P. aeruginosa (108/mL) treated with 272 μM Nano-MgB2 (12.5 μg/mL) or 544 μM H3BO3 + Mg2+ for 3 h were stained with SYTO9 (Thermo Fisher Scientific Cat 2266591 6 μM) and Propidium iodide (Beyotime Cat ST511, 15 μM) for 15 min at room temperature in the dark. For S. aureus, S. aureus (1 × 108) were incubated with 10.88 mM Nano-MgB2 (500 μg/mL) or 21.76 mM H3BO3 + Mg2+ for 3 h. The live and dead cells were visualized with confocal laser microscopy (Nikon A1 + R-980). For the Flow cytometry assay, bacteria treated with Nano-MgB2 and H3BO3 + Mg2+ for 3 h were added with 15 μM Propidium iodide and incubated for 30 min in the dark. Flow cytometry was performed using FACScan (BD LSRFortessa X-20). The gating strategy for the Flow cytometry was listd as follows. First, using Forward Scatter Area (FSA) and Side Scatter Area (SSA) to find the main group of bacteria; second, using SSA and SSH(Side Scatter High) to remove adherent bacterial populations; last, using SSA and PE to display the positive cell population of dead bacteria (Supplementary Fig. 23 ).
St511
Manufactured by Beyotime
Sourced in China
The ST511 is a laboratory equipment product. It serves as a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Syto9, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Sta 350, by Cell Biolabs (1 mentions)
Ts2 s sm, by Nikon (1 mentions)
Facscalibur flow cytometry, by BD (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Ab137346, by Abcam (1 mentions)
6 well plate, by Corning (1 mentions)
Ab150077, by Abcam (1 mentions)
A1r hd25 confocal microscope, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole dapi c1002, by Beyotime (1 mentions)
Rnase a, by Takara Bio (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Quanteon flow cytometer, by Agilent Technologies (1 mentions)
9 protocols using st511
1
Antibacterial Efficacy of Nano-MgB2 and H3BO3+Mg2+
2
Measuring DNA Damage using Comet Assay
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DNA damage in individual cells was measured by the comet assay or single-cell gel electrophoresis assay (STA-350, Cell Biolabs). Briefly, porcine SCs were digested by trypsin, washed twice with ice-cold PBS, and then resuspended at 1 × 105 cells/ml in ice-cold PBS. Then, comet agarose was pipetted onto the OxiselectTM comet slide to form a base layer; cells were mixed with Oxiselect comet agarose at 37°C, and then the mixture was pipetted on top of the base layer. The slide was soaked in prechilled lysis buffer for 1 h at 4°C in the dark, and after lysis, the slides were treated with alkaline electrophoresis solution for 30 min and electrophoresed for 15 min under alkaline conditions at 4°C. Porcine SCs were stained with propidium iodide (ST511, Beyotime) for 15 min in the dark, and slides were viewed by epifluorescence microscopy (TS2-S-SM, Nikon). DNA damage was quantified by measuring the displacement between the genetic material of the nucleus (“comet head”) and the resulting “tail”. Tail moment was the parameter selected to analyze the comet assay results in this study and was measured by Open Comet in ImageJ software.
3
Annexin V and Propidium Iodide Staining
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Cells at a density of 1 × 106/ml were suspended in the binding buffer (A21009‐100 ml, Alpha Applied Bioscience), subsequent to which FITC Annexin V (5 μl; C1062S, Beyotime Biotechnology) and 1 μl propidium iodide (100 μg/ml; ST511, Beyotime Biotechnology) solution and the binding buffer (300 μl) were cultured with the cell suspension for 15 min at room temperature. Finally, a FACS Calibur TM flow cytometry (BD, Franklin Lakes) was adopted for the analysis of results.
4
Cell Cycle Analysis After KIF18B Overexpression
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The cell cycle after transfection of KIF18B overexpression plasmid was assessed by a flow cytometer (CytoFLEX, Beckman, USA). In short, the transfected cells were cultured until its confluence is over 70%. Subsequently, cells were collected, fixed with 70% ethyl alcohol (459836, Sigma‐Aldrich, USA) for 24 h, and then stained with propidium iodide (ST511, Beyotime, China) at 37°C for 30 min. Then, the flow cytometer was employed for calculation and analysis.
