Flow cytometry analysis was employed to detect the effect of PCs on the cell cycle of A549 cells. The cells were treated with DMSO and procyanidins (25, 50, 100 µM) for 24 hours. The trypsinized cells were washed with cold PBS, and the heart was centrifuged at 2,000 rpm for 5 minutes. The cells were slowly recovered with 50 µL of PBS, and then 950 µL of 75% ethanol was added to fix the cells at 4 °C overnight. The fixed cells were washed with cold PBS, centrifuged at 2,000 rpm for 5 minutes, and incubated with 500 µL propidium iodide (PI)/RNase staining buffer (BD Biosciences, Franklin Lakes, NJ, USA) for 15 minutes in the dark at room temperature. The above cells were detected by flow cytometry (BD-FACSAria II, Franklin Lakes, NJ, USA). The cell cycle was analyzed with Modfit-LT (VeritySoftwareHouse, USA), and the experiment was repeated three times.
Propidium iodide rnase staining buffer
Manufactured by BD
Sourced in United States
Propidium iodide/RNase staining buffer is a laboratory reagent used in flow cytometry applications. It is a solution that contains propidium iodide, a fluorescent dye that intercalates with DNA, and RNase, an enzyme that degrades RNA. The primary function of this buffer is to stain cellular DNA for the purpose of cell cycle analysis and DNA content quantification.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (7 mentions)
Facscalibur flow cytometer, by BD (5 mentions)
Facscanto 2, by BD (4 mentions)
Software, by FlowJo (4 mentions)
Accuri c6, by BD (3 mentions)
Facsdiva software, by BD (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (2 mentions)
Lsrfortessa x 20, by BD (2 mentions)
Facsaria illu flow cytometer, by BD (2 mentions)
Pe annexin 5 apoptosis detection kit, by BD (2 mentions)
Cellquest software, by BD (2 mentions)
Stain buffer, by BD (2 mentions)
Accuri c6 flow cytometer, by BD (2 mentions)
Pe annexin 5 apoptosis detection kit 1, by BD (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
Modfit lt, by Verity Software House (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Antibody for β actin, by Merck Group (1 mentions)
72 protocols using propidium iodide rnase staining buffer
1
Procyanidins Modulate Cell Cycle in A549 Cells
2
Erianin Induces Apoptosis and Autophagy
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Erianin was obtained from ChemFaces Biochemical (Wuhan, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and chloroquine were bought from Sigma-Aldrich (St. Louis, MO, USA). Ad-mCherry-p62 and Ad-mCherry-GFP-LC3B were purchased from Beyotime (Nantong, China). Apoptosis detection kit and propidium iodide (PI)/RNase staining buffer were purchased from BD Pharmingen (San Diego, CA, United States). Antibodies for Cyto C, caspase 9, cleaved caspase 3, GSDMD, CDC25C, CDC2, phospho-CDC2 (p-CDC2, Thr161), cyclin B1 and phospho-4EBP1 (p-4EBP1) were obtained from Cell Signaling Technology (Danvers, MA, United States); antibodies for PARP, NLRP3, 4EBP1, mTOR, phospho-mTOR (p-mTOR), RHEB, LC3B, beclin-1 and p62 were obtained from Proteintech Group (Wuhan, China); antibodies for cleaved-caspase 1, GSDME and PPT1 were from Abcam (Cambridge, MA, United States); and the antibody for β-actin was from Sigma-Aldrich. The secondary antibodies were purchased from Zhong Shan Golden Bridge Biotech Co. (Beijing, China).
3
Autophagy and Inflammation Modulation in Cells
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Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Lipofectamine™ 3000 transfection reagent, TRIzol reagent, LysoTracker Red and the high capacity cDNA reverse transcription kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Rapa and 3-MA were obtained from Sigma-Aldrich Co. (St Louis, MO, USA). CQ and BAY-11-7082 were obtained from Selleck (Houston, Texas, USA). Mouse anti-human monoclonal antibodies against PreS2/S, β-actin, and histone 3 were purchased from Abcam (C ambridge, UK). Anti-human monoclonal antibodies against LC3, Beclin-1, SQSTM/P62, NF-κB, p-NF-κB/p65, IκB, p-IκB, Vimentin, E-cadherin and Protein Disulfide Isomerase (PDI) were purchased from Cell Signaling Technology (Boston, MA, USA). The Immobilon Western Chemiluminescent HRP substrate was purchased from EMD Millipore (Billerica, MA, USA). The cell counting kit-8 was purchased from Beyotime (Shanghai, China). Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L), Alexa Fluor 594-conjugated goat anti-rabbit IgG (H + L), DyLight 405-labeled goat anti-mouse IgG (H + L) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (H + L) were purchased from ZSGB-BIO (Beijing, China). Propidium Iodide (PI)/RNase staining buffer was purchased from BD Biosciences (Franklin, NJ, USA).
4
Flow Cytometric Analysis of H. pylori-Induced DNA Damage
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GES-1 cells were cultured in four chamber slides and infected with H. pylori (100: 1 bacteria to host cell ratio, MOI=100) for 24 h. The bacterial media was removed and the cells were rinsed with PBS then, cells were harvested by trypsinization, washed once with PBS and pelleted. The pellet was resuspended by adding 70% ice-cold ethanol dropwise while vortexing. Cells were fixed overnight at −20 °C. The cells were incubated with primary phospho-γH2AX antibody (Millipore, Billerica, MA, USA; 05-636) 1:500 overnight at 4 °C. Following the incubation, cells were washed twice with PBS and incubated with anti-mouse secondary antibody conjugated to FITC 1:500 for 1 h at RT. Cells were washed twice with PBS and resuspended in 500 μl propidium iodide (PI)/RNase staining buffer (BD Pharmingen, Franklin Lakes, NJ, USA). Fluorescence was analyzed by flow cytometry using the BD FACSCalibur (Franklin Lakes, NJ, USA) and also by using FlowJo 8.8.6 software (Ashland, OR, USA).
