All pancreas samples were fixed and cryo-protected in 30% sucrose overnight before freezing, as described before.25 (link) Fast red staining was performed with the chromogen system (Dako, Carpinteria, CA, USA). For fluorescent staining, mCherry was detected by direct fluorescence. Primary antibodies used were: guinea pig polyclonal anti-insulin (Dako), goat polyclonal anti-PlGF (R&D Systems), rat polyclonal anti-Ki-67 (Dako) and rat anti-F4/80 (Invitrogen). Secondary antibodies were all purchased from Dako. Nuclear staining (Hoechst 33342 (HO)) was performed with Hoechst 33 342 (Becton-Dickinson Biosciences, San Jose, California, USA). Quantification of beta-cell proliferation was based on the percentage of Ki-67+ cells in total beta cells, as previously described.26 (link) Beta-cell mass was analyzed as described before.21 (link) Briefly, the mouse pancreata were trimmed of all non-pancreatic tissue, weighed, fixed, cryoprotected in 30% sucrose overnight before freezing in a way to allow longitudinal sections from tail to head of the pancreas to be obtained. Sections at 150 µm intervals from whole pancreas were immunostained for insulin. The beta-cell mass per pancreas was estimated as the product of the relative cross-sectional area of beta cells per total tissue and the weight of the pancreas.
Hoechst 33342
Manufactured by BD
Sourced in United States
Hoechst 33342 is a fluorescent dye used for labeling and visualizing DNA in various biological applications. It binds to the minor groove of DNA, emitting a blue fluorescence when excited by ultraviolet light. Hoechst 33342 is commonly used for cell staining, flow cytometry, and fluorescence microscopy.
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Lab products found in correlation
Hoechst 33342, by Merck Group (11 mentions)
Hoechst 33342, by Thermo Fisher Scientific (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Facsaria 3, by BD (3 mentions)
Bacterial collagenase, by Merck Group (2 mentions)
Caspase 9 total and cleaved, by Cell Signaling Technology (2 mentions)
Caspase 7 total and cleaved, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated anti rabbit igg and anti mouse igg antibodies, by Merck Group (2 mentions)
Cox 2, by Cell Signaling Technology (2 mentions)
P ampkα, by Cell Signaling Technology (2 mentions)
Caspase 8, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Pycr1, by Abnova (2 mentions)
Pycrl, by Abnova (2 mentions)
Diclofenac, by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Trypsin edta, by Merck Group (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Thermo Fisher Scientific (2 mentions)
79 protocols using hoechst 33342
1
Immunohistochemical Analysis of Pancreatic Beta Cells
2
Flow Cytometry Analysis of Side Population Cells
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After reaching the logarithmic growth phase (24 hours after replating), the cells were analysed by FACS. The cells were digested with 0.25% trypsin (Sigma, St. Louis, MO, USA), washed twice with calcium/magnesium-free PBS, and resuspended in ice-cold RPMI 1640 culture (supplemented with 2% FBS) at a concentration of 1 × 106 cells/mL. The DNA binding dye Hoechst 33342 (Sigma, St. Louis, MO, USA) was then added to obtain a final concentration of 5 mg/mL, and the samples were incubated for 70–90 min in the dark with periodic mixing. The cells were then washed twice with PBS. Then, 1 mg/mL propidium iodide (Sigma, St. Louis, MO, USA) was added to the cells, and the cells were maintained at 4°C in the dark prior to sorting by flow cytometry (BD FACSAria).
Because Hoechst 33342 extrudes from cells treated with verapamil (a calcium ion tunnel antagonist) through sensitive ABC transporters, a subset of the cells were incubated with 50 mmol/L verapamil for 30 min at 37°C before the addition of Hoechst 33342 to determine whether this treatment blocks the fluorescent efflux of SP cells.
Because Hoechst 33342 extrudes from cells treated with verapamil (a calcium ion tunnel antagonist) through sensitive ABC transporters, a subset of the cells were incubated with 50 mmol/L verapamil for 30 min at 37°C before the addition of Hoechst 33342 to determine whether this treatment blocks the fluorescent efflux of SP cells.
3
Evaluating Mitochondrial Membrane Potential and Nuclear Morphological Changes
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Δψm was determined by membrane-permeable JC-1 dye (Beyotime). Brie y, HepG2 and BEL-7404 cells were treated with different concentrations of CTPG (0, 200, 400 and 600 μg/mL) for 24 h. All cells were collected and washed with JC-1 washing buffer. Cells were stained with the JC-1 uorescent probe according to manufacturer`s Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js instruction for 20 min at RT. After washing with PBS twice, all samples were analyzed by ow cytometry (BD FACSCalibur).
