino) Pyridine was purchased from TCI. Fibrin glue was purchased from Shanghai yuanye Bio-Technology Co., Ltd. Gelatin type A (from porcine skin, 300 Bloom), Live/Dead Cell Double Staining Kit, CORM-3, and GSNO were purchased from Sigma. CCK-8 was purchased from Beyotime. CD31, IL-6, TNF-a, IL-1b, and all other antibodies are purchased from Abcam. IR@dye 680-NHS ester and IR@dye 800-NHS ester were purchased from LI-COR.
7
Histological Analysis of Liver Samples
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Liver samples were fixed in 10% phosphate-buffered (PBS) formalin and paraffin embedded for pathological assessment. The liver sections were stained with haematoxylin and eosin (H&E) and picro-sirius red and immunohistochemistry following the routine protocols as described, respectively (12 (link),13 (link)). Antibodies were as below: NLRP3, IL1b, caspase 1 (Abcam, Cambridge, MA, USA).
8
Inflammatory Markers Immunohistochemistry
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We performed an immunohistochemical assay for CD68, TNF-a, IL-1b, inducible nitric oxide synthase (iNOS), and nuclear factor kappa B (NF-kB). Antigenic recuperation was performed by heat (18 minutes) in a citrate solution with a pH of 6.0. The slides were submitted to peroxidase blocking with a 3% H 2 O 2 solution diluted in PBS (phosphate-buffered saline) or in a methanol (NF-kB) solution for 30 minutes. Subsequently, protein blocking was performed (albumin)
(1 hour). The fragments were incubated with primary antibodies CD68 (Dako â , 1:500, overnight, Dopenhage, Denmark), TNF-a (Abcam â , 1:50, 1 hours), IL-1b (Abcam â , 1:100, 1 hours, Cambridge, UK), iNOS (Abcam â , 1:200, overnight), and NF-kB (SantaCruz â , 1:200, overnight, Finnell Street Dallas, TX, USA).
Universal immune-peroxidase polymer (Histofineâ for Dako â or Abcam â primary antibodies; 30 minutes, Nicherei Biosciences Inc., Tokyo, Japan) or secondary biotinylated anti-rabbit IgG (for primary antibodies Santa Cruz â ; 30 minutes) plus ABC system (Santa Cruz â ; 30 minutes) was used. The revelation system in all cases was 5,5diaminobenzidine tetrahydrochloride (DAB) (Dako â ).
(1 hour). The fragments were incubated with primary antibodies CD68 (Dako â , 1:500, overnight, Dopenhage, Denmark), TNF-a (Abcam â , 1:50, 1 hours), IL-1b (Abcam â , 1:100, 1 hours, Cambridge, UK), iNOS (Abcam â , 1:200, overnight), and NF-kB (SantaCruz â , 1:200, overnight, Finnell Street Dallas, TX, USA).
Universal immune-peroxidase polymer (Histofineâ for Dako â or Abcam â primary antibodies; 30 minutes, Nicherei Biosciences Inc., Tokyo, Japan) or secondary biotinylated anti-rabbit IgG (for primary antibodies Santa Cruz â ; 30 minutes) plus ABC system (Santa Cruz â ; 30 minutes) was used. The revelation system in all cases was 5,5diaminobenzidine tetrahydrochloride (DAB) (Dako â ).
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