The primary antibodies for immunofluorescence staining were rabbit polyclonal anti-ionized calcium binding adapter molecule 1 (Iba1) (1:100; Fujifilm Wako Pure Chemical Co.), rabbit polyclonal anti-S100β (1:1000; abcam), rabbit polyclonal anti-CD163 (1:100; Bioss), rabbit polyclonal anti-inducible nitric oxide synthase (iNOS) (1:50; BD Biosciences), rat monoclonal anti-myelin basic protein (MBP, aa-82-87) (1:100; AbD Serotec), rat monoclonal anti-Lamp2 (1;100; abcam). Alexa Fluor 488-conjugated species-specific secondary antibodies (1:500; Thermo Fisher Scientific) with 1 µg/ml Hoechest 33342 (Thermo Fisher Scientific) for labeling nuclei.
Hoechest 33342
Manufactured by Thermo Fisher Scientific
Sourced in United States
Hoechst 33342 is a fluorescent dye used for nucleic acid staining. It binds to double-stranded DNA and emits blue fluorescence when exposed to UV or blue light. This product is commonly used in various applications, such as cell counting, flow cytometry, and microscopy, to detect and visualize DNA content in cells.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Rabbit polyclonal anti s100β, by Abcam (1 mentions)
Alexa fluor 488 conjugated species specific secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Snap cell tmr star, by New England Biolabs (1 mentions)
X tremegene hp reagent, by Roche (1 mentions)
Dmi6000, by Leica (1 mentions)
Superblock pbs blocking buffer, by Thermo Fisher Scientific (1 mentions)
Mitotracker, by Thermo Fisher Scientific (1 mentions)
Nuclei pure prep isolation kit, by Merck Group (1 mentions)
Edu apollo 567 in vitro imaging kit, by RiboBio (1 mentions)
Axio imager z2, by Zeiss (1 mentions)
Secondary goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Biosesang (1 mentions)
Cellquest software, by BD (1 mentions)
Arrayscan vti hcs, by Thermo Fisher Scientific (1 mentions)
Tcs sp5 confocal laser scanning microscope, by Leica (1 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Krebs henseleit buffer, by Merck Group (1 mentions)
Chloromethyl h2dcfda, by Thermo Fisher Scientific (1 mentions)
22 protocols using hoechest 33342
1
Immunofluorescence Staining of Microglial Markers
2
Fluorescent Labeling and Microscopy of SNAP-tagged BAP1 Variants
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HeLa-Kyoto cells were regularly maintained in DMEM with 10% FBS and 1% penicillin/streptomycin and plated in the chamber-slide (µ-Slide; Ibidi) before transfection. The DNA sequences corresponding to BAP1 variants were subcloned into the pSNAP-tag vector, with an N-terminal SNAP-tag for fluorescence labeling.
Plasmids of SNAP-tagged BAP1 variants were transfected into cells using X-tremeGENE HP reagent (Roche). 2 d after transfection, exogeneous SNAP-tagged BAP1s were live-labeled with SNAP-Cell TMR-star (New England BioLabs), and nuclei were stained with Hoechest33342 (Thermo Fisher Scientific) for 30 min. Labeled cells were transferred into the fresh medium and used for epifluorescent live-cell microscopy. Images were taken using a Leica DMI6000 microscope with an HCX PL FL 20×/0.40-NA CORR objective lens and an Andor Luca R EMCCD sensor. Integral fluorescent intensities of labeled BAP1 variants in the separate cellular compartments were processed and counted based on double-stained monochrome images using MetaMorph Multi-Wavelength cell scoring software application (Molecular Devices).
