Pi rnase staining solution

Manufactured by Cell Signaling Technology

Sourced in United States

PI)/RNase Staining Solution is a laboratory reagent used to stain and identify cellular DNA. It contains propidium iodide (PI), a fluorescent dye that binds to DNA, and RNase, an enzyme that degrades RNA, allowing for the specific detection of DNA in cell samples.

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Lab products found in correlation

Facsaria 2 cytometer, by BD (5 mentions) Annexin 5 apoptosis detection kit, by BD (3 mentions) Propidium iodide, by Cell Signaling Technology (3 mentions) Crystal violet, by Merck Group (2 mentions) Facscalibur, by BD (2 mentions) Facsdiva 6, by BD (2 mentions) Facscalibur flow cytometer, by BD (2 mentions) Apc annexin 5 apoptosis detection kit, by BD (1 mentions) Fitc annexin 5 apoptosis detection kit, by BD (1 mentions) Facscanto 2 flow cytometer, by BD (1 mentions) Facscelesta cell analyzer, by BD (1 mentions) Annexin 5 fitc, by Thermo Fisher Scientific (1 mentions) Alexa fluor 647 anti h2a x phosphorylated ser139 antibody, by BioLegend (1 mentions) Facscanto 2 cytometer, by BD (1 mentions) Facs caliber flow cytometer, by BD (1 mentions) Cellquest pro, by BD (1 mentions) Facs accuri c6 plus, by BD (1 mentions) Alexa fluor 488 conjugated annexin 5, by Thermo Fisher Scientific (1 mentions) Hoechst 33342, by Cell Signaling Technology (1 mentions) Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

PI staining solution (2 citations)

41 protocols using pi rnase staining solution

Apoptosis and mitochondrial function analysis

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For annexin V APC staining the APC annexin V Apoptosis Detection Kit (BD Pharmingen, U.S.A.) was used according to the manufacturer's instructions. For PI staining, cells were resuspended in 300 μl PBS and fixated by adding 1000 μl ice-cold ethanol prior to incubation over night at 4ºC. Then the cells were centrifuged at 1800 rpm, the supernatant was removed and 400 μl PI/RNase staining solution (Cell signaling technology, Danvers, MA, U.S.A.) were added prior to incubation for 15 min at RT and flow cytometric analysis.
To detect intrinsic apoptosis staining for JC-1 was performed according to the manufacturer's instructions using the MitoProbe^TM JC-1 assay kit for flow cytometry (Molecular Probes^®, Life Technologies) [38 (link)]. The data were analysed with the FlowJo software (version 8.7.1; Tree Star, Ashland, OR, U.S.A.).

Karpel-Massler G., Shu C., Chau L., Banu M., Halatsch M.E., Westhoff M.A., Ramirez Y., Ross A.H., Bruce J.N., Canoll P, & Siegelin M.D. (2015). Combined inhibition of Bcl-2/Bcl-xL and Usp9X/Bag3 overcomes apoptotic resistance in glioblastoma in vitro and in vivo. Oncotarget, 6(16), 14507-14521.

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Apoptosis and Cell Cycle Analysis

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Lymphoma primary cells and cell lines were treated with GSK-591 and Venetoclax. After 72 h, apoptosis and cell death were determined using FITC Annexin V apoptosis detection kit according to the manufacturer’s instruction (BD Biosciences, cat. 556547).
To determine the effect of GSK-591 and Ro combination on cell cycle, cell lines were incubated with these compounds for 72 h. 1 ×10⁶ cells were collected, fixed with 70% ethanol and then stained with propidium iodide (PI)/RNase staining solution (Cell Signaling Technology, cat.4087) at room temperature for 15 minutes. Flow cytometric data were acquired using a FACSCalibur (BD Biosciences, San Jose, CA). Greater than 10,000 events were acquired. Doublets were excluded by gating out high FL3-W (width) cells.

Erazo T., Evans C.M., Zakheim D., Chu K.L., Refermat A.Y., Asgari Z., Yang X., Da Silva Ferreira M., Mehta S., Russo M.V., Knezevic A., Zhang X.P., Chen Z., Fennell M., Garippa R., Seshan V., de Stanchina E., Barbash O., Batlevi C.L., Leslie C.S., Melnick A.M., Younes A, & Kharas M.G. (2022). TP53 mutations and RNA-binding protein MUSASHI-2 drive resistance to PRMT5-targeted therapy in B-cell lymphoma. Nature Communications, 13, 5676.