5
Molecular Mechanisms of Autophagy Regulation
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SBI‐0206965 was from Selleck (Houston, TX, USA), and MRT68921 was from Sigma (St. Louis, MS, USA). ULK1 antibody (#8054), LC3B antibody (#3868), the cell‐cycle regulation antibody sampler kit II (#9870) and the horseradish peroxidase‐linked anti‐rabbit and anti‐mouse IgG were all from Cell Signaling Technology (Danvers, MA, USA). The anti‐β‐Tubulin, anti‐(glyceraldehyde 3‐phosphate dehydrogenase) and anti‐β‐actin Ig and TransStart FastPfu DNA Polymerase were from Beijing TransGen Biotech (Beijing, China). Nocodazole was from Selleck. Propidium iodide (PI)/RNase staining buffer was from BD Pharmingen (San Diego, CA, USA). The siRNAs were ordered from Genepharm (Shanghai, China), and primers were from Sangon Biotech (Shanghai, China). HiPerFect was from Qiagen (Dusseldorf, Germany). Protease inhibitor and phosphatase inhibitor cocktails were from Roche (Basel, Switzerland), and the polyvinylidene fluoride membrane was from Millipore (Billerica, NJ, USA). GlutaMAX was from Gibco (Carlsbad, CA, USA). The secondary fluorescently conjugated antibodies, Antifade ProLong Gold, were from Molecular Probes (Eugene, OR, USA). M‐MLV Reverse Transcriptase and TRIzol were from Invitrogen (Waltham, MA, USA). Prestained Protein Ladder (26616) and M‐PER were from Thermo Pierce (Waltham, MA, USA). CellTiter‐Glo (#G7570) was from Promega (San Luis Obispo, CA, USA).
6
Obatoclax Mesylate Induced Apoptosis
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Obatoclax mesylate (OBAT) was purchased from Selleck Chemicals (Munich, Germany). The OBAT was dissolved in DMSO and stored as 5 mM stock solutions at −20 °C. RPMI 1640 medium and fetal calf serum were obtained from GIBCO BRL Life Technologies (Gaithersburg, MD, USA). L-glutamine, antibiotic antimycotic solution (AAS), dimethyl sulfoxide (DMSO), NBT, phorbol 12-myristate 13-acetate (PMA) and anti-β-actin antibody (cat. #A5316) were purchased from Sigma Aldrich ( St. Louis, MO, USA). The FITC Annexin V Apoptosis Detection Kit I, propidium iodide (PI)/RNase staining buffer and anti-CD-11b were obtained from BD Biosciences (San Jose, CA, USA). The CellEvent™ Caspase 3/7 Green Flow Cytometry Assay kit was purchased from Molecular Probes (Eugene, OR, USA). The Hemacolor® Rapid Staining kit was obtained from Merck Millipore (Darmstadt, Germany). The antibody against Bcl-2 (cat. #2876) and horseradish peroxidase-conjugated secondary antibody (cat. #7074) were purchased from Cell Signaling Technology (Danvers, MA, USA).
7
Cell Apoptosis and Cycle Analysis
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Flow cytometric analyses of apoptosis and cell cycle were performed with Annexin V-FITC Apoptosis Detection Kits and propidium iodide (PI)/RNase staining buffer (BD Biosciences, USA), respectively, according to the manufacturer’s instructions.
8
Cell Cycle Analysis of Treated Cell Lines
9
Cell Cycle Analysis of Lung Cancer Cells
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Cells were resuspended in RPMI-1640 at a density of 3×105 cells/well in 6-well plates (Costar; Corning Incorporated). Drug exposure was conducted after culturing for 24 h and at 1% FBS. H1975 cells were treated with BFD (0–30 mg/ml) or MBFD (0–15 mg/ml) for 24 h; H292 cells were treated with BFD (0–30 mg/ml) or MBFD (0–20 mg/ml) for 24 h at 37°C in an atmosphere containing 5% CO2. Cells were then collected, fixed with cold 75% ethanol and stored at −20°C for ≥24 h. Subsequently, after washing twice with cold PBS, cells were incubated with propidium iodide (PI)/RNase staining buffer (BD Biosciences, San Diego, CA, USA) for 15 min at room temperature. DNA content was analyzed by flow cytometry using BD Accuri C6 (version 1.0.264.15; BD Biosciences). The percentage of cells in the various phases of the cell cycle was determined using ModFit LT 4.1 software (BD Biosciences).
10
Evaluation of Paclitaxel and Birinapant Effects
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Paclitaxel, sulforhodamine B sodium salt (SRB), trichloroacetic acid, dimethylsulfoxide (DMSO), and Tris were from Sigma-Aldrich (St. Louis, MO). Propidium iodide (PI)/RNase Staining Buffer and the AnnexinV-phycoerythrin (PE)/7-aminoactinomycin D (7-AAD) Apoptosis Detection Kit were from BD Pharmingen (San Diego, CA). The ACCUTASE cell detachment solution was from EMD Millipore (Temecula, CA). Birinapant was a gift of TetraLogic Pharmaceuticals (Malvern, PA). Stock solutions of PTX (10 mM) and BRP (30 mM) were prepared in DMSO and stored at − 20 °C until use. When diluted to final concentrations in cell culture medium, the DMSO concentration was below 0.1% (v/v) and did not perturb cell growth.
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