Cloned embryos were washed in PBS containing 0.1% polyvinyl alcohol (PVA) and then fixed in 4% (w/v) paraformaldehyde at room temperature for 30 min. The oocytes were then washed in PBS/PVA and incubated overnight in PBS containing 1% BSA and 0.1% Triton X-100 at 4°C. Following this, the oocytes were washed three times in PBS-0.1% BSA and incubated with a 1:200 dilution of H3K9me3 antibody (07–442, Millipore) at the 1- and 2-cell stages and purified mouse anti-OCT-3/4 (611203, BD Bioscience) at the blastocyst stage for 2 h at room temperature. The cloned embryos were then washed three times in PBS-0.1% BSA and incubated with a 1:200 dilution of goat anti-mouse antibody (ab150113, Abcam) for 1 h at room temperature. Following this, they were washed in PBS-0.1% BSA three additional times. The DNA was visualized by staining the oocytes with 4′,6-diamidino-2-phenylindole (DAPI; D5942, Sigma-Aldrich). The embryos were mounted on glass slides with a drop of fluorescent mounting medium and then observed using fluorescence confocal microscopy (Zeiss LSM880; Zeiss, Jena, Germany).
D5942
Manufactured by Merck Group
Sourced in Germany, United States, Switzerland
D5942 is a laboratory equipment produced by Merck Group. It is designed for conducting scientific experiments and analysis in a controlled environment. The core function of this product is to provide a reliable and precise measurement tool for researchers and scientists.
Automatically generated - may contain errors
Lab products found in correlation
Ab150113, by Abcam (1 mentions)
Lsm 880, by Zeiss (1 mentions)
H3k9me3 antibody, by Merck Group (1 mentions)
Purified mouse anti oct 3 4, by BD (1 mentions)
S1460, by Selleck Chemicals (1 mentions)
P8765, by Merck Group (1 mentions)
T3434, by Merck Group (1 mentions)
Pd98059, by Merck Group (1 mentions)
Galaxy 48r, by Eppendorf (1 mentions)
Y 27632, by STEMCELL (1 mentions)
Perfection v700 photo scanner, by Epson (1 mentions)
Ab108937, by Abcam (1 mentions)
Notch1, by Cell Signaling Technology (1 mentions)
N1icd, by Cell Signaling Technology (1 mentions)
Sc5594, by Santa Cruz Biotechnology (1 mentions)
Pd98059 p215, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Annexin 5 propidium iodide kit, by BD (1 mentions)
Bromosporine, by Merck Group (1 mentions)
M0896, by Merck Group (1 mentions)
10 protocols using d5942
1
Immunofluorescence Analysis of Cloned Embryos
2
Manipulating chordate embryonic development
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Phallusia mammillata embryos were treated with 150 ng/ml recombinant zebrafish BMP4 protein (1128-BM, R&D Systems Inc, 100 μg/mL stock solution in HCl 4 mM + BSA 0,1%) or recombinant mouse BMP2 protein (355-BM, R&D Systems Inc, 100 μg/mL stock solution in HCl 4 mM + BSA 0,1%) complemented with 0.1% BSA from either the eight-cell stage (St. 4) or initial gastrula stage (St. 10) (both proteins being as potent in our hands), with the BMP receptor inhibitors Dorsomorphin (S7306, Euromedex, 10 mM stock solution in water) and DMH1 (S7146, Euromedex, 10 mM stock solution in DMSO) at 20 and 2.5 μM respectively from eight-cell stage or initial gastrula stage (St. 10), or with 25 μM of the γ-secretase inhibitor DAPT (D5942, Sigma-Aldrich, 10 mM stock solution in DMSO) from early neurula stage (St. 14). For B. lanceolatum, embryos and explants were treated with the same molecules but at 250 ng/ml for zebrafish BMP4 and 20 μM for Dorsomorphin. In addition, B. lanceolatum embryos were treated with 50 μM of the γ-secretase inhibitor DAPT, 10 μM of the Gsk3β inhibitor 1-azakenpaullone (A3734, Sigma-Aldrich, 10 mM stock solution in DMSO) and 10 μM of the porcupine inhibitor C59 (M3131, Euromedex, 10 mM stock solution in DMSO). Doses and timings of treatments were determined following pilot experiments.
