According to the procedure previously outlined by Alqahtani et al. [48 (link)] and Nasr et al. [49 (link)], the cell cycle distribution was evaluated. PC-3 cells were cultured for 24 h after being treated with B. aegyptiaca extract at the previously estimated IC50 (92 µg/mL). The cells were then removed, thrice rinsed in cold PBS, fixed in cold ethanol (70%), and kept at 4 °C for four hours. PBS was used to rehydrate the fixed cells followed by the addition of RNase A (100 g/mL) and propidium iodide (100 g/mL, Abcam, Boston, MA, USA) for DNA staining. Using BD FACSCalibur flow cytometer with CellQuest software (BD Biosciences, San Diego, CA, USA), the DNA content was calculated after 30 min of incubation. propidium iodide fluorescence intensity was collected on FL2 of a flow cytometer and 488 nm laser excitation.
Rnase a
Manufactured by Abcam
Sourced in United States
RNase A is a ribonuclease enzyme that catalyzes the hydrolysis of single-stranded RNA. It cleaves the phosphodiester bonds of RNA, resulting in the production of 3'-phosphomononucleotides.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Abcam (3 mentions)
Facscan flow cytometer, by BD (3 mentions)
Mod fit lt software v2, by BD (3 mentions)
Dnase 1, by Promega (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Cellquest software, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Abcam (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Facscan, by BD (1 mentions)
Modfit lt version 2, by Verity Software House (1 mentions)
Phospho ampkα, by Cell Signaling Technology (1 mentions)
5 aminoimidazole 4 carboxamide ribonucleotide, by Merck Group (1 mentions)
Poly adp ribose polymerase parp, by Cell Signaling Technology (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Phospho acc, by Cell Signaling Technology (1 mentions)
Acetyl coa carboxylase, by Cell Signaling Technology (1 mentions)
P glycoprotein p gp, by Cell Signaling Technology (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Thermo Fisher Scientific (1 mentions)
11 protocols using rnase a
1
Cell Cycle Analysis of PC-3 Cells
2
Cell Cycle and Apoptosis Analysis by Flow Cytometry
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For cell-cycle analysis by flow cytometry, cells were harvested in 1× trypsin (Invitrogen) and centrifuged, then washed in PBS, and fixed in ice cold 70% ethanol overnight. Cells were washed again in 1× PBS before staining with 1× PBS containing 200 µg/mL RNAse A and 20 µg/mL propidium iodide (PI; Abcam) for 4 hours in the dark at 4°C. Stained cells were analyzed by flow cytometry on a Beckman Coulter CytoFlex flow cytometer to determine cell-cycle distribution. For cell-cycle analyses of YFP positive cells, cells were gated based on untransfected control cells and only YFP-positive cells analyzed for cell-cycle distribution using PI. DNA peaks resulting from PI staining were gated manually based on the n and 2n populations of control untreated samples and kept constant across repeat experiments. To determine the percentage of cells in apoptosis, cells were grown in 6 cm dishes. All cells were harvested and washed in fresh prewarmed complete growth media, and allowed to recover at 37°C 5% CO2 for 30 minutes. Cells were then pelleted by centrifugation and washed in PBS. Cells were stained using the Abcam Annexin-V FITC Apoptosis Detection Kit (Ab14085) according to manufacturer's instructions and incubated for 15 minutes at room temperature in the dark. Stained cells were analyzed by flow cytometry on a CytoFlex flow cytometer (Beckman Coulter).
