The MIN6 cell line was purchased from Fenghbio Biological Ltd. (Fenghbio, Changsha, China) and tested regularly for mycoplasma contamination using a one-step mycoplasma detection kit (YiseMed, Shanghai, China). The MIN6 murine beta-cell line was cultured at 37 °C in an incubator containing 5% CO2 with RPMI 1640 medium supplemented with 10% FBS and 100 units/mL penicillin/streptomycin; 0.25% trypsin (Thermo Fisher Scientific, Waltham, MA, USA) was used to detach the cells, and they were passaged at a 1:3 ratio when the cell density reached around 80–90%. The hypoxia parameters were 1% O2 + 5% CO2. Various doses of 4-OI (Selleck, Houston, TX, USA), FX-11 (MCE, Monmouth Junction, South Brunswick Township, NJ, USA), and N-acetyl-l-cysteine (NAC) (Selleck, Houston, TX, USA) were used to pretreat cells for 4 h before incubation under hypoxic conditions.
N acetyl l cysteine nac
Manufactured by Selleck Chemicals
Sourced in United States, China
N-acetyl-L-cysteine (NAC) is a chemical compound commonly used in laboratory settings. It is a derivative of the amino acid cysteine and serves as a precursor to the antioxidant glutathione. NAC has versatile applications in various research and analytical procedures.
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Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
Goat anti rabbit igg, by Proteintech (2 mentions)
Histone h3, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Goat anti mouse igg, by Proteintech (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by GE Healthcare (1 mentions)
Hpaepic, by ScienCell (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Nlrp3, by Abcam (1 mentions)
Fitc conjugated secondary antibody, by Proteintech (1 mentions)
Mmp 9, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
7 protocols using n acetyl l cysteine nac
1
Hypoxia-induced cytotoxicity in MIN6 cells
2
Inflammasome Regulation in Alveolar Epithelial Cells
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HPAEpiC were obtained from ScienCell Research Laboratories (San Diego, CA, USA); Dulbecco's modified Eagle's medium (DMEM; GE Healthcare Life Sciences, Logan, UT); Lipofectamine 2000 (Invitrogen, USA); 1% penicillin-streptomycin mixture (Gibco, Grand Island, NY); N-Acetyl-L-cysteine (NAC; Selleck, Houston, TX, USA); dexamethasone (DEX; Sigma-Aldrich, St. Louis, MO, USA); Cell Counting Kit-8 (CCK-8; Signaling Antibody, College Park, MD, USA); fluorescent 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA) probe (Beyotime, China); IL-1β and IL-18 ELISA kits (Nanjing Jiancheng Bioengineering Institute); TRIzol (Thermo Fisher Scientific,, USA); NLRP3 (Abcam, Cambridge, MA, USA; ab263899); Zonula Occludens Protein 1 (ZO-1; Abcam; ab96587); Claudin 4 (CLDN4; Proteintech; 16195-1-AP); Caspase-1 (Abcam; ab207802); GAPDH (Proteintech; 60004-1-1G); anti-Horseradish peroxidase- (HRP-) conjugated secondary antibody (Beyotime Biotechnology; A0208 and A0216).
3
Investigating Molecular Mechanisms in Cell Signaling
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The following primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): GAPDH, histone H3, β-actin, E-cadherin, N-cadherin, vimentin, matrix metalloproteinase 9 (MMP9), PARP, Bax, Bcl2, P62, light chain 3B (LC3B), VEGFR2, nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), and SOD2. The following secondary antibodies were provided by Proteintech (Wuhan, China): goat anti-rabbit IgG, goat anti-mouse IgG, and FITC-conjugated secondary antibody. Apatinib and TBHQ (an Nrf2-specific activator) were purchased from MCE, China. N-acetyl-L-cysteine (NAC), a reactive oxygen species (ROS) scavenger, was purchased from Selleck, China.
4
Osteosarcoma Cell Line Culture Protocol
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The two human osteosarcoma cell lines were obtained from were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai) and were cultured in RPMI 1640 medium (Hyclone) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco) at 37 °C in 5% CO2. Human normal primary osteoblast (Lonza) was cultured using the Osteoblast Growth Medium BulletKit (Catalog No. CC-3207). Narasin was obtained from Sigma. Doxorubicin and acetylcysteine (N-acetyl-l-cysteine, NAC) were obtained from Selleck Chemicals. All were reconstituted according to manufacture’s recommendations and stored at aliquots in -200 C.