Hoechst 33342 staining Hoechst 33342 staining was done according to our previous study [16] . The morphological changes of nuclei were examined using membrane-permeable DNA-binding dye Hoechst 33342 (Beyotime). Brie y, the cells were inoculated in 6-well plate at the concentration of 1×10 5 cells/well in 2 mL medium. When reaching 70%~80% con uence, the cells were treated with 200, 400 and 600 μg/mL of CTPG or cisplatin for 24 h. The cells were washed with PBS and xed with 4% ice-cold paraformaldehyde at 4℃ for 10 min. After washing with PBS, cells were stained with Hoechst 33342 at 4℃ for 10 min. Samples were observed by uorescence inverted microscope (Nikon Eclipse Ti-E, Japan).
Hoechst 33342 staining Hoechst 33342 staining was done according to our previous study [16] . The morphological changes of nuclei were examined using membrane-permeable DNA-binding dye Hoechst 33342 (Beyotime). Brie y, the cells were inoculated in 6-well plate at the concentration of 1×10 5 cells/well in 2 mL medium. When reaching 70%~80% con uence, the cells were treated with 200, 400 and 600 μg/mL of CTPG or cisplatin for 24 h. The cells were washed with PBS and xed with 4% ice-cold paraformaldehyde at 4℃ for 10 min. After washing with PBS, cells were stained with Hoechst 33342 at 4℃ for 10 min. Samples were observed by uorescence inverted microscope (Nikon Eclipse Ti-E, Japan).
4
Molecular Mechanisms of MCF-7 Cell Apoptosis
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MCF-7 cells (ATCC® HTB-22™) were obtained from ATCC (Manassas, VA, USA). MEM Eagle’s medium, fetal bovine serum, puromycin, Pen/Strep mix, and PBS were products of Gibco (Waltham, MA, USA). Propidium iodide (PI), annexin V-CF488A conjugate, and annexin V binding buffer were provided by the Biotium Company (Fremont, CA, USA). NC-Slide A2™ was purchased from Chemometec (Allerod, Denmark). Horseradish peroxidase conjugated anti-rabbit IgG and anti-mouse IgG antibodies, bacterial collagenase, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2′,7′-dichlorofluorescin diacetate (DCFDA), indomethacin, and diclofenac were purchased from Sigma Aldrich (Saint Louis, MO, USA). Primary antibodies against COX2, p-AMPKα, mTor, caspase 8, caspase 9 total and cleaved, caspase 7 total and cleaved, BID, PARP, Beclin 1, AGT5, ATG 7, and B-actin were products of Cell Signaling Technology (Danvers, MA, USA). Primary antibodies for PRODH/POX, PPARγ, GLUD 1/2, and prolidase were products of Santa Cruz Biotechnology (Dallas, TX, USA). PYCR1, PYCRL, and PPARδ primary antibodies were obtained from Abnova (Taipei, Taiwan). Hoechst 33342 was obtained from Becton Dickinson (Franklin Lakes, NJ, USA). CRISPR All-In-One Non-Viral Vector and lipofectamine were products of Applied Biological Materials Inc. (Richmond, BC, Canada).
5
Live Cell Cycle Analysis of Larval Eye Discs
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Live GFP-labeled 3rd instar eye-imaginal disc cells were dissected in 1× PBS and simultaneously dissociated with gentle agitation and stained (wrapped in foil) with Hoechst 33342 (Cell Signaling, 500 g/ml) for 2 h using a solution of 450 µl 10× Trypsin-EDTA (Sigma), 50 µl 10× PBS, and 0.5 µl Hoechst 33342. Cells were then passed through a 40 μm cell strainer prior to FACS analysis. Hoechst 33342 expression was analyzed for a minimum of 10,000 GFP-positive cells by flow cytometry on a Becton Dickinson FACS Canto II cytometer using FACSDiva software. Elimination of dead cells and the distribution of cells within G1, S, and G2/M phases of the cell cycle was determined using FlowJo software.
6
FACS-based Identification of Side Population Cells
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Eca109 and Kyse 510 cells were subjected to FACS analysis during the logarithmic growth phase. The cells were digested with 0.025% trypsin and 1 mM EDTA and rinsed in PBS. The cells were then resuspended in DMEM (with 2% FBS added) at a concentration of 1×106 cells/mL and incubated for 10 minutes at 37 °C, 5% CO2. Thereafter, the DNA-binding dye Hoechst 33,342 (Sigma) was added to the cells and they were incubated for 90 minutes under dark conditions. After incubation, the cells were washed with cold PBS and filtered through a 40 μm cell filter to obtain a single-cell suspension. After completion of FACS (FACSDiva Option; Becton Dickinson) cell analysis and sorting, Hoechst 33,342 was excited by 350 nm UV rays. 405/BP209 (Hoechst blue) and 570/BP20 (Hoechst red) fluorescence were measured using corresponding filters. Hoechst 33,342 negative cells, ie, side population (SP) cells, and Hoechst 33,342 positive cells (non-SP cells) were finally sorted into two cell subpopulations, which was used to analyze the proportion of the SP groups and verify the expression of important candidate genes.
