The Porcine jejunal epithelial cell line (IPEC-J2) was originally obtained from the Cell Resource Center, Shanghai Institute of Life Sciences, Chinese Academy of Sciences. All reagents for the cell culture were procured from Life Technologies (Grand Island, NY, USA). Interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) Assay Kits were purchased from R&D Systems (Minneapolis, MN, USA). Total superoxidase (T-SOD), glutathione peroxidases (GPx) and malondialdehyde (MDA) Assay Kits were obtained from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). The Occludin, Claudin-1 and β-actin primary antibodies and secondary HRP antibodies were purchased from Abcam Biotechnology (Cambridge, MA, USA). The Bicinchoninic acid (BCA) protein assay Kit and RIPA lysis buffer were procured from Solarbio Life Sciences Co. (Beijing, China). The Hoechst 33342 Staining Kit, adenosine triphosphate (ATP) Assay Kit, mitochondrial membrane potential (MMP) Assay Kit, reactive oxygen species (ROS) Assay Kit and the Cell Counting Kit-8 (CCK-8) were procured from Beyotime Biotechnology (Shanghai, China). Sodium selenite and fluorescein isothiocyanate-dextran (FITC-dextran, 4 kDa) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Hoechst 33342 staining kit
Manufactured by Beyotime
Sourced in China
Hoechst 33342 staining kit is a fluorescent dye used for the detection and quantification of DNA in biological samples. It binds specifically to the minor groove of double-stranded DNA, emitting a blue fluorescence upon excitation. The kit provides the necessary components for the staining procedure.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Jc 1 kit, by Beyotime (2 mentions)
Horse serum, by Thermo Fisher Scientific (2 mentions)
Adenosine triphosphate atp assay kit, by Beyotime (1 mentions)
Mitochondrial membrane potential assay kit, by Beyotime (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Cell counting kit 8 cck 8, by Beyotime (1 mentions)
Reactive oxygen species assay kit, by Beyotime (1 mentions)
Bicinchoninic acid bca protein assay kit, by Solarbio (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Annexin 5 pi assay kit, by Keygen Biotech (1 mentions)
Fetal bovine serum (fbs), by Capricorn (1 mentions)
Cck 8 kit, by Keygen Biotech (1 mentions)
Adenosine, by Merck Group (1 mentions)
Cytochrome c, by Cell Signaling Technology (1 mentions)
13 protocols using hoechst 33342 staining kit
1
IPEC-J2 Cell Line Characterization
2
Hoechst 33342 Staining for Cell Viability
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Hoechst 33342 staining measurements were performed with a Hoechst 33342 staining kit (C1022, Beyotime, Shanghai, China). The cells were seeded in confocal dishes at a cell density of 3 × 104 cells/mL (200 μL/dish) and incubated at 37 °C and 5% CO2. After 24 h, the previous medium was discarded, and 200 μL of the prepared P6 solutions at different concentrations were added to the treatment group; 200 μL of complete medium was added to the control group. After 24 h of P6 treatment, the previous medium was aspirated and the cells were carefully rinsed 2–3 times. Two hundred microliters of Hoechst 33342 dye was added to each well, and after incubation in the dark for 15 min, the dye was discarded and the cells were carefully rinsed with PBS buffer 2–3 times. The photographs of the samples were recorded using a fluorescence microscope (Olympus, Tokyo, Japan).
3
Synthesis and Characterization of N-acylated CM4 Peptides
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CM4, N-acylated CM4,
and FITC-labeled N-acylated CM4 were synthesized using solid-phase
Fmoc methods by Synpeptide Inc. (Nanjing, China). The synthesized
peptides were all >95% homogeneous as indicated by C18 reverse-phase
HPLC and electrospray ionization (ESI) mass spectroscopy analyses.
RPMI-1640 and Dulbecco’s modified Eagle’s medium
(DMEM) were purchased from Thermo Fisher Scientific (Waltham, MA),
fetal bovine serum (FBS) was purchased from Capricorn Scientific (Hessen,
Germany). DMEM/F12 and horse serum were purchased from Invitrogen.
TFE was purchased from Sigma-Aldrich (St. Louis, MO). Melittin was
purchased from Synpeptide, Inc. (Nanjing, China). Hoechst 33342 staining
kit was purchased from Beyotime Institute of Biotechnology (Shanghai,
China). CCK-8 kit, Annexin V/PI assay kit, and MitoRed (mitotracker
red) were purchased from KeyGEN BioTECH, Inc. (Nanjing, China). A
prestained protein marker (3.1–20.1 kDa) was purchased from
Solarbio (Beijing, China). All other reagents were of analytical grade
and produced in China. All of the reagents were used by the rules
of standard biosecurity and safety procedures of Nanjing Normal University.
and FITC-labeled N-acylated CM4 were synthesized using solid-phase
Fmoc methods by Synpeptide Inc. (Nanjing, China). The synthesized
peptides were all >95% homogeneous as indicated by C18 reverse-phase
HPLC and electrospray ionization (ESI) mass spectroscopy analyses.
