Melanoma cells (A375) were seeded with 1 × 105 cells per well in a 6-well plate, incubated for 24 h, and treated with derivative 4j and sorafenib for 48 h. Then, adherent and floating cells were collected from the cultures, washed twice with PBS (phosphate buffered saline), fixed in ice-cold 60% ethanol at 40 °C, and re-washed in PBS. Subsequently, the cells were resuspended in 500 μL propidium iodide (PI) with RNase staining buffer, BD (Franklin Lakes, NJ, USA), and incubated for 30 min. Lastly, FACS analyses were performed utilizing an ACEA Novocyte™ flow cytometer (ACEA Biosciences Inc., San Diego, USA). For every sample, the data from 12 000 cells were collected and the distribution of cell cycle phases was analyzed using the ACEA Novo Express™ software (ACEA Biosciences Inc., San Diego, USA).64 (link)
Rnase staining buffer
Manufactured by BD
Sourced in United States
RNase staining buffer is a solution used in molecular biology applications to detect the presence of RNase enzymes. It is designed to provide a consistent and reliable method for visualizing RNase activity in samples.
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Lab products found in correlation
9 protocols using rnase staining buffer
1
Cell Cycle Analysis of Melanoma Cells
2
Cell Cycle Analysis by Flow Cytometry
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Cells were harvested, washed twice in PBS, resuspended in 70% ethanol overnight, and then diluted in propidium iodide, RNase staining buffer (BD Pharmingen) and incubated for 15 min at 37 °C. Samples were analyzed with flow cytometry (BD LSRFortessa Cell analyzer) and Flowjo software (FLOWJO, LLC).
3
Cell Cycle Analysis by Flow Cytometry
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Cells were trypsinized, resuspended and fixed in 70% ethanol at 4 °C for 30 min. Cells were then rinsed by PBS, resuspended and incubated in PI and RNase staining buffer (BD) at 37 °C for 30 min. Cell cycle distribution was analysed in BD FACSCalibur flow cytometer.
4
Cell Cycle Analysis of DOX-resistant Breast Cancer
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DOX-resistant
breast cancer cells (MCF-7ADR) were treated with the precalculated
IC50 of hypophyllanthin (PN4 ) and phyllanthin
(PN5 ) alone or in combination with DOX for 48 h. Then,
the cells were harvested by trypsinization, twice washed with PBS
(phosphate-buffered saline), fixed in ice-cold 60% ethanol at 40 °C,
and re-washed in PBS. After that, the cells are resuspended in 500
μL of propidium iodide (PI) with RNase staining buffer, BD (Franklin
Lakes, NJ, USA), and incubated for 30 min. Last, FACS analyses were
executed utilizing the ACEA Novocyte flow cytometer (ACEA Biosciences
Inc., San Diego, USA). For every sample, data from 12,000 cells were
collected, and the distribution of cell cycle phases was analyzed
using the ACEA Novo Express software (ACEA Biosciences Inc., San Diego,
USA).26 (link)
breast cancer cells (MCF-7ADR) were treated with the precalculated
IC50 of hypophyllanthin (
(
the cells were harvested by trypsinization, twice washed with PBS
(phosphate-buffered saline), fixed in ice-cold 60% ethanol at 40 °C,
and re-washed in PBS. After that, the cells are resuspended in 500
μL of propidium iodide (PI) with RNase staining buffer, BD (Franklin
Lakes, NJ, USA), and incubated for 30 min. Last, FACS analyses were
executed utilizing the ACEA Novocyte flow cytometer (ACEA Biosciences
Inc., San Diego, USA). For every sample, data from 12,000 cells were
collected, and the distribution of cell cycle phases was analyzed
using the ACEA Novo Express software (ACEA Biosciences Inc., San Diego,
USA).26 (link)
5
Cell Cycle Analysis by Flow Cytometry
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The cells were collected at pre-determined intervals, washed twice in phosphate-buffered saline, and fixed in 75% ethanol at 4 °C overnight. Ethanol was removed, and the cells were stained with propidium iodide in RNase staining buffer (BD Pharmingen, San Diego, CA, USA) for 15 min. The BD Biosciences Accuri C6 flow cytometer detected the cell cycle and analyzed it by the Modfit software (LT 5.0, Verity Software House Inc, Topsham, ME, USA).
6
Metformin Induces Apoptosis and Cell Cycle Arrest
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Cell apoptosis detection kit (propidium iodide (PI), RNase staining buffer and FITC-labeled Annexin V) were purchased from BD Pharmingen (San Diego, CA, USA). Cells were seeded 2.5 × 105 per well in 6-well plates for 24 h. Then the medium was replaced by culture medium containing metformin 0, 20 or 40 mM for 24 or 48 h. The cells were harvested for analysis of cell cycle and apoptosis, respectively. The cell cycle was analyzed using PI staining, according to the manufacturer’s instructions. Briefly the cells were fixed in 70 % ethanol, stained with PI, and the amount of PI-labeled DNA in a cell was measured by a flow cytometer (Accuri C6, Becton Dickinson, San Jose, CA, USA). The acquired data were analyzed by FlowJo software (Ashland, OR, USA). To determine the apoptotic cells, the cells were stained with Annexin V-FITC and PI immediately after harvesting, and analyzed by flow cytometry, as described by the manufacturer’s instructions.
7
Cell Cycle Analysis by Flow Cytometry
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Cell cycle progression was assessed 48 h after siRNA transfection by flow cytometry. Cells were detached from the plates using trypsin-EDTA and subsequently treated with 0.2% Triton X-100 and stained with propidium iodide with RNase staining buffer (BD Biosciences, San Jose, CA, United States). Flow cytometry data were acquired using a BD Accuri C6 plus flow cytometer (BD Biosciences) to assess DNA content in at least 10000 cells.
8
Cell Cycle Analysis by PI Staining
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The cells were collected and fixed overnight in 75% cold ethanol at -20 ℃. Then the cells were washed twice with cold phosphate-buffered saline solution and labeled with propidium iodide (PI) /RNase Staining Buffer (BD, Cat# 550825) according to the manufacturer’s instructions. The PI labeled cells were analyzed immediately after staining with a flow cytometer.
9
Cell Cycle Analysis by Flow Cytometry
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The cells were collected and xed overnight in 75% cold ethanol at -20 ℃. Then the cells were washed twice with cold phosphate-buffered saline solution and labeled with propidium iodide (PI) /RNase Staining Buffer (BD, 550825) according to the manufacturer's instructions. The PI labeled cells were analyzed immediately after staining with a ow scan ow cytometer.
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