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Phallusia mammillata embryos were treated with 150 ng/ml recombinant zebrafish BMP4 protein (1128-BM, R&D Systems Inc, 100 μg/mL stock solution in HCl 4 mM + BSA 0,1%) or recombinant mouse BMP2 protein (355-BM, R&D Systems Inc, 100 μg/mL stock solution in HCl 4 mM + BSA 0,1%) complemented with 0.1% BSA from either the eight-cell stage (St. 4) or initial gastrula stage (St. 10) (both proteins being as potent in our hands), with the BMP receptor inhibitors Dorsomorphin (S7306, Euromedex, 10 mM stock solution in water) and DMH1 (S7146, Euromedex, 10 mM stock solution in DMSO) at 20 and 2.5 μM respectively from eight-cell stage or initial gastrula stage (St. 10), or with 25 μM of the γ-secretase inhibitor DAPT (D5942, Sigma-Aldrich, 10 mM stock solution in DMSO) from early neurula stage (St. 14). For B. lanceolatum, embryos and explants were treated with the same molecules but at 250 ng/ml for zebrafish BMP4 and 20 μM for Dorsomorphin. In addition, B. lanceolatum embryos were treated with 50 μM of the γ-secretase inhibitor DAPT, 10 μM of the Gsk3β inhibitor 1-azakenpaullone (A3734, Sigma-Aldrich, 10 mM stock solution in DMSO) and 10 μM of the porcupine inhibitor C59 (M3131, Euromedex, 10 mM stock solution in DMSO). Doses and timings of treatments were determined following pilot experiments.
3
Cochlear Modiolus Primary Cells Treated with DAPT
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Primary cells from the cochlear modiolus prepared as above, were divided into two groups, one as a control group, and the other as the DAPT treated group. DAPT (D5942; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at a final concentration of 5 µM as in a previous study (19 (link)) was added to the culture medium and the cells treated for 24 h to 48 h to the time of sample preparation.
4
Hypoxic Injury Mechanisms in Neonatal Rat Cardiomyocytes
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Neonatal rat CMs were plated in 6-well plates. To mimic the hypoxic injury of myocardium, cells were maintained in a 5% O2 incubator (Galaxy 48R, Eppendorf, Germany) for various periods of time, and then harvested for associated experiments. Before hypoxic treatment, hypoxia (5% O2)-balanced DMEM/F-12 medium was changed. To further understand the mechanisms underlying hypoxia-induced Jagged1 expression in CMs, the following inhibitors were used: YC-1 (HIF-1α inhibitor, 10 μmol/L; Y102, Sigma), PD98059 [extracellular-regulated signal kinase (ERK) inhibitor, 50 nmol/L; sc-3532, Santa Cruz Biotechnology, Dallas, TX, USA], PTDC [nuclear factor κB (NF-κB) inhibitor, 10 μmol/L; P8765, Sigma), DAPT (Notch inhibitor, 10 μmol/L; D5942, Sigma), SP600125 [c-Jun N-terminal kinase (JNK) inhibitor, 25 μmol/L; S1460, Selleck Chemicals, Houston, TX, USA], AG490 [Janus kinase (JAK) inhibitor, 25 μmol/L; T3434, Sigma), and Stattic [signal transducer and activator of transcription 3 (STAT3) inhibitor,10 μmol/L; S7949, Sigma]. Dimethyl sulfoxide (DMSO; 0.1%, v/v) was used as a solvent control.
5
Collagen Contraction Assay for Cancer-Associated Fibroblasts
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To assess force-mediated collagen contraction, 3 × 104 CAFs were embedded in 100 μl of a 2.5-mg/ml collagen solution (Corning, #354249) and seeded in triplicates in 96-well plates. After polymerization, culture medium was added on top of the gels and in the surrounding empty wells to limit evaporation. To assess DAPT (Sigma, #D5942), BLEBBISTATIN (Sigma, #B0560) and Y27632 (STEMCELL Technologies, #72302) impact on CAF contractility, 10 μM of each drug was added to the medium, DMSO as control. Gel contraction was assessed 4 days after plating: plates were scanned (EPSON Perfection V700 Photo scanner) and quantification was performed on Fiji software. Percentage of contraction was calculated using the formula:
. Six CAF-S1 and CAF-S4 pairs were tested in four independent experiments. For DAPT experiments, three independent experiments were performed, including four different CAF-S4 cell lines.