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The two human osteosarcoma cell lines were obtained from were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai) and were cultured in RPMI 1640 medium (Hyclone) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco) at 37 °C in 5% CO2. Human normal primary osteoblast (Lonza) was cultured using the Osteoblast Growth Medium BulletKit (Catalog No. CC-3207). Narasin was obtained from Sigma. Doxorubicin and acetylcysteine (N-acetyl-l-cysteine, NAC) were obtained from Selleck Chemicals. All were reconstituted according to manufacture’s recommendations and stored at aliquots in -200 C.
5
Punicalagin Inhibits HPV E6/E7 Expression
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Punicalagin was purchased from Macklin (purity ≥ 98% determined using high-performance liquid chromatography; Shanghai, China). The anti-HPV type 16/18 E6 and anti-HPV type 16/18 E7 antibodies were purchased from Novus and Genetex, respectively. The horseradish peroxidase (HRP)-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies were purchased from Zhongshan Goldenbridge Biotechnology (Beijing, China). All other antibodies were purchased from Cell Signaling Technology. LipoRNAi transfection reagent, MG-132, and radioimmunoprecipitation assay (RIPA) lysis buffer were purchased from Beyotime (Shanghai, China). TRIzol was purchased from Invitrogen. E64d, pepstatin A, SP600125 (JNK inhibitor), and 3-methyladenine (3-MA) (PI3KC3 inhibitor) were purchased from Sigma-Aldrich. The reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) was obtained from Selleck. Cycloheximide (CHX) was purchased from Absin. The details of the antibodies and reagents are listed in Table S1 and Table S2.
6
Investigating Cellular Responses to Oxidative Stress
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The following antibodies were purchased: primary antibodies to SIRT5 (Cell Signaling Technology, Danvers, MA), H2A histone family member X phosphorylated on S139 (γ-H2AX, Cell Signaling Technology), nuclear factor erythroid-2-related factor 2 (Nrf2; Proteintech, Wuhan, China), heme oxygenase 1 (HO-1; Proteintech), manganese-dependent superoxide dismutase (MnSOD)/SOD2 (Proteintech), breast cancer gene 1 (BRCA1, Cell Signaling Technology), histone H3 (Cell Signaling Technology), and β-actin (Cell Signaling Technology); and secondary antibodies, specifically goat anti-rabbit IgG (Proteintech), goat anti-mouse IgG (Proteintech), and tetramethylrhodamine (TRITC)-conjugated secondary antibody (Proteintech).
Cisplatin and ML385 (an Nrf2-specific inhibitor) were purchased from MCE, China. N-acetyl-L -cysteine (NAC), a reactive oxygen species (ROS) scavenger, was purchased from Selleck, China.
Cisplatin and ML385 (an Nrf2-specific inhibitor) were purchased from MCE, China. N-acetyl-
7
Autophagy Quantification in PBMCs
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Autophagy was measured using CYTO-ID autophagy detection (Enzo Life Sciences, Farmingdale, NY) kit following the manufacturer’s instructions. PBMCs of HS and PD were incubated with rapamycin (500 nM) and chloroquine (10 μM) in the presence or absence of N-acetyl-L-cysteine (NAC, 1 mM) (Selleckchem, Houston, TX) or Resatorvid (TAK-242, 10 μM) (Selleckchem) for 18 hours.
After stimulation, cells were collected by centrifugation and resuspended in 5% fetal bovine serum-PBS. CYTO-ID Green staining solution (1:1000 in 5% fetal bovine serum-PBS) was added to the cells and incubation was performed for 30 minutes at room temperature in the dark. Cells were collected by centrifugation, washed with 5% fetal bovine serum-PBS, and resuspended in 500 μL of 5% fetal bovine serum-PBS and analyzed by an FACSCanto II cytometer (Becton Dickinson, San Jose, CA).
The cells were gated based on forward and side scatter on living cells and mean fluorescence was recorded from the green (FL1 or FITC) channel.
After stimulation, cells were collected by centrifugation and resuspended in 5% fetal bovine serum-PBS. CYTO-ID Green staining solution (1:1000 in 5% fetal bovine serum-PBS) was added to the cells and incubation was performed for 30 minutes at room temperature in the dark. Cells were collected by centrifugation, washed with 5% fetal bovine serum-PBS, and resuspended in 500 μL of 5% fetal bovine serum-PBS and analyzed by an FACSCanto II cytometer (Becton Dickinson, San Jose, CA).
The cells were gated based on forward and side scatter on living cells and mean fluorescence was recorded from the green (FL1 or FITC) channel.
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