ATG5 and HMOX-1 siRNA (RiboBio; siB12531154855-1-5 and siG0931881756-1-5, respectively) transfections were performed as previously described (60 (link)) using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol. The siRNA and Lipofectamine complexes were prepared in Opti-MEM (Gibco) at a 1:1 ratio and added to the predifferentiated THP-1 macrophages for 24 h in a dropwise manner. The medium was replaced after 36 h, and scrambled siRNA was used as a negative control. HO-1 expression was also inhibited by 2.5 μM zinc protoporphyrin (Sigma) treatment. The efficiency of knockdown was determined in both cases by quantitative reverse transcription-PCR (RT-qPCR) and/or Western blotting.
Zinc protoporphyrin
Manufactured by Merck Group
Sourced in United States
Zinc protoporphyrin is a lab equipment product used for analytical purposes. It is a metalloporphyrin compound that can be used as a fluorescent marker in various biochemical and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Hmox 1 sirna, by RiboBio (1 mentions)
Deferoxamine mesylate salt dfo, by Merck Group (1 mentions)
4 hydroperoxy cyclophosphamide, by Santa Cruz Biotechnology (1 mentions)
Ferrostatin 1 fer 1, by Merck Group (1 mentions)
Methylene blue, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem f12, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rhodamine 123, by Thermo Fisher Scientific (1 mentions)
Deferoxamine, by Merck Group (1 mentions)
Cobalt protoporphyrin 9, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Cerulein, by Merck Group (1 mentions)
Z ietd fmk, by Merck Group (1 mentions)
Keap1, by Santa Cruz Biotechnology (1 mentions)
Sanglifehrin a, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Anti hmgb1 antibody, by Abcam (1 mentions)
6 protocols using zinc protoporphyrin
1
Modulating Macrophage Autophagy Regulators
2
Antioxidant Modulation in Oxidative Stress
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The antibodies used in this study included NRF2 (16396-1-AP), Histone H3 (17168-1-AP), SLC7A11 (26864-1-AP), HMOX-1 (10701-1-AP), and GAPDH (16396-1-AP) (ProteinTech Group, IL, United States). 4-Hydroperoxy cyclophosphamide (4HC) was purchased from TCR (39800-16-3, Santa Cruz Biotechnology, TA, United States). Cyclophosphamide (CTX), Hemin, zinc protoporphyrin (ZNPP), ferrostatin-1 (Fer1), and deferoxamine mesylate salt (DFO) were purchased from Sigma–Aldrich (Sigma–Aldrich, MO, United States).
3
In-vitro Neurodegeneration Assay
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All procedures were performed in accordance with the International Guiding Principles for Biomedical Research Involving Animals. MPP+, recombinant α-synuclein, cobalt protoporphyrin IX (CoPPIX), zinc protoporphyrin (ZnPP), deferoxamine (DFO), methylene blue (MB) and primary antibodies against microtubule-associated protein 2 (MAP2) and GFAP were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Primary HO-1 antibody was purchased from Stressgen Bioreagents (Ann Arbor, MI, USA). Primary mitochondria ferritin (MtFt) was purchased from Abcam (Cambridge, UK). Primary cytochrome c oxidase subunit IV (COX4) was purchased from Takara Biomedical Technology (Beijing, China). Rhodamine 123 was purchased from Invitrogen (Madrid, Spain). Caspase-3 Kit was from BD Biosciences, Dulbecco’s modified Eagle’s medium (DMEM)/F12, B27 and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). All other chemicals and reagents were of the highest grade available and were obtained from local commercial sources.
4
Luteolin-based Pancreatitis Treatment Protocol
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Purified luteolin (>99%, CAS: 491-70-3) was purchased from Shanghai Tauto Biotech Co., Ltd. (Shanghai, China) and its quality was analyzed by high-performance liquid chromatography (HPLC) (Fig. 1 ). Cerulein, LPS and zinc protoporphyrin (ZnPP) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Luteolin was dissolved in 35% propanediol to the concentration of 20 mg/ml. Cerulein and LPS were dissolved in 0.9% NaCl to the concentration of 10 μg/ml and 1 mg/ml, respectively. The ZnPP solution was prepared as follows: first, 25 mg ZnPP were dissolved in 3.3 ml NaOH (0.2 M) in a dark room and 0.2 M HCl was added to adjust the pH to 7.0. Finally, saline was added to 50 ml (0.5 mg/ml). The resulting solution was stored away from light.
5
Nrf2 Activation and Inhibition Assay
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D3T, H2O2 z-LEHD-fmk, z-IETD-fmk, sanglifehrin A (SfA) and Zinc protoporphyrin (ZnPP, the HO-1 inhibitor) were obtained from Sigma (St. Louis, MO). Rapamycin and LY294002 were purchased from Calbiochem (Darmstadt, Germany). The antibody against tubulin was purchased from Sigma (St. Louis, MO). KEAP1, Nrf2 and HO-1 antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA). P-Nrf2 (Ser 40) antibody was purchased from Abcam (Shanghai, China). All other antibodies utilized in this study were described previously6 (link).
6
AMPK and HO-1 Modulation in RAW 264.7 Cells
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RAW 264.7 cells were cultured in Rosewell Park Memorial Institute-1640 Medium supplemented with 10% fetal bovine serum, penicillin, and 100 μg/ml streptomycin (Gibco BRL Gaithersburg, MD). Antibodies against AMPK, p-AMPK, acetyl-CoA carboxylase (ACC), p-ACC, p38, and phospho-p38 (p-p38) were purchased from Cell Signaling Technology (Beverly, MA). Anti-HMGB1 antibody was purchased from Abcam (Cambridge, MA). Anti-HO-1 antibody, anti-β-actin antibody, scrambled siRNAs, and siAMPKα1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The enhanced chemiluminescence western blotting detection reagent was purchased from Amersham (Buckinghamshire, UK). The p38 MAPK inhibitors (PD98059, SB203580, and SP600125) were purchased from Calbiochem (San Diego, CA). All other chemicals, including LPS (Escherichia coli 0111:B4), naringin, zinc protoporphyrin (ZnPP; an inhibitor of HO-1 enzyme), and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) were purchased from Sigma-Aldrich (St. Louis, MO). LPS and inhibitors were treated on cultured cells with appropriate concentration: 1 μg/ml of LPS, 10 μM of PD989052, SB203580, SP600125, and ZnPP.
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