Babco, Signet, Sternberger Monoclonals, Senetek, Covance

other

Helix NP™ Blue

425305

250 μL

211 USD

425305

Helix NP™ Blue

250 μL

211 USD

NONAB_CHMPRB

Helix NP™ Blue is a cell-impermeant nucleic acid probe suitable for use as a viability dye and for assessing DNA content and cell cycle status following cell fixation and permeabilization in flow cytometry. It can also be used as a viability dye in live cell imaging or as a nuclear counterstain on fixed and permeabilized cells and tissues. It is a blue-emitting dye with an excitation/emission max of 430 nm/470 nm that can be detected in the Brilliant Violet 421™ or Pacific Blue™ channel.Protocol for flow cytometric viability staining using Helix NP™ Blue:;Isolate cells following protocol of choice.;Optional For multicolor flow cytometry experiments, surface-stain cells as recommended. Helix NP Blue should be added as the last step prior to sample acquisition.;Note: The use of Helix NP for viability staining is incompatible with cell fixation and permeabilization protocols. If cells are to be fixed and permeabilized, use a fixable viability dye, such as a Zombie™ Fixable Viability kit.;;Dilute Helix NP to required concentration. We have observed good flow cytometric viability staining in a final concentration of 5 - 200 nM Helix NP Blue, but recommend titrating the reagent to determine the optimal concentration for cells of interest.;For example, to stain cells in a final concentration of 20 nM Helix NP Blue, prepare a 1:5000 dilution of the 5.0 mM stock in Cell Staining Buffer (or equivalent). Then, add 10 µL of diluted reagent to 500 µL of cell suspension.;;Do not wash the cells after adding Helix NP™ Blue. Samples are ready for acquisition.;Analyze cells on a cytometer equipped with a 405 nm violet laser.;Protocol for fixed cell cycle analysis using Helix NP™ Blue:;Isolate cells following protocol of choice.;Wash cells twice with phosphate-buffered saline (PBS). Using 70% ethanol (EtOH) chilled to -20°C, slowly add to cells while vortexing. Following the addition of 70% EtOH, continue vortexing for an additional 30 seconds. Incubate fixed cells at -20°C for a minimum of 1 hour. Prior to staining, wash cells once with phosphate-buffered saline (PBS), followed by another wash with Cell Staining Buffer (Cat. No. 420201).;Dilute Helix NP™ Blue to required concentration. We have observed good cell cycle analysis results using a final Helix NP Blue concentration of 0.2 - 20 nM, but recommend titrating the reagent to determine the optimal concentration for cells of interest.For example, to stain cells in a final concentration of 20 nM Helix NP Blue, prepare a 1:5000 dilution of the 5.0 mM stock in Cell Staining Buffer (or equivalent). Then, add 10 µL of diluted reagent to 500 µL of cell suspension.;Do not wash cells after adding Helix NP™ Blue. Samples are ready for acquisition.;Analyze cells on a cytometer equipped with a 405 nm laser.;Protocol for nuclear counterstaining fixed and permeabilized cell/tissue specimens using Helix NP™ Blue:;Fix specimens with 1%-4% Paraformaldehyde (PFA) for 10 minutes at room temperature.1% for cultured cells;4% for frozen tissue;Wash the cells/tissue two times with 1X PBS.;Permeabilize the cells/tissue with 0.5% Triton X-100 for 10 minutes at room temperature.;Wash the cells/tissue two times with 1X PBS.;Block cells with 5% fetal bovine serum for 30 minutes at room temperature.;Complete any additional blocking steps, antibody staining and washes prior to the addition of the Helix NP™ Blue counterstain.;Prepare the working solution.It is best to try a range of dye concentrations to determine the optimal concentration for cells/tissues of interest to achieve the best image. We have obtained good results using a starting range of 2.5 - 50 µM for IHC-F and 2.5 - 25 µM for IF/ICC.;Stain the cells/tissues with working solution for 20 minutes at 4°C or room temperature in dark.Protect from light prior to imaging.;No wash step is needed after staining.;Mount the slides with an antifade medium.;Image slides with a filter ideal for ex/em max of 430 nm/470 nm (either Alexa 488 or BV510 filter);