RPMI-1640 and Dulbecco’s modified Eagle’s medium
(DMEM) were purchased from Thermo Fisher Scientific (Waltham, MA),
fetal bovine serum (FBS) was purchased from Capricorn Scientific (Hessen,
Germany). DMEM/F12 and horse serum were purchased from Invitrogen.
TFE was purchased from Sigma-Aldrich (St. Louis, MO). Melittin was
purchased from Synpeptide, Inc. (Nanjing, China). Hoechst 33342 staining
kit was purchased from Beyotime Institute of Biotechnology (Shanghai,
China). CCK-8 kit, Annexin V/PI assay kit, and MitoRed (mitotracker
red) were purchased from KeyGEN BioTECH, Inc. (Nanjing, China). A
prestained protein marker (3.1–20.1 kDa) was purchased from
Solarbio (Beijing, China). All other reagents were of analytical grade
and produced in China. All of the reagents were used by the rules
of standard biosecurity and safety procedures of Nanjing Normal University.
4
Investigating Ovarian Cancer Cell Lines
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The human ovarian cancer cell lines A2780 and SKOV3 were purchased from the American Type Culture Collection (ATCC). Adenosine (>99% purity) was provided by Sigma-Aldrich (Cat NO: A9251, Milwaukee, WI). DMEM, fetal bovine serum (FBS) and trypsin were obtained from Gibco (Grand Island, NY, USA). MTT was derived from Sigma-Aldrich (Milwaukee, WI), and the Annexin V-FITC/7-AAD apoptosis detection kit was offered by BD Biosciences (San Jose, CA). The Hoechst 33342 staining kit, ROS kit, JC-1 kit, and BCA protein assay kit were provided by the Beyotime Institute of Biotechnology (Jiangsu, China). The antibodies against β-actin, caspase-3, cleaved caspase-3, Bax, Cytochrome C, β-Tubulin, VDAC and poly (ADP-ribose) polymerase (PARP) were derived from Cell Signaling Technology (Danvers, MA, USA).
5
Visualizing and Quantifying Apoptosis in HBEpC
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To visualize the morphological changes of HBEpC during apoptosis, a Hoechst 33342 staining kit (Beyotime Institute of Biotechnology) was used, as described by previous methods (22 (link)). At the end of each experiment, the cells were stained with Hoechst 33342 (1 mg/ml) for 15 min. The cells were then observed under an IX71 inverted fluorescence microscope (Olympus, Tokyo, Japan).
To quantitatively analyze the rate of apoptosis of the cells, Annexin V-propidium iodide (PI) flow cytometry was performed using an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA). Following the treatment of the cells with RES, with or without CSE for 24 h, ~1×105 cells were collected and resuspended in 500 μl Annexin V binding buffer (1X). Annexin V-FITC (5 μl) and PI (5 μl) were then added to each sample, followed by an incubation for 15 min at room temperature (25°C) in the dark. The cells were then quickly subjected to fluorescence-activated cell sorting analysis (FACSCalibur; BD Biosciences).
To quantitatively analyze the rate of apoptosis of the cells, Annexin V-propidium iodide (PI) flow cytometry was performed using an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA). Following the treatment of the cells with RES, with or without CSE for 24 h, ~1×105 cells were collected and resuspended in 500 μl Annexin V binding buffer (1X). Annexin V-FITC (5 μl) and PI (5 μl) were then added to each sample, followed by an incubation for 15 min at room temperature (25°C) in the dark. The cells were then quickly subjected to fluorescence-activated cell sorting analysis (FACSCalibur; BD Biosciences).
6
Signaling Pathway Modulation Protocol
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The reagents used in our research were: a BCA protein assay kit (Solarbio, #PC0020), BeyoECL Plus (Beyotime, #P0018S, Shanghai, China), CQ (Sigma Aldrich, #C6628, St. Louis, MO, USA), crystal violet (Beyotime, #C0121), DCFH-DA (Beyotime, #S0033S), DMSO (Solarbio, #30072418, Beijing, China), EBSS (Gibco, #24010043, Waltham, UK), GSH (AAT, #22810, Sunnyvale, California, USA), a Hoechst33342 staining kit (Beyotime, #C1025), Lipofectamine 2000 reagent (Invitrogen, #11668027), matrigel (BD, #356234, New York, USA), MTT (Solarbio, #M8180, Beijing, China), a CCK-8 kit (Yeasen,#40203ES60, Shanghai, China), Opti-MEM (Gibco, #11095080), and RAPA (Beyotime, #S1842).
The antibodies used in the experiments were from Cell Signaling Technology (CST): α-Tubulin (CST, #2144), β-actin (CST, #58169), Beclin-1 (CST, #3495), ERK (CST, #4695), GAPDH (CST, #2118), LC3 (CST, #3868), mTOR (CST, #2983), phospho-ERK (Thr202/Tyr204, CST, #4370), phospho-mTOR (Ser2448, CST, #5536), phospho-S6 (Ser235/236, CST, #2211), phospho-VEGFR2 (Tyr1175, CST, #2478), S6 (CST, #2217), TSC2 (CST, #4308), and VEGFR2 (CST, #2479). The fluorescence antibody was PE-VEGFR2 (BD, #89106). Other antibodies included goat anti-rabbit/mouse IgG-HRP (Fdbio science, FDR007 and FDM007, Hangzhou, China) and FITC goat anti-rabbit IgG (Beyotime, A0562).