. Six CAF-S1 and CAF-S4 pairs were tested in four independent experiments. For DAPT experiments, three independent experiments were performed, including four different CAF-S4 cell lines.
6
Notch Signaling Pathway Characterization
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Antibodies against Notch1, cleaved Notch1 (N1ICD), ERK1/2, phospho-ERK1/2 (p-ERK), cleaved caspase-3, and β-actin were purchased from Cell Signaling Technology: Notch1 (cat. no. 3608), N1ICD (cat. no. 4147), ERK1/2 (cat. no. 4695), p-ERK (cat. no. 4370), cleaved caspase-3 (cat. no. 9664), and β-actin (cat. no. 4970). Antibodies against Notch2, Notch3, and Hes1 were purchased from Abcam: Notch2 (ab8926), Notch3 (ab23426), and Hes1 (ab108937). Antibodies against Notch4 were purchased from Santa Cruz Biotechnology (sc5594). Antibodies against Hey2 were purchased from Proteintech (10597-1-AP). The ERK inhibitor PD98059 (P215) and γ-secretase inhibitor DAPT (N-[N-(3,5-difluorophenacetyl-l -alanyl)]-S-phenylglycinet-butyl ester; GSI-IX; D5942) were obtained from Sigma-Aldrich. The Annexin V + propidium iodide (PI) kit was purchased from BD Biosciences.
7
Spheroid Formation Assay Protocol
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For preparation, we diluted material (corning #354236) to 50 μg/mL with 0.02N acetic acid, added 300–500 μL diluted material into 24‐well plate and incubate at room temperature for 1 h. After that, the remaining solution should be carefully aspirated, using PBS rinse well to remove acid for 2–3 times. Then the needed number of cells (250–300) and the appropriate volume of 1640 medium were mixed to make the cell concentration at 250 cells/500 μL in one tube. Another tube contained the 1640 medium, material and NaOH at the ratio of 5:1:0.0235. Mixed two tubes on ice and then plated the mixture in the well. After one‐week incubation, spheroid numbers were counted under a phase‐contrast microscope. Cells treated with DAPT (Notch inhibitor, Sigma #D5942) at the indicated concentrations were also used for spheroid formation assay.
8
Screening for Epigenetic Modulators
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Supplementary Figure 1
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9
Macrophage and Epithelial Cell Infection
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RAW 264.7 murine macrophages and HCT116 human colonic epithelial cells were purchased from ATCC (Manassas, VA). RAW cells were grown in DMEM+10% FBS. HCT116 cells were grown in McCoy’s 5a Medium Modified+10% FBS. No antibiotics were added to the culture media. Cells were grown at 37°C in a humidified incubator with 5% CO2. Prior to the day of infection, 8x105 cells were plated in a 6-well plate. Cells were treated with DSV at various MOI and times. For DAPT treatment (D5942, Sigma Aldrich), cells were pretreated overnight with various DAPT concentrations followed by infection with DSV. In control cells, equivalent volume of DMSO was added as a vehicle control for DAPT. DMSO itself was found to have no effect on the expression of Notch proteins when compared to no DMSO control (data not shown).
10
Osteogenic Differentiation of Cxcl12-GFP+ Dental Pulp Cells
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Cxcl12-GFP+ dental pulp cells were isolated from molars and incisors of PN15 Cxcl12-GFP mice. Dental pulps were minced to small pieces and digested in Collagenase P (2.5 mg/mL) for 30 min at 37 °C. Cell suspensions were then filtered through a 70 μm cell strainer. Sorted Cxcl12-GFP+ dental pulp cells were cultured in DMEM supplemented with 30% FBS. Osteogenic differentiation was induced by culturing the cells in DMEM supplemented with 10% FBS, Ascorbic Acid (200 μM), β-Glycerolphosphate (10 mM), Dexamethasone (10 nM) (Sigma-Aldrich/Merck, Darmstadt, Germany), and Amphotericin B (0.25 μg/μL) (ThermoFisher Scientific, Switzerland). After 10 days of treatment, cells were harvested and snap frozen in liquid nitrogen. To inhibit the Notch signaling pathway, Cxcl12-GFP+ dental pulp cells were cultured in DMEM supplemented with 30% FBS and 2.5 μM DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; D5942, Sigma-Aldrich, Buchs, Switzerland). After 48 h of treatment, cells were harvested and snap frozen in liquid nitrogen.
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