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Cell Cycle Analysis of Colon Cancer Cells

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Sensitive and chemo-resistant colon cancer cells were seeded into six-well plates and then exposed for 72 h to testing agents. Thereafter, cells were harvested by trypsinization and fixed with 70% ice-cold ethanol. After washing twice with 1X ice-cold PBS, cells were resuspended in propidium iodide (PI)/RNase staining solution (Cell Signaling Technology; Danvers, MA) for 30 min in the dark at 37 °C. Cell cycle progression was analyzed on a Becton–Dickinson FACScanto II flow cytometer and further analyzed with BD FACSDiva 6 software (Becton–Dickinson). The PI fluorescence signal at FL2-A peak versus counts was used to determine cell cycle distribution and the data were analyzed using the Modfit software.

Riahi-Chebbi I., Souid S., Othman H., Haoues M., Karoui H., Morel A., Srairi-Abid N., Essafi M, & Essafi-Benkhadir K. (2019). The Phenolic compound Kaempferol overcomes 5-fluorouracil resistance in human resistant LS174 colon cancer cells. Scientific Reports, 9, 195.

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Cell Cycle Analysis by Flow Cytometry

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To determine the effect of ASN007 treatment on cell cycle, cell lines were cultured with or without the ERK1/2 inhibitor for 24 to 72 hours. Cells were collected and then stained with propidium iodide (PI)/RNase staining solution (Cell Signaling, #4087) at room temperature for 15 minutes. Flow cytometric data was acquired on a BD FACSCalibur (BD Biosciences, San Jose, CA) using CellQuest Pro Version 6.0. Propidium iodide was excited by the 488 nm laser and fluorescence emission was measured in fluorescence parameter 3 (FL3) – with the standard 670LP filter. Greater than 10,000 events were acquired. Data Analysis: Doublets were excluded by gating out high FL3- W (width) cells.

Portelinha A., Thompson S., Smith R.A., Da Silva Ferreira M., Asgari Z., Knezevic A., Seshan V., de Stanchina E., Gupta S., Denis L., Younes A, & Reddy S. (2021). ASN007 is a selective ERK1/2 inhibitor with preferential activity against RAS-and RAF-mutant tumors. Cell Reports Medicine, 2(7), 100350.

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Cell Cycle Analysis of NF1-MPNST Cells

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Modulation of the cell cycle was determined in NF1-MPNST cells treated with DMSO, TNO155, ribociclib, or their combination for 48 hours, following incubation with culture medium containing 0.1% FBS for 24 hours to synchronize cells. After trypsinization, cells were fixed in ice-cold 70% ethanol for at least 30 minutes and were then labeled with propidium iodide (PI)/RNase staining solution (#4087, Cell Signaling Technology) and further incubated for 15 minutes at 37°C. Finally, cells were analyzed using the FACSCelesta^™ Cell Analyzer (BD Biosciences). Data analysis was performed using FlowJo 10.8, and cell cycle distribution was assigned by using the implemented models.

Wang J., Calizo A., Zhang L., Pino J.C., Lyu Y., Pollard K., Zhang X., Larsson A.T., Conniff E., Llosa N., Wood D.K., Largaespada D.A., Moody S.E., Gosline S.J., Hirbe A.C, & Pratilas C.A. (2023). CDK4/6 inhibition enhances SHP2 inhibitor efficacy and is dependent upon restoration of RB function in malignant peripheral nerve sheath tumors. bioRxiv.

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Live Cell Imaging and Cell Viability Analysis

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Live-cell imaging was performed using a Leica SP8 confocal microscope with spectral detection and an HC FLUOTAR L 25×/0.95 VISIR water immersion objective. Dead cells or cells with compromised plasma membranes were stained by adding 200 μL of propidium iodide (PI)-RNase staining solution (200 μL PI solution to 300 μL of serum free cell culture medium) (Cell Signaling, Inc.; catalog no. 4087S) for 5 min at room temperature prior to imaging. PI labels only cells with compromised plasma membranes and dead cells. Such cells were excluded from the analysis. PI was excited at 561 nm, and emission was detected between 600 and 700 nm. GFP was excited at 488 nm, and emission was detected between 500 and 550 nm. Fluorescence and transmission images were acquired simultaneously. Maximum-intensity projections of acquired z-stacks (sampling of 0.08 by 0.08 by 5 μm) were generated using the open-source software Fiji (96 (link)).

