For Ki67-Hoechst assays, CD34+ cells with or without editing were first stained with fixable viability stain 660 (1:1000, BD) for 5 min in 37° C and were fixed with Cytofix/Cytoperm buffer (BD) for 15 min in 4° C. Cells were stained with FITC-anti-KI67 (1:25, BD, 556027) for 2hours-overnight in Permwash buffer (BD), then with Hoechst 33342 (1:5000; Life Technologies) for 5 min at RT. Samples were sorted on a Aria Fusion Cell Sorter (BD) or analyzed on a LSR Fortessa cytometer (BD). For assessment of immunophenotypic markers together with cell cycle analysis, human CD34+ cells with or without editing were stained with Percp-Cy5.5-anti-CD34 (1:50) and PE-Cy7-anti-CD38 (1:50) for 30 min in 4° C before they were stained with fixable viability stain and fixed. For assessment of cell cycle status without fixation (live cell cycle status), cells with or without editing were stained with Hoechst 33342 (1:1000, Invitrogen) for 45min in 37° C, and then were stained with Pyronin Y (1:20,000, Invitrogen) for additional 15 min in 37° C or were just stained with Pyronin Y for 15 min in 37° C. Samples were then sorted on Aria Fusion Cell Sorter (BD) or analyzed on LSR Fortessa cytometer (BD).
Pyronin y
Manufactured by Thermo Fisher Scientific
Pyronin Y is a fluorescent dye used in biological and biochemical applications. It is a nucleic acid stain that binds to RNA, allowing for the visualization and quantification of RNA in various samples.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (7 mentions)
Pyronin y, by Merck Group (2 mentions)
Annexin 5 apc pi apoptosis kit, by MultiSciences Biotech (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Hoechst 33342 h3570, by Thermo Fisher Scientific (1 mentions)
P1304mp, by Thermo Fisher Scientific (1 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
Lsrfortessa, by BD (1 mentions)
Anti ki67 fitc, by BD (1 mentions)
Fixable viability stain 660, by BD (1 mentions)
Lsr fortessa cytometer, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Cytofix cytoperm buffer, by BD (1 mentions)
Aria fusion cell sorter, by BD (1 mentions)
6 protocols using pyronin y
1
Ki67-Hoechst Assay for Cell Cycle Analysis
2
Comprehensive Cell Surface Profiling
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The PE-conjugated CD133 antibody (Clone 7), APC-conjugated CD44 antibody (IM7), and PE-conjugated H-2Dd antibody (34-2-12) were obtained from eBioscience. To stain cell surface antigens, 1 × 106/ml cells were stained with 5 μg/ml each antibody at 4 °C for 30 min. For cell cycle analysis, 5 × 105 cells were fixed in 70% ethanol at −20 °C for 1 h. After that, cells were stained with 2 μg/ml Hoechst33342 (Thermo Fisher Scientific) and 4 μg/ml Pyronin Y (Thermo Fisher Scientific) for 1 h. For Ki67 staining, 1 × 106/ml cells were fixed in 3% paraformaldehyde for 15 min, followed by incubation in 90% methanol-PBS for 30 min. Cells were then stained with 5 μg/ml APC-conjugated Ki-67 antibody (SolA15, eBioscience). For apoptosis detection, cells were treated with the Annexin V-APC/PI apoptosis kit (Multisciences) based on the supplier's brochure. A BD FACSCalibur™ Flow Cytometer (BD Biosciences) was used for analysis.
3
Cell Cycle Analysis of Organoids
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The cell cycle was assessed by RNA and DNA staining. Organoids were harvested at 7 days post seeding and dissociated to a single-cell suspension. Cells were fixed for least 2 h using 70% ethanol. Cells were then stained (50 μg/ml propidium iodide, P1304MP, ThermoFisher Scientific; 4 μg/ml Pyronin Y, 418630010, ThermoFisher Scientific; 2 μg/ml Hoechst 33342, H3570, ThermoFisher Scientific) and the cell cycle phase was assessed using a BD Fortessa flow cytometer and FlowJo software.
4
DAPI and Pyronin Y Staining of Embryos
5
Myoblast Cell Cycle Analysis
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Freshly isolated human myoblasts or human 48 h-MuRC were resuspended in PBS at a concentration of 1 × 106 cell/ml and incubated first with Hoechst 33,342 at 5 μg/ml (Thermo Fisher) for 45 min at 37 °C to avoid non-specific binding of pyronin Y to DNA. Cells were then incubated with pyronin Y (Sigma-Aldrich) at 1 μg/ml for 45 min at 37 °C and then kept at 4 °C until flow cytometry acquisition using a BD LSRFortessa.
6
Hematopoietic Stem Cell Isolation
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Antibodies CD34 and CD38 were obtained from Beckton Dickinson (Breda, the Netherlands). For Hoechst and Pyronin Y staining cells were washed and resuspended in HPGM, stained in 5 μg/ml Hoechst 33342 (Invitrogen) at 37°C for 45 minutes after which 1.0 μg/ml Pyronin Y (Sigma) was added at 37°C for an additional 45 minutes. Cells were washed in the solution containing Hoechst and Pyronin Y, followed by FcR blocking at 4°C for 10 minutes. After staining with CD34-APC and CD38-Alexa700 at 4°C for 20 minutes, cells were washed and analyzed. All FACS analyses were performed on an LSRII (Becton Dickinson) or MACSquant (Milenyi Biotech) flowcytometer and data was analyzed using WinList 3D (Topsham, USA) or FlowJo (Tree Star, Oregon, USA) software.
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