BGC-823 and SGC-7901 cells were collected by centrifugation at low speed for five minutes, followed by adequate washing with phosphate buffer, and then, the cells were fixed with 70% ethanol, incubated for 12 h at 4°C in an ice bath, and stained with Ribonuclease A (ST576, Beyotime, Shanghai, China) and PI (P4170, Sigma Aldrich, USA) staining buffer. The cells were suspended in staining solution (P4170, Sigma Aldrich, MS, USA), and the cell cycle was analyzed by flow cytometry (BD, Biosciences, NJ, USA). The Modfit software (Verity Software House, GA, USA) provided the estimation of the percentage of cells in G0/G1, S, and G2/M phases of the cycle.
P4170
Manufactured by Merck Group
Sourced in United States, Germany, Spain
The P4170 is a specialized laboratory equipment product. It serves a core function within the research and development process, though the specific intended use is not provided.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (13 mentions)
Facscalibur, by BD (11 mentions)
Rnase a, by Merck Group (7 mentions)
F7378, by Merck Group (7 mentions)
B2261, by Merck Group (5 mentions)
Facsdiva software, by BD (3 mentions)
Hoechst 33342, by Merck Group (3 mentions)
G2025, by Merck Group (3 mentions)
Modfit lt, by Verity Software House (3 mentions)
Dm4000 led, by Leica (3 mentions)
Axiovert 40 cfl, by Zeiss (3 mentions)
H3570, by Thermo Fisher Scientific (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Axio observer a1, by Zeiss (3 mentions)
Sp8 confocal microscope, by Leica (2 mentions)
Dylight 488, by Abcam (2 mentions)
R5125, by Merck Group (2 mentions)
Fc500 mpl, by Beckman Coulter (2 mentions)
Bal amca, by Biotrend (2 mentions)
Bp0352, by Biotrend (2 mentions)
151 protocols using p4170
1
Cell Cycle Analysis of BGC-823 and SGC-7901 Cells
2
Confocal Imaging of Cell Death
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Confocal images were taken after 30 hours (MEC1/OVA) or 48 hours (B16F10/OVA) of coculture by automated spinning disc confocal microscopy (BD Pathway 855 High-Content Bioimager; Olympus UApo/340 20x/0.75). For GFP signal detection, the 488/ 10 excitation filter and 520/35 emission filter were used. For propidium iodide (PI; Sigma-Aldrich, P4170) signal detection, the 548/20 excitation filter and 645/75 emission filter were used. To discriminate dead from live cells, PI (10 mL/well; 0.5 mg/mL in PBS; Sigma-Aldrich, P4170) was automatically added 20 minutes before imaging.
3
High-Throughput Live Cell Imaging Assay
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After 72 hours of treatment with the drugs, the cells were stained with Hoechst (BDBiosciences #BD 561908) and Propidium Iodide (Millipore Sigma #P4170) at a final concentration of 1 μg/ml and 2 μg/ml to stain all cells and dead cells, respectively. After 30 minutes, the wells were imaged using the TagBFP (Excitation-390/18 nm, Emission-447/60 nm) and RFP filters (Excitation-531/40 nm, Emission-593/40 nm) in a Cytation 5 (Biotek). Each image was flatfield corrected and background subtracted using CellProfiler. The nuclei were then identified using the IdentifyPrimaryObjects feature, and a pseudo image was generated. The number of all counts of cell nuclei stained with Hoechst and Propidium Iodide were compiled by CellProfiler and exported as csv files (provided in the code). The Propidium Iodide-stained nuclei counts were subtracted from the Hoechst stained nuclei counts for each well, and this was taken as the live cell counts which were the primary data.
4
Cdc42-Deficient Enteroid Assay
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Enteroid cultures from Cdc42-deficient or Cdc42-WT mice were conducted based on previously described methods (119 (link)). To induce Cdc42 deletion in iKO enteroids, 100–200 WT or Cdc42iKO crypts were seeded in Matrigel with ENR medium and allowed to develop for 3 days. On day 4, 500 nM 4-OHT was added to the enteroids for 24 hours. Viable enteroids were counted under a bright-field microscope 1–7 days after 4-OHT treatment. For propidium iodide staining, enteroids were incubated with 500 nM propidium iodide (P4170, MilliporeSigma) in PBS solution at 37°C for 10 minutes and washed with PBS before imaging. For 3D cell viability assay, 100 organoids were seeded in each well on a 96-well plate and cultured in ENR medium for 4 days before addition of 4-OHT. Cell viability was determined typically at 72 hours after 4-OHT treatment using the Cell Titer-Glo 3D Cell Viability Assay (G9681, Promega) by Glomax system (E9032, Promega).
