293 T and Hela cells were transfected with 2 μg wild type ESX1 or equivalent ESX1 variants using Lipofectamine 3000 18 (link). Twelve hours later, cell synchronization and releasing were performed as aforementioned. Then, cells were harvested and fixed with 70% ethanol overnight at − 20 °C. Samples were analyzed by flow cytometry (BD Accuri C6 Plus, BD Company) after staining with PI, complexed with RNase solution (Invitrogen, F10797). The data were analyzed using the FlowJo V10 analysis program (Treestar). FlowJo has a Cell Cycle Platform that uses multiple models to fit the data, constrains the fitting parameters, and automatically calculates the percentage of cells in the G1, S, or G2 phases. Briefly, the FCS data was dragged onto the interface. Then, cells along the most dense diagonal were gated to exclude potential debris or agglomerates via two serial gatings. Finally, the subpopulation was analyzed by selecting the Cell Cycle task. The ratios of phase distribution automatically generated by FlowJo from at least three independent experiments were analyzed statistically via GraphPad 5.
Rnase solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
The RNAse solution is a laboratory reagent designed to degrade ribonucleic acid (RNA) molecules. It functions by catalyzing the hydrolysis of RNA, thereby removing any unwanted RNA from a sample. The solution is commonly used in various molecular biology and biochemistry applications where the removal of RNA is necessary for downstream processing or analysis.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Thermo Fisher Scientific (6 mentions)
Accuri c6 plus, by BD (1 mentions)
Cold water fish gelatin, by Merck Group (1 mentions)
Flow cytometry, by BD (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Sytox green, by Thermo Fisher Scientific (1 mentions)
Macsquant, by Miltenyi Biotec (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
15 ml tube, by Corning (1 mentions)
Facsdiva software, by BD (1 mentions)
24 well plate, by Corning (1 mentions)
Modfit lt software, by Verity Software House (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Np 40, by Merck Group (1 mentions)
Np 40, by Thermo Fisher Scientific (1 mentions)
Ethanol, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
8 protocols using rnase solution
1
Cell Cycle Analysis of ESX1 Variants
2
Telomere Length Analysis by ImmunoFISH
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ImmunoFISH was performed with some changes to the protocol45 . Human VSMCs were grown on coverslips and fixed and permeabilized with a 1:1 methanol to acetone solution. Subsequently, cells were washed, incubated with blocking buffer (2% Cold Water Fish Gelatin (Sigma) and 5% BSA in PBS), and stained with primary or isotype-control antibodies overnight at 4 °C. Cells were then washed and incubated with secondary antibody for 1 h at RT. All primary and secondary antibodies were diluted using a blocking buffer. Following secondary antibody incubation, cells were washed once with blocking buffer before being re-fixed with methanol/acetone for 2 min, treated with 0.1 mg ml−1 RNase solution (Invitrogen), and incubated for 20 min at 37 °C. After dehydration in ice-cold 70%, 85%, and 100% ethanol, coverslips were air-dried prior to hybridization with a Cy3-TelC PNA probe, following the manufacturer’s instructions (PNA Bio inc). Next, cells were then washed with PBS, incubated with DAPI, and mounted for imaging with confocal microscopy.
3
Cell Cycle Analysis by Flow Cytometry
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After 72 h transfection, the cells were obtained via centrifugation, rinsed with Phosphate buffer saline (PBS) and added with 300 μl PBS for resuspension. Then, 700 μl 100% ethanol was added into each group for cell fixation at 4°C for 24–48 h. The cells were subsequently cleaned and stained using 100 μg/ml propidium iodide solution and 50 μg/ml RNase solution (Invitrogen, USA) at 37°C for 30 min avoiding light. Finally, flow cytometry (BD) was utilized for cell detection following the removal of cell clumps; then, CELL Quest and ModFit LT softwares were utilized to analyze the result [15 (link)].