The antibodies used in the experiments were from Cell Signaling Technology (CST): α-Tubulin (CST, #2144), β-actin (CST, #58169), Beclin-1 (CST, #3495), ERK (CST, #4695), GAPDH (CST, #2118), LC3 (CST, #3868), mTOR (CST, #2983), phospho-ERK (Thr202/Tyr204, CST, #4370), phospho-mTOR (Ser2448, CST, #5536), phospho-S6 (Ser235/236, CST, #2211), phospho-VEGFR2 (Tyr1175, CST, #2478), S6 (CST, #2217), TSC2 (CST, #4308), and VEGFR2 (CST, #2479). The fluorescence antibody was PE-VEGFR2 (BD, #89106). Other antibodies included goat anti-rabbit/mouse IgG-HRP (Fdbio science, FDR007 and FDM007, Hangzhou, China) and FITC goat anti-rabbit IgG (Beyotime, A0562).
7
6-OHDA-Induced Parkinson's Disease Model
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BBR with the purity greater than 99%, 6-OHDA, dimethyl sulfoxide (DMSO), nomifensine (Nom), and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich Co (St. Louis, MO, USA). F-12K medium, phosphate buffered saline (PBS), penicillin-streptomycin (PS) were purchased from Gibco (Maryland, USA). Fetal bovine serum (FBS) and horse serum (HS) were obtained from Invitrogen (Carlsbad, CA, USA). Antibodies against phosphor-phosphoinositide 3-kinase (p-PI3K), PI3K, Bcl-2, phosphor-protein kinase B (p-AKT), AKT, nuclear factor-E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and GAPDH, and secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA) or Proteintech (Chicago, IL, USA). Secondary antibody fluorescein isothiocyanate (FITC) was obtained from Abcam Inc (Cambridge, MA, USA). Hoechst 33342 staining kit, LY294002, Annexin V-FITC/propidium iodide (PI) apoptosis detection kit, caspase-3 activity assay kit, cell cycle and apoptosis analysis kit, and the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) cell apoptosis detection kit were obtained from Beyotime Institute of Biotechnology (Nanjing, Jiangsu, China). Zinc protoporphyrin IX (ZnPP) was obtained from Enzo Life Sciences (Farmingdale, NY, USA).
8
Cytotoxicity and Apoptosis Analysis
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CVB-D (C26H46N2O, FW 402.66, purity 66; YuanYe Biotechnology Co., Ltd.), the Rhodamine 123 (Rh123) kit (BB-41051-1), the Annexin V-FITC/PI apoptosis test kit (BB-4101-2) and the cell cycle test kit (BB-4104-2) were purchased from Shanghai BestBio Biotechnology Co., Ltd. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. Fetal bovine serum (100 kU/l) and penicillin and streptomycin (100 mg/l) were purchased from Gibco-BRL Invitrogen; Thermo Fisher Scientific, Inc. The BCA Protein Assay Kit (P0010), and the reagent kits for the protein measurements were purchased from Beyotime Institute of Biotechnology. Bax (cat no. ab69643), Bcl-2 (cat. no. ab32124), caspase-3 (cat. no. ab32351) and cleaved caspase-3 (cat. no. 9664S), beta-actin (cat. no. 4970S) antibodies were purchased from Cell Signaling Technology and Abcam, Inc. Horseradish peroxidase-conjugated anti-rabbit (cat. no. ab44171) and anti-mouse (cat. no. ab21172) antibodies were obtained from Bioworld Technology, Inc. Immobilon western chemiluminescent HRP Substrate and PVDF membranes were obtained from EMD Millipore. The Hoechst 33342 staining kit (C1028) was purchased from Beyotime Institute of Biotechnology.
9
Mitochondrial Dysfunction and Oxidative Stress
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Dioscin was sourced from Chengdu Must Bio-technology Co., Ltd. Porphyromonas gingivalis-LPS was procured from InvivoGen. The ROS Assay Kit, Mito-Tracker Red CMXRos, Hoechst 33342 staining kit, and Mitochondrial Isolation kit were obtained from Beyotime. MitoSOX™ Red was from Thermo Fisher. DAPI Fluoromount-G™ was from Yeasen. Mitoquinone (MitoQ) was bought from MCE.
10
Hoechst 33342 Apoptosis Assay for Glioma Cells
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Apoptotic cells were observed using the Hoechst 33342 staining kit (Beyotime Biotechnology) according to the manufacturer's instructions. Briefly, glioma U87 and U251 cells were seeded in a 12-well plate for 24 h, and then transfected with siRNAs. At 48 h after transfection, the cells were fixed using 4% polyoxymethylene and incubated with 100 µl of 1X Hoechst 33342 solution for 30 min in the dark. After a PBS wash, the cells were visualized and photographed under a fluorescence microscope (Leica Microsystems CMS GmbH, Wetzlar, Germany).
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