Cells were seeded at 6000 cells/well in a 96-well plate. After attachment and syncytialization, the cells were incubated with 5 mM, 10 mM, and 25 mM glucose. The medium was updated every 24 h for a total of 96 h. Thereafter, all groups were treated with a mixture of DCF-DA (20 μM, Sigma-Aldrich, St. Louis, MO, USA) and Hoechst 33342 (HO, 2.5 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) for 30 min to detect intracellular ROS and the corresponding number of viable cells. The fluorescence intensity was measured by a microplate reader after discarding the supernatant and PBS rinse. The excitation/emission wavelengths were 490/530 nm for DCF-DA and 340/425 nm for HO. The results were calculated as the ratio of DCF-DA/HO signals per well. All samples were performed in triplicate. The placental explants were cultured and digested into single-cell suspensions, and then, the ROS level was measured according to the manufacturer’s standard (E004, Nanjing Jiancheng Bioengineering Institute).
Hoechst 33342 ho
Manufactured by Merck Group
Sourced in United States
Hoechst 33342 (HO) is a fluorescent dye used for labeling and visualizing nucleic acids, particularly DNA, in various biological applications. It binds to the minor groove of DNA, emitting a blue fluorescence upon excitation. Hoechst 33342 is commonly used in cell biology, microscopy, and flow cytometry techniques.
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Lab products found in correlation
Propidium iodide, by Merck Group (4 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Ab7842, by Abcam (2 mentions)
Ab10988, by Abcam (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Anti γ h2a x ser139 antibody, by Cell Signaling Technology (1 mentions)
Hct116, by American Type Culture Collection (1 mentions)
Cell counting kit 8 cck 8, by Merck Group (1 mentions)
In situ cell death detection kit, by Merck Group (1 mentions)
Oxaliplatin, by Merck Group (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Merck Group (1 mentions)
D luciferin sodium, by Merck Group (1 mentions)
P3566, by Thermo Fisher Scientific (1 mentions)
Ultra sensitive mouse insulin elisa kit, by Crystal Chem (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
2 7 dichlorodihydrofluorescein diacetate dcfh da, by Thermo Fisher Scientific (1 mentions)
12 protocols using hoechst 33342 ho
1
Glucose-Induced Oxidative Stress in Placental Cells
2
Multiparametric Evaluation of DNA Damage
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Propidium iodide (PI), PBS, and RPMI1640 medium were purchased from Thermo Fisher Scientific. DMSO and Hoechst 33 342 (Ho) were purchased from Sigma Aldrich. Anti–poly ADP‐ribose polymerase 1 (PARP) antibody, anti–cleaved caspase‐3 antibody, anti–γ‐H2A.X (ser139) antibody, anti–mutS homolog 2 (MSH2) antibody, anti–mutS homolog 6 (MSH6) antibody, anti–mutL homolog 1 (MLH1) antibody, anti–PMS2 antibody, anti–RAD23 homolog A (RAD23A) antibody, anti–Fanconi anemia group D2 (FANCD2) antibody, anti–O6‐methylguanine‐DNA methyltransferase (MGMT) antibody, and anti–rabbit secondary antibody were purchased from Cell Signaling Technology. Anti–dynein cytoplasmic 2 heavy chain 1 (DYNC2H1) antibody was purchased from Abcam, TMZ and ACNU (dissolved in DMSO) were purchased from Fujifilm Wako Pure Chemical Corporation, and CCNU (dissolved in DMSO) was purchased from Tokyo Chemical Industry.
3
Viability Assay for Mouse Islets
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The percentages of viable cells were determined using the DNA-binding dyes propidium iodide (PI, 5 µg/mL, Sigma-Aldrich) and Hoechst 33342 (HO, 5 µg/mL, Sigma-Aldrich), as described [65 (link)]. For mouse islets, the percentages of dead cells were evaluated in a minimum of 10 islets per condition. All assessments were performed by two independent researchers one of whom was unaware of the identity of the samples.
4
Cell Viability Assay with PI and Hoechst
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Cells were stained with 15 µg/mL propidium iodide (PI, Sigma Aldrich) and 15 µg/mL Hoechst 33,342 (HO, Sigma-Aldrich) for 10 min. The percentage of viable and dead cells was determined as already described [9 (link)]. A minimum of 500 cells was counted for each experimental condition.