4
Cell Cycle Analysis of Simvastatin Treatment
5
Yeast Cell Viability Assay Protocol
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For each of the 182 strains, cells from the frozen collection (temperature: −80 °C) were grown in tubes for 36 h at 30 °C under agitation in 3 mL of YPD medium (1% yeast extract, 2% peptone, 2% dextrose). We then collected 1 mL of culture (about 1 × 107 cells/mL) in 2 mL Eppendorf tubes; cells were collected by centrifuging (5 min at 3500 r.p.m.) and resuspended in 300 µL of sterile water. We slowly added 700 µL of pure ethanol, repeatedly inverted tubes and incubated overnight at 4 °C. After centrifuging 5 min at 3500 r.p.m., cells were washed once with 1 mL of sterile water, resuspended in 0.5 mL of RNase solution (40 µg/mL; Thermo Fisher) and incubated for 4 h at 37 °C. Then, cells were collected by centrifuging (5 min at 3500 r.p.m.) and resuspended in 0.5 mL of 50 mM Tris-HCl (pH 8.0). 50 µL of suspension were transferred in hemolysis tubes with 0.5 mL of SYTOX Green (Invitrogen) staining solution (1 µM SYTOX Green in 50 mM Tris-HCl buffer, pH 8.0). Finally, samples (60,000 cells) were analyzed using a MACSQuant (Miltenyi) flow cytometer, with a 488 nm laser to excite SYTOX Green and a bandpass filter 500–550 nm to detect fluorescence.
6
Cell Cycle Analysis of Calcitriol and Tacalcitol
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Cells (105 cells/well) were plated in 24-well plates (Corning Incorporated, Corning, NY, USA) in 2 mL of media. After 24 h, 10 nM of calcitriol and tacalcitol were added. Cells were harvested at 24, 48, 72, 96, and 120 h in 15 mL tubes (Corning Incorporated, Corning, NY, USA), centrifuged at 400× g at 4 °C for 10 min. The supernatant was removed, and cells were washed with PBS and counted. One million cells were resuspended in 0.7 mL of 70% cold ethanol and stored at −20 ° C for a minimum of 24 h. On the day of the cell cycle analysis, the 70% ethanol was removed by centrifuging the samples at 400× g at 4 °C for 10 min. Then the cell pellet was washed by adding 0.5 mL PBS and resuspended in an 8 µg/mL RNAse solution (ThermoFisher Scientific, Waltham, MA, USA), transferred to cytometer tubes (Corning Incorporated, Corning, NY, USA), and incubated for 1 h at 37 °C. After incubation, 50 µL of 0.1 mg/mL propidium iodide (ThermoFisher Scientific, Waltham, MA, USA) was added to the cells. After 30 min incubation at room temperature, samples were analyzed using a BD LSRFortessa flow cytometer using FACS Diva software (Becton Dickinson, San Jose, CA, USA). The percentage of cells in particular phases of the cell cycle was determined using ModFit LT software (Verity Software House, Topsham, ME, USA).
7
Cell Cycle Analysis with PTX and PD98059
8
Holmium-Based Nanoparticle Synthesis and Characterization
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Holmium acetate hydrate (Ho(CH3COO)3 • xH2O, Sigma Aldrich, 99.99%) and holmium nitrate hydrate (Ho(NO3)3•5H2O, Sigma Aldrich, 99.99%) were used as holmium precursors. Phosphoric acid (Sigma Aldrich, 85%) and sodium phosphate monobasic (NaH2PO4, Sigma Aldrich ≥99.0%) were used as phosphate source. The following polyols were used as solvents: butylene glycol (BG, Fluka, 99.5%), ethylene glycol (EG, Sigma Aldrich, 99.8%), glycerol (Gly, Sigma Aldrich, ≥99.5%) diethylene glycol (DEG, Sigma, ≥99%).Polyacrylic acid (PAA, Sigma Aldrich, Mw 1800) was used for the NPs functionalization. Phosphate Buffered Saline (PBS, Sigma Aldrich). 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT, Sigma Aldrich, ≥97.5%), 4′,6-diamidino-2-phenylindole (DAPI, Sigma Aldrich, ≥98%), and propidium iodide (PI, Sigma Aldrich, ≥94%). TO-PRO-3 Iodide and RNAse solution were purchased from Thermo Fisher. Dimethyl sulfoxide (DMSO, 99%)) was supplied by Acros organics.
Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), L-glutamine and penicillin/streptomycin solution were obtained from Gibco. All chemicals were used as received, except PBS, which was applied the following procedure: one tablet of PBS was dissolved in 200 mL water to obtain 137 mM NaCl, 2.7 mM KCl and 10 mM phosphate Buffer, pH 7.4 at 25 ºC).
Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), L-glutamine and penicillin/streptomycin solution were obtained from Gibco. All chemicals were used as received, except PBS, which was applied the following procedure: one tablet of PBS was dissolved in 200 mL water to obtain 137 mM NaCl, 2.7 mM KCl and 10 mM phosphate Buffer, pH 7.4 at 25 ºC).
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