7
Quantifying Cell Cycle Changes
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Primary fibroblasts were seeded at a density of 4,000 cells/cm2. Parallel cultures were grown in DMEM with GlutaMAX (Gibco) and supplemented with 15% FBS (PAN). For flow cytometry, 48 h cultures were left untreated or exposed to 10 ng/ml MMC (Medac) or initially irradiated with 1.5 Gy from a linear accelerator. Cells were detached using 1× (0.05%) trypsin (diluted from trypsin 0.5%-EDTA 0.2% solution 10×, PAA), pelleted, and stained in medium containing 15 µg/ml Hoechst dye 33342 (Molecular Probes) for 30 min in the dark. Gates were set on vital cells via propidium iodide (PI, 1 µg/ml) exclusion. Split samples were stained with 1 µg/ml 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes) in buffer containing 154 mM NaCl, 0.1 M Tris pH 7.4, 1 mM CaCl2, 0.5 mM MgCl2, 0.2% BSA, and 0.1% NP40 in dH2O. Univariate flow histograms were recorded on a triple-laser equipped LSRII flow cytometer (Becton Dickinson) using UV excitation of Hoechst 33342 or DAPI, and 488-nm excitation of PI. Resulting cell cycle distributions reflecting cellular DNA content were quantified using the MPLUS AV software package (Phoenix Flow Systems).
8
Synthesis and Characterization of Gold Nanoparticles
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Ultrapure water (18.2 MΩ) was used throughout the study. All glassware and magnetic stir bars were washed with aqua regia, rinsed with abundant ethanol and ultrapure water, and dried in an oven before use. All chemicals were commercially available and used as received: gold (III) chloride trihydrate (HAuCl4·3H2O), p-mercaptobenzoic acid (MBA), sodium borohydride (NaBH4), ammonium acetate (NH4OAc), agar, lysozyme, lysostaphin, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), paraformaldehyde (PFA), and 2,5-dihydroxybenzoic acid (DHB) were purchased from Sigma-Aldrich; sodium hydroxide (NaOH) was from Merck; Luria−Bertani (LB) was from Becton Dickinson; Hoechst 33342, SYTOX® Green nucleic acid stain and ProLong® Gold antifade reagent with DAPI were purchased from Life Technologies; lipid peroxidation (MDA) assay kit was purchased from Abcam.
9
Side Population Analysis of LMNA-KD Cells
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To analyze the side population phenotype, LMNA-KD cells were stained according to the protocol of Goodell et al. [42 ]. Briefly, 5.0 × 106 cells/ml were suspended in pre-warmed DMEM. Hoechst 33342 (Sigma) was added at a final concentration of 5 μg/ml in the presence or absence of 50 μM verapamil (Sigma), and the cell samples were incubated for 90 min at 37°C with intermittent shaking. After incubation, the cells were washed with ice-cold PBS, resuspended in ice-cold PBS, and analyzed for Hoechst 33342 efflux with a FACS Aria II (Becton Dickinson). The Hoechst 33342 dye was excited at 375 nm, which is near-ultraviolet, and the resultant fluorescence was measured at two wavelengths, using 450/40 BP and 670 LP filters for the detection of Hoechst blue and red, respectively. All data were analyzed using the Diva 6.1 Software (Becton Dickinson).
10
Molecular Signaling in MCF7 Breast Cancer
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MCF7 (ATCC® HTB-22™) were obtained from the ATCC. Horseradish-peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG antibodies, Alexa488 conjugated anti-rabbit IgG, bacterial collagenase, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DCFDA, tetrahydro-2-furoic acid (THFA), glycyl-L-proline, ibuprofen, indomethacin, diclofenac, and sulindac were purchased from Sigma-Aldrich. The celecoxib and troglitazone were products of Targetmol. Solution 5 (VB-48™ PI–AO) was purchased from ChemoMetec. The primary antibodies against: COX2; p53; p-AMPKα; m-TOR; caspase-8; caspase-9, total and cleaved; caspase-7, total and cleaved; PARP, total and cleaved; and B-actin were products of Cell Signaling Technology. The PRODH/POX primary antibodies and PPARγ were products of Santa Cruz Biotechnology. The PYCR1, PYCRL, and PPAR delta primary antibodies were obtained from Abnova. The Hoechst 33342 was obtained from Becton Dickinson.
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