5
Colorectal Cancer Cell Line Assays
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Human colon cancer cell lines HCT116, DLD-1and HT29 and the nonmalignant colonic epithelial cell line NCM460 were obtained from the ATCC (Manassas, VA, USA) and cultured in McCoy's 5A medium (modified), DMEM and RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin, respectively. All cells were cultured in the optimal environment of 37 °C with 5% CO2. Cells treated with DMSO or PBS were used as the negative control (Ctrl). Oxaliplatin, D-Luciferin sodium, DCF-DA, Hoechst 33342 (HO), the Annexin V-FITC PI Apoptosis Detection Kit, the In Situ Cell Death Detection Kit, and the Cell Counting Kit-8 (CCK-8) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). All antibodies were purchased from Cell Signaling Technology (Beijing, China). A new H2S-specific near-infrared fluorescence enhanced probe was donated by Beijing University of Chemical Technology. A Total GSH Detection Kit was purchased from Beyotime Biotechnologies (Jiangsu, China).
6
Tomato-lectin Induced Immune Response
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Ten minutes after tomato-lectin infusion through the tail vein, the mice were sacrificed. The pancreas and the injured skin were dissected out and fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich, St. Louis, MO, USA) for 6 hours. After incubation in 30% sucrose for 24 hours, the samples were frozen and embedded. Fluorescent immunostaining was done as described before (10 (link)). Tomato-lectin was detected by direct red fluorescence and virus-transduced macrophages were detected by GFP. Immunofluorescent staining for glucagon and insulin was performed with a monoclonal antibody against glucagon (Ab10988, Abcam) and a guinea pig polyclonal antibody against insulin (Ab7842, Abcam), respectively. Hoechst 33342 (HO, Sigma-Aldrich) was applied to stain the nucleus.
7
Fluorescence Microscopy of Adherent Cells
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Fluorescence microscopy of unfixed/unpermeabilized adherent cells stained with 1 µg/mL 2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2.50-bi-1H-benzimidazole (Hoechst 33342, HO; Sigma-Aldrich, B-2261) and 5 µg/mL propidium iodide (PI, Invitrogen, P-3566) was performed as previously described [15 (link)].
8
Islet Isolation and Glucose-Stimulated Insulin Secretion
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For islet isolation, mouse pancreases were digested by collagenase and incubated in a water bath at 37 °C. The islets were handpicked under a stereomicroscope. The isolated islets were cultured and treated as described. Glucose-stimulated insulin secretion (GSIS) was performed in freshly isolated islets Insulin was quantified using the Ultra-Sensitive Mouse Insulin ELISA Kit (Crystal Chem, Downers Grove, USA). The GSIS experiments were performed and measured in triplicates.
The percentages of viable cells were determined using the DNA-binding dyes Propidium Iodide (PI, 5 µg/mL, Sigma-Aldrich) and Hoechst 33342 (HO, 5 µg/mL, Sigma-Aldrich), as described [54 (link)]. The percentages of dead islets β-cells were evaluated in a minimum of 10 islets per condition. All assessments were performed by two independent researchers, including one in a blind manner.
The percentages of viable cells were determined using the DNA-binding dyes Propidium Iodide (PI, 5 µg/mL, Sigma-Aldrich) and Hoechst 33342 (HO, 5 µg/mL, Sigma-Aldrich), as described [54 (link)]. The percentages of dead islets β-cells were evaluated in a minimum of 10 islets per condition. All assessments were performed by two independent researchers, including one in a blind manner.
9
Immunostaining of Pancreas and Skin Tissues
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After FITC-lectin perfusion, mouse pancreas or limb skin tissue was dissected out and fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich, St. Louis, MO, USA) for 6 hours. After overnight incubation in 30% sucrose, samples were frozen in liquid nitrogen and embedded in tissue freezing medium. Fluorescent immunostaining was done as described before [11 (link)]. FITC-lectin was detected by direct green fluorescence. Insulin and glucagon staining used guinea pig polyclonal antibody against insulin (1:500, Ab7842, Abcam, Cambridge, MA, USA) and monoclonal antibody against glucagon (1:400, Ab10988, Abcam), respectively. Hoechst 33342 (HO, Sigma-Aldrich) staining was done for visualizing nucleus.
10
Quantitative Analysis of Apoptosis and Necrosis
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