3
HTLV-1 EV Subpopulations Modulate Immune Responses
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Various HTLV-1 EV subpopulations (2 k, 10 k, and 100 k HUT102 EVs; 1:10,000 cells) were subjected to RNase A (Abcam) or DNase I (Promega, M6101) digestion for 1 h at 37 °C to remove any DNA or RNA outside of EVs. The RNase A and DNase I enzyme activity in the EV samples were removed by addition of TF and cleaned over G-50 spin column. These EVs and untreated EVs (control) were used to treat astrocytes (CCF-STTG1) and mDCs (THP-1 cells + GM-CSF (50 ng/mL) + IL-4 (1000 U/mL) + TNF-α (20 ng/mL) + ionomycin (200 ng/mL)). Cells (5 × 105 cells/mL) were plated and treated with EVs at a ratio of 1:10,000. Supernatants were collected 5 days later and enriched for EVs using nanotrap nanoparticles (NT80/82). Western blot analysis was performed to assess the presence of proinflammatory cytokines (IL-8 and RANTES), GAPDH, and Actin.
4
Cell Cycle Analysis of RPMI8226 Cells
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RPMI8226 cells in the logarithmic phase were harvested and fixed in ice-cold 70% ethanol on ice for 20 min. The cells were rinsed in PBS, and stained using 20 mg/ml propidium iodide (PI) and 10 U/ml RNase A (Abcam) in a 37°C water bath for 30 min. Cell cycle distribution was determined using flow cytometry (FACScan; BD Biosciences) and the data were analyzed using ModFit LT™ version 2.0 (Verity Software House, Inc.) to determine the percentage of cells in each phase of the cell cycle.
5
Metabolic Regulation in Cancer Cells
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Fetal bovine serum (FBS), Dulbecco’s modified Eagle medium DMEM, and Dulbecco’s phosphate-buffered saline (DPBS) were obtained from Invitrogen (Carlsbad, CA, USA). Penicillin-streptomycin was purchased from GenDEPOT (Katy, TX, USA). Sodium butyrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide (MTT), and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) were supplied by Sigma-Aldrich (St. Louis, MO, USA). Antibodies against p21; cyclin A; cyclin D1; cyclin E; Bad; acetyl-CoA carboxylase (ACC); ATP citrate lyase (ACLY); fatty acid synthase (FASN); LC3B; Beclin-1; p62; phospho-AMPKα (Thr172); AMPKα; mammalian target of rapamycin (mTOR); phospho-mTOR (Ser2448); Akt; phospho-Akt (Ser473); phospho-ACC (Ser79); carnitine palmitoyltransferase 1A (CPT1); breast cancer resistance protein (BCRP); Bax; P-glycoprotein (P-gp); and poly ADP ribose polymerase (PARP) were purchased from Cell Signaling Biotechnology (Beverly, MA, USA). Actin, short-chain acyl-CoA dehydrogenase (SCAD), and p53 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Long-chain acyl-CoA dehydrogenase (LCAD) antibody, propidium iodide, and RNase A were purchased from Abcam (Cambridge, UK). All other chemicals and reagents were of analytical or HPLC grade and were used without further purification.
6
Evaluating Cell Cycle and Apoptosis after PDT
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For evaluating changes in cell cycle progression and PCD-induced DNA fragmentation after PDT, the cells were harvested 4 h after 5-ALA-PDT, fixed and permeabilized with 70% cold ethanol, and then stained with propidium iodide (PI) solution (50 µg/ml PI in PBS with 550 U/ml RNaseA; Abcam, USA). Cellular DNA content was analysed by flow cytometry using a BD FACSCalibur (BD Biosciences, San Jose, CA, USA).35 The data were analysed using FlowJo (FlowJo LLC, OR). Western blot analysis was conducted to detect PCD markers (cleaved PARP, pro-caspase 3 and cleaved caspase 3) using the apoptosis western blot cocktail (ab136812, Abcam).36 (link) The amount of cellular ROS was measured using the OxiSelectTM Intracellular ROS assay kit (Cell Biolabs Inc. San Diego, CA, USA) following the manufacturer’s instructions. Briefly, 5-ALA-PDT-treated cells were incubated with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) for 30 min at 37 °C, and the fluorescence was measured using a Synergy Mx Fluorescence plate reader (BioTek Instruments Inc., VT) at 480 nm/530 nm. The amount of ROS was determined by comparison with a 2′,7′-dichlorodihydrofluorescein (DCF) standard curve.