Plasmids of SNAP-tagged BAP1 variants were transfected into cells using X-tremeGENE HP reagent (Roche). 2 d after transfection, exogeneous SNAP-tagged BAP1s were live-labeled with SNAP-Cell TMR-star (New England BioLabs), and nuclei were stained with Hoechest33342 (Thermo Fisher Scientific) for 30 min. Labeled cells were transferred into the fresh medium and used for epifluorescent live-cell microscopy. Images were taken using a Leica DMI6000 microscope with an HCX PL FL 20×/0.40-NA CORR objective lens and an Andor Luca R EMCCD sensor. Integral fluorescent intensities of labeled BAP1 variants in the separate cellular compartments were processed and counted based on double-stained monochrome images using MetaMorph Multi-Wavelength cell scoring software application (Molecular Devices).
3
Measurement of Cell Proliferation in HCEC-1CT Cells
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HCEC-1CT cells were plated on chamber slides and allowed to culture in the supernatant of HBSS or poly P-treated PRP for 6 h. Slides were then fixed for 15 min in 4% paraformaldehyde, washed extensively with PBS, permeabilized with 0.2% Triton X-100 for 30 min, and blocked in SuperBlock Blocking Buffer (PBS) (Thermo Fisher) for 1 h at room temperature. Slides were then sequentially incubated with primary antibodies Ki-67 (Cell Signaling Technology) for 24 h at 4°C, washed with PBS, and then incubated with anti-rabbit antibody conjugated with Alexa Fluor 488 (Thermo Fisher) for 1 h. The nuclei were counterstained with Hoechest33342 (Thermo Fisher) for 3 min. The cells were mounted with an antifade mounting medium, and immunofluorescence was visualized using a confocal microscope. The Ki-67-positive rate (%) was calculated based on the proportion of the Ki-67-positive area to the Hoechst-positive area, which was measured using the ImageJ software program.
4
Isolation and Characterization of Functional Nuclei
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Functional nuclear isolation was performed using commercially available nuclei isolation kit (Nuclei Pure Prep Isolation Kit; Sigma‐Aldrich) as reported previously [15 (link)]. Purity of nuclei was assessed by western blot using anti‐OXPHOS, anti‐GAPDH, total histone antibodies, and imaging with nuclear marker Hoechest33342 (Thermo Fisher Scientific Inc.) and mitochondrial marker (MitoTracker; Thermo Fisher Scientific Inc.). For functional experiments, isolated nuclei were used immediately.
5
MA2 Inhibits Cell Proliferation
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Cell proliferation was detected using an EdU Apollo 567 In Vitro Imaging Kit (RiboBio, C10310–1). Briefly, the cells were seeded into 96-well plates (1 × 104 cells per well) and cultured overnight. After incubating with 0, 40, 80 and 120 μmol/L MA2 for 48 h, cells were labeled with 10 μmol/L EdU at 37 °C, 5% CO2 for 2 h, fixed in 4% paraformaldehyde for 30 min, followed by staining with fluorescent dye. Cell nuclei were counterstained by Hoechest 33342 (Thermo, 62249). EdU positive cells were observed and counted under an inverted fluorescence microscope (Olympus, IX71).
6
Immunocytochemistry Analysis of Myogenic Markers
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After proliferation for 6 days and differentiation for 4 days, the cells were fixed in 4% paraformaldehyde (Biosesang, Seongnam, Republic of Korea) for 10 min and treated with blocking solution (2% goat serum + 2% horse serum + 0.3% triton X-100 + 1X PBS) for 1 h. Then, the cells were treated with MyoD antibody (1:100, Thermo Fisher Scientific) and MyoG antibody (1:100, Thermo Fisher Scientific) at 4 °C overnight. After washing, cells were incubated with secondary goat anti-mouse lgG (1:500, Thermo Fisher Scientific) and secondary goat anti-rabbit IgG (1:500, Thermo Fisher Scientific) for 1 h at room temperature. Nucleus staining was performed using Hoechest 33342 (Thermo Fisher Scientific). Cell images were measured using Zeiss Axio Imager Z2 (Carl Zeiss, Jena, Germany) under a 10× objective lens.