5
Cardiomyocyte Death Assessment Protocol
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Human-induced pluripotent stem cell-derived cardiomyocytes were separated and placed in 6-well plates (Corning, New York, USA). The combined staining of α-actinin (ab137346, Abcam), immunoglobulin G (IgG) H&L (Alexa Fluor® 488) (ab150077, Abcam), and propidium iodide (PI) 1 μg/ml (ST511, Beyotime, Shanghai, China) was used to detect cardiomyocyte death. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) (C1002, Beyotime, Shanghai China) and the dead cells were labeled with PI to pass through the damaged cell membrane. A Nikon A1R HD25 confocal microscope was used to capture images. The total number of cells in the PI-positive and five randomly selected fields was counted using ImageJ software by a researcher blinded to the treatment assignments. A manual pipeline (CellProfiler, Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard in Cambridge) was used to determine the cell surface area (22 (link)). Briefly, 5 to 6 random pictures were taken with 20X magnification and 150–200 cells/per condition were analyzed in order to determine cell size following the instructions of the software.
6
Cell Cycle Analysis by Flow Cytometry
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Cells (2×106/mL) were fixed with 70% cold ethanol overnight, then incubated in ice-cold PBS containing 50 mg RNase A (Takara, Japan) at 37°C for 1 hour. Cells were treated with propidium iodide at the final concentration of 5μg/ml (ST511, Beyotime Biotechnology, Hangzhou, China), and incubated for 20 minutes at room temperature. Then, cells were subjected to flow cytometry for analysis.
7
Investigating SARS-CoV-2 ORF3a Impact on MHV-A59 Infection
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MHV-A59 was propagated by infecting 17Cl-1 cells at a multiplicity of infection (MOI) of 0.05. To infect 17Cl-1 cells, the viral inoculum in DMEM was incubated with cells for 1 h with occasional swirling. The inoculum was then removed and replaced with fresh DMEM plus 10% fetal bovine serum. The titer of MHV-A59 was determined by plaque assay on monolayer 17Cl-1 cells. All titrations were performed in duplicate by incubating cells with serially diluted cell culture supernatants for 36 h. Plaques were counted and calculated as plaque forming units (PFU) per milliliter of supernatant. To determine the effect of SARS-CoV-2 ORF3a on MHV-A59 production, 17Cl-1 cells were transfected with pEGFP-C1 vector or pEGFP-ORF3a plasmid. At 24 h post-transfection, cells were infected with MHV-A59 at a MOI of 3. Supernatants were collected at 16 h post-infection and virus production was quantified by plaque assay.
For propidium iodide (PI) staining, 17Cl-1 cells were cultured on glass-bottom dishes (801001, NEST) and transfected with the indicated plasmids. At 24 h post-transfection, cells were infected with MHV-A59 at a MOI of 3. After 16 h infection, cells were treated with 10 μg/ml propidium iodide (ST511, Beyotime) and 1 μg/ml Hoechst 33342 (C1022, Beyotime).
For propidium iodide (PI) staining, 17Cl-1 cells were cultured on glass-bottom dishes (801001, NEST) and transfected with the indicated plasmids. At 24 h post-transfection, cells were infected with MHV-A59 at a MOI of 3. After 16 h infection, cells were treated with 10 μg/ml propidium iodide (ST511, Beyotime) and 1 μg/ml Hoechst 33342 (C1022, Beyotime).
8
DAPI/PI Staining for Cell Death Analysis
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4',6‐diamidino‐2‐phenylindole/propidium iodide (DAPI/PI) double satining was performed to evaluate cell death.13 RAW264.7 cells were suspended with 1 mL cell staining buffer, followed by staining with 5 μL DAPI solution (C1005; Beyotime) and 5 μL PI staining solution (ST511; Beyotime), at 4°C for 30 min. Finally, the stained cells were observed under a fluorescence microscope (TE2000; Nikon).
9
DNA Content Analysis by FACS
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DNA content was analyzed with fluorescence-activated cell sorting (FACS). For the FACS analysis, the cells were first fixed with cold 70% ethanol and kept on ice for >30 min before pelleting at 12,000 rpm. Cells were then resuspended in 50 mM sodium citrate with 10 µg/mL RNase A for over 3 h. DNA was then stained with 2 μg/mL propidium iodide (PI) (Beyotime Biotechnology, ST511) before brief sonication. At least 20,000 cells were acquired per sample on a Quanteon flow cytometer (ACEA Biosciences) for PI detection (FL2-A). DNA content is shown on a linear scale after gating for single cells in FlowJo (version 10) software.
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