7
Cell Cycle Analysis by Flow Cytometry
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Harvested cells were fixed in 66% Ethanol at a concentration of 106 cells/ml for a minimum of 2 h at 4 °C. Cells were washed with phosphate buffer saline and incubated with 200 µl staining solution consisting of 50 µg/ml propidium iodide and 550 U/ml RNase A (Abcam) in the dark at 37 °C for 20 to 30 min. Measurements were taken at excitation wavelength of 488 nm using the FACSVerse flow cytometer (BD Biosciences). Gating was carried out using FlowJo 10.5.3 software (FlowJo) and calculated using the software’s Watson Pragmatic algorithm to correct for overlaps between the peaks.
8
Quantifying DNA in Extracellular Vesicles from HUT102 Cells
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DNA was isolated from 2 k (6.27 × 1011 particles/mL), 10 k (6.37 × 1011 particles/mL), and 100 k (8.47 × 1011 particles/mL) HUT102 EVs and HUT102 cell lysates (controls and standards) using the GoScript Reverse Transcription System (Promega) and concentration determined using NanoDrop 1000 Spectrophotometer (Thermo Scientific). EVs were evaluated in titration at volumes of 0.5, 1, and 2 μL for each EV. To evaluate if DNA bound to histones was present in the membrane of EVs or encapsulated, EV samples were treated with Proteinase K (Sigma, Cat# P4850) alone at 95 °C for 3 min, or with DNase I (Promega, Cat# M6101) and RNase A (Abcam, Cat# G117) at 37 °C for 1 h prior to addition of DNase Stop Solution (Promega, Cat# M199A) 65 °C for 5 min. DNA was isolated using the Wizard® Genomic DNA Purification Kit according to the manufacturer’s protocol (Promega Corporation) and quantified via qPCR using primers (IDT Technologies) specific for GAPDH (GAPDH-Forward 5′-TGT AGT TGA GGT CAA TGA AGG G-3′, Tm = 64.5 °C; and GAPDH Reverse 5′-ACA TCG CTC AGA CAC CAT G-3′, Tm = 64.5 °C). Serial dilutions of DNA from HUT102 cells were used as quantitative standards. PCR conditions were as follows: for GAPDH primers 50 °C for 2 min, 95 °C for 2.5 min, then 39 cycles of: 95 °C for 15 s, 50 °C for 30 s, and 72 °C for 40 s. The quantification of samples and reactions were carried out as described above.
9
Cell Cycle Analysis by Flow Cytometry
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Cell cycle analysis was performed using flow-cytometry. For fixation, 5×105 cells were incubated with 70% ice-cold ethanol at −20°C overnight. The next day, the fixed cells were centrifuged at 1,200 × g for 1 min at 4°C and washed twice with PBS. The cells were resuspended in 200 µl RNaseA (1 mg/ml) for 10 min at 37°C, 300 µl PI (BioVision, Inc.) was added, and the cells were incubated at room temperature for 20 min to stain the DNA. The cells were protected from light during the staining procedure. The cells were analyzed for cellular DNA content using Mod Fit LT software V2.0 (Becton Dickinson and Company) with a FACScan flow cytometer (Becton, Dickinson, and Company).
10
Flow Cytometry Cell Cycle Analysis
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Cell cycle analysis was performed by flow cytometry. To be more specific, 5×105 cells with 70% cold ethanol were cultured -20°C overnight. The following day, the fixed cells were centrifuged at 1,200 × g for 1 min at 4°C, and washed twice with PBS. The cells were treated with 200 μl RNase A (1 mg/ml) for 10 min at 37°C in suspension, following which 300 μl PI (100 μl/ml, BioVision, Inc.) was added to stain the DNA in cells in the dark. Following incubation at room temperature for 20 min, the cellular DNA content of the cells was analyzed in a FACScan flow cytometer (BD Biosciences) using ModFit LT software V2.0 (BD Biosciences).
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