7
Quantifying Apoptosis in Cell Cultures
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Cells were inoculated in a 96-well plate and grown to 75% confluence. Cells in the logarithmic growth phase were collected. After washing with RPMI-1640 medium without serum, cells were incubated in 100 μl RPMI-1640 medium without serum [50 mg/ml propidium iodide (Sigma-Aldrich, St Louis, Missouri, USA), 5 μg/ml Hoechest 33342 (Thermo, Hom Bridge City, Massachusetts, USA)]. Cells were incubated for 10 m at 37°C and the percentage of apoptotic cells was determined by Thermo Scientific ArrayScan VTI HCS (Thermo). Cellquest software (Becton Dickinson, Franklin, New Jersey, USA) was used for analysis. This experiment was repeated three times.
8
Imaging Cellular Uptake of Fluorescent Polymer Micelles
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L929 cells were seeded at a density of 50,000 cells/cm2. The medium was replaced with DMEM supplemented with low serum 24 hours before the experimental assay. 2 hours before PM addition, the medium was replaced with piperazine-N,N′-bis (2-ethanesulfonic acid) (PIPES) buffer supplemented with 0.5% serum. Fluorescein-loaded PMs were added (5 × 1012 PMs ml−1) to the cells and images were taken every 60 seconds over 60 minutes using a TCS SP5 laser scanning confocal microscope (Leica, Milton Keynes, UK). Cell nuclei were stained with Hoechest 33342 (Thermo Fisher Scientific, Basingstoke, UK). A 63 × 1.30 glycerol immersion objective was used. Negative controls were, PBS-loaded PMs (5 × 1012 PMs) and a 10 mM solution of fluorescein. Image analysis was performed by randomly selecting 20 cells of interest and measuring the mean fluorescence intensity at each time point.
9
Intracellular ROS Measurement by CM-H2DCFDA
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Intracellular ROS production was determined by chloromethyl-H2DCFDA (Thermo Fischer Scientific, C6827). First, 4 × 104 cells were plated in 96 VIEWPLATE black and flat bottom plates (Perkin Elmer, 6005225) and let growth for 24 h; cells were incubated with 10 uM of CM-H2DCFDA (Thermo Fischer Scientific, C6827) in KREBS-Henseleit buffer (Sigma Aldrich, K3753) for 30 min at 37 °C. ROS production was measured in control conditions and after treatment with or without 2DG, with/without MG in presence or absence of liraglutide treatment. Excitation filter for the CM-H2DCFDA was set at 495 and the emission filter was set at 529 nm. The fluorescence intensity data obtained have been normalized for the cell number using Hoechest 33342 (Thermo Fischer Scientific, H3570) at 350 nm as excitation and 461 nm as emission. The probe’s fluorescence was monitored with a Perkin Elmer Envision 2104 multilabel reader (Perkin Elmer).
10
Cell Viability and Apoptosis Assay
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All DNA were synthesized and purified by Sangon Biotechnology Company, Ltd. (Shanghai). Sequences of all the DNA were listed in Supplementary Table S1 . Dulbecco’s phosphate buffered saline (D-PBS) was purchased from Corning. 0.1 M PB buffer was prepared using NaH2PO4 and Na2HPO4. Binding buffer was D-PBS containing 5 mM MgCl2, 4.5 g/L glucose, 1 mg/ml BSA and 0.1 mg/ml yeast tRNA. Methylthiazolyldiphenyl-tetrazolium bromide (MTT), yeast tRNA, bovine serum albumin (BSA) and propidium iodide (PI) were purchased from Sigma-Aldrich. Calcein-AM, SYBR Gold and Hoechest 33,342 were purchased from Thermo Fisher Scientific. Doxorubicin (Dox) was purchased from Dingguo Biotechnology Company, Ltd. (Beijing). Caspase 3 detection kit was purchased from Beyotime Biotechnology Company, Ltd. (Shanghai). Annexin-V FITC apoptosis detection kit was purchased from Sangon Biotechnology Company, Ltd. (Shanghai). Other chemicals were of analytical grade and used without further purification.
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