5
Hippocampal Tissue Sampling After PI
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PI (P4170, MilliporeSigma, 1 mg/mL dissolved in saline) was injected into bilateral ventricles of mice. By 2 hours after PI injection, mice were anesthetized by an i.p. injection of 10% chloral hydrate, and hippocampal tissue samples were frozen at –80°C or fixed in 4% paraformaldehyde for further analysis.
6
Cell Cycle Analysis of Drug-Treated LN229 Cells
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LN229 cells (5 × 105) were seeded in culture plates with complete medium, and after 24 h, they were treated with 6 nM 9 , 100 nM 12 , 400 nM 18bNP, and 100 μM 21bNP and incubated for a further 24 h in standard conditions. Cells were harvested, centrifuged, and treated with ice-cold 70% w/v ethanol at 4 °C for 20 min. Then, cells were washed twice with phosphate buffer saline (PBS) and resuspended in a final volume of 300 μL of PBS containing 0.1 μg mL−1 RNAse (Merck R6513) and 36 μg mL−1 propidium iodide (PI, Merck P4170). The analysis of the DNA content was performed by FACSAria III flow cytometry, and the data were analyzed by BD FACSDiva software.
7
Cell-Cycle Profile Analysis by PI Staining
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Cell‐cycle profile was evaluated by propidium iodide (PI) staining and fluorescence‐activated cell sorting (FACS) analysis as described previously.33 , 34 Briefly, cells were treated with MLN4924 or dimethyl sulfoxide (DMSO) then fixed with 70% ethanol at −20°C overnight, stained with PI (36 μg/mL; Sigma‐Aldrich, P4170, Merck KGaA, Darmstadt, Hesse, Germany) containing RNase A (10 μg/mL; Sigma‐Aldrich, R6513) at 37°C for 15 min and analysed for cell‐cycle profile by CyAn ADP (Beckman Coulter, Brea, CA, USA). Data were analysed with ModFit LT software (Verity Software House, Topsham, ME, USA).
8
Cytotoxic Effects of Compound on A2780 Cells
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A2780 cells (2 × 105) were seeded in culture dishes with complete medium and incubated at 37°C in a 5% CO2 humidified atmosphere. After 24 h, the cells were treated with the test compound at 0.1, 0.2, and 0.3 μM concentrations and incubated for 48 h in standard conditions. Cells were then harvested, centrifuged, and 3 × 105 cells for each sample were treated with ice-cold 70% w/v ethanol at 4°C for 20 min. Then, cells were washed twice with PBS and resuspended in a final volume of 300 μL PBS containing 0.1 mg mL−1 RNAse (Merck R6513) and 36 μg mL−1 PI (Merck P4170). The analysis of the DNA content was performed by the FACSAria III flow cytometer, and data were analyzed by BD FACSDiva software.
9
Live/Dead Cell Staining Assay
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Before live/dead staining, channels were washed three times using 100 μL PBS+. Afterwards, 100 μL of staining solution with 27 μg mL -1 of fluorescein diacetate (FDA; F7378, Sigma-Aldrich) for labeling viable cells and 135 μg mL -1 of propidium iodide (PI; P4170, Sigma-Aldrich) for labeling dead cells in PBS+ was filled into the channels. The staining solution was incubated for 5 min at 37 °C. Chips were washed three times using 100 μL PBS+ and imaged immediately. Images of stained HUVECs were acquired at 37 °C using a fluorescence microscope with heated enclosure (Leica DMi8, Leica Microsystems).
10
Cell Viability Assay with PI
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After being seeded in 24-well cell plates, cells were treated with PD at the indicated concentrations and times. Propidium iodide (PI) (Sigma-Aldrich, P4170) was added at a final concentration of 50 μg/mL to measure the dead cells with damaged cell membrane integrity.
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