Thapa H.B., Kohl P., Zingl F.G., Fleischhacker D., Wolinski H., Kufer T.A, & Schild S. (2023). Characterization of the Inflammatory Response Evoked by Bacterial Membrane Vesicles in Intestinal Cells Reveals an RIPK2-Dependent Activation by Enterotoxigenic Escherichia coli Vesicles. Microbiology Spectrum, 11(4), e01115-23.

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Cell Cycle Analysis after JQ1 Treatment

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Cell cycle analysis was performed 24 hours after JQ1 treatment. Cells were fixed in cold ethanol and resuspended in Propidium Iodide (PI)/RNase Staining Solution (Cell Signaling Technology). After incubation for 15 minutes at room temperature in the dark, flow cytometric analysis was performed on a FACS AriaII cytometer (BD Biosciences). Flow cytometry data was analyzed by using FlowJo software and the cell cycle was plotted as histogram after excluding doublets.

Zhang Z., Ma P., Jing Y., Yan Y., Cai M.C., Zhang M., Zhang S., Peng H., Ji Z.L., Di W., Gu Z., Gao W.Q, & Zhuang G. (2016). BET Bromodomain Inhibition as a Therapeutic Strategy in Ovarian Cancer by Downregulating FoxM1. Theranostics, 6(2), 219-230.

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Cell Cycle and Apoptosis Analysis

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Cell cycle analysis was performed 24 hours after drug treatment. Cells were fixed in cold ethanol, resuspended in Propidium Iodide (PI)/RNase Staining Solution (Cell Signaling Technology) and incubated for 15 minutes at room temperature in the dark. For apoptosis analysis,cells were digested and collected with trypsin without EDTA, washed with PBS, incubated with Annexin V-FITC (Life Technologies) in room temperature for 15 minutes in dark and then incubated with PI for another 5 minutes. Flow cytometric analysis was performed on a FACS AriaII cytometer (BD Biosciences). Flow cytometry data was analyzed by using FlowJo software and the cell cycle was plotted as histogram after excluding doublets.

Jing Y., Zhang Z., Ma P., An S., Shen Y., Zhu L, & Zhuang G. (2015). Concomitant BET and MAPK blockade for effective treatment of ovarian cancer. Oncotarget, 7(3), 2545-2554.

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Phospho-H2A.X Flow Cytometry Protocol

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For flow cytometry analyses a minimum of 10⁵ cells from each condition was washed with PBS and then fixed in pre-cooled 70% absolute ethanol and incubated at −20°C for 60 min. Fixed cells were washed 3 times with cold PBS and then stained with Alexa Fluor 647 anti-H2A.X-phosphorylated (Ser139) antibody (no. 613408; BioLegend, Inc., San Diego, CA, USA). Finally, the cells were washed once with PBS and resuspended in 50 μl of propidium iodide (PI)/RNase staining solution (no. 4087; Cell Signaling Technology, Inc.) 15 min before fluorescence acquisition with BD FACS Canto II cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Guenat D., Merla G., Deconinck E., Borg C, & Rohrlich P.S. (2017). DNA damage response defect in Williams-Beuren syndrome. International Journal of Molecular Medicine, 39(3), 622-628.

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Cell Cycle Analysis of ECA-109 Cells

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A total of 1×10⁶ ECA-109 cells treated with 0, 20, 40 or 80 mg/l COE for 24 h were harvested, and then fixed in 70% ethanol at 4°C for 2 h. After 24 h, the cells were washed twice with ice-cold PBS, stained with enough neat propidium iodide (PI)/RNase Staining Solution (Cell Signaling Technology, Inc., Danvers, MA, USA) at 25°C for 15 min in the dark. The measurements were performed using a FACSCaliber flow cytometer and Cell Quest Pro software version 349226 (BD Biosciences, Franklin Lakes, NJ, USA). The data were analyzed using FlowJo 7.6 software (Tree Star, Inc., Ashland, OR, USA). The tests were performed ≥3 independent times.

Jin F., Zhu G., Li D., Ni T., Dai X., Wang H., Feng J., Qian Y., Yang L., Guo S., Hisamitsu T, & Liu Y. (2017). Celastrus orbiculatus extracts induce cell cycle arrest and apoptosis in human esophageal squamous carcinoma ECA-109 cells in vitro via the PI3K/AKT/mTOR signaling pathway. Oncology Letters, 15(2), 1591-1599.

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