FxCycle™ Violet stain (F10347) is used for flow cytometric analysis of DNA content in fixed cells. This dye is excited with a 405⁄407 nm laser with emission collected in the 450⁄50 BP filter or equilavent. This dye is DNA selective so RNAse is not required. With multicolor cell cycle studies possible using flow cytometry, it is often necessary to analyze DNA content on alternative lasers to preserve the common 488 nm laser and detection channels for other markers. Well suited for the popular violet laser line, FxCycle™ Violet stain is a good choice for DNA content analysis in multicolor cell cycle studies. |
| 图1.Dual parameter plot of Click-iT® EdU Alexa Fluor® 488 and FxCycle™ Violet.Jurkat (human T-cell leukemia) cells were treated with 10 µM EdU for 2 hours and detected according to the recommended staining protocol using Alexa Fluor® 488 azide. Cells were then stained with FxCycle™ Violet Stain (F10347). Data was collected and analyzed using an Attune® Acoustic Cytometer using 488 nm excitation and a 530/30 bandpass filter for detection of the EdU Alexa Fluor® 488 azide and 405 nm excitation and 450/40 bandpass filter for detection of the FxCycle™ Violet fluorescence. Cells in region 1 (dual positive cells) are in the S-phase, cells in region 2 are in G0/G1 phase, and cells in region 3 are in G2/M phase of the cell cycle. 图2.Histogram of HL-60 promyeloblast cells stained with FxCycle™ Violet stain showing DNA content distribution.HL-60 cells were fixed overnight with alcohol, washed, and then resuspended in 0.1% Triton® X-100/PBS/1% BSA before staining with FxCycle™ Violet stain for 30 minutes at room temperature. G0/G1 and G2/M phase histogram peaks areseparated by the S-phase distribution. Analysis was performed at 405 nm excitation with a 450/50 bandpass filter. |
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