Helix NP™ Green

Product Details

Preparation

Helix NP™ Green is supplied at 250 µl per vial.

Concentration

5.0 mM

Storage & Handling

Upon receipt, store at -20°C.

Application

FC - Quality tested
IF, IHC-F - Verified

Recommended Usage

For use in viability assays, the optimal concentration can range from 1 - 10 nM. For immunofluorescence microscopy and immunohistochemical staining on frozen tissue sections, the suggested use of this reagent is 0.5 - 10 µM. It is recommended that the reagent be titrated for optimal performance for each application.

Application Notes

Helix NP™ Green is a cell-impermeant nucleic acid probe suitable for use as a viability dye in flow cytometry. It can also be used for viability in microscopy on live cells or as a nuclear counterstain on fixed and permeabilized cells and tissues. It is a green-emitting dye with an excitation/emission max of 495 nm/519 nm that can be detected in the Alexa Fluor® 488 or FITC channel.

Protocol for flow cytometric viability staining using Helix NP™ Green:

1. Isolate cells following protocol of choice.
2. Optional— For multicolor flow cytometry experiments, surface-stain cells as recommended. Helix NP Green 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.
3. Dilute Helix NP Green to required concentration. We have observed good flow cytometric viability staining in a final concentration of 1 - 10 nM, but recommend titrating the reagent to determine optimal concentration for cells of interest.
   • For example, to stain cells in a final concentration of 10 nM Helix NP Green, prepare a 1:5000 dilution of the 5.0 mM stock in Cell Staining buffer (or equivalent). Then, add 5 µL of diluted reagent to 500 µL of cell suspension.
4. Do not wash the cells after adding Helix NP™ Green. Samples are ready for acquisition.
5. Analyze cells on a cytometer equipped with a 488 nm blue laser.

Protocol for nuclear counterstaining fixed and permeabilized cell /tissue specimens using Helix NP™ Green:

1. Fix specimens with 1%-4% Paraformaldehyde (PFA) for 10 minutes at room temperature.
   • 1% for cultured cells
   • 4% for frozen tissue
2. Wash the cells/tissue two times with 1X PBS.
3. Permeabilize the cells/tissue with 0.5% Triton X-100 for 10 minutes at room temperature.
4. Wash the cells/tissue two times with 1X PBS.
5. Block cells with 5% fetal bovine serum for 30 minutes at room temperature.
6. Complete any additional blocking steps, antibody staining, and washes before adding the Helix NP™ Green counterstain.
7. 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 range of 0.5 - 10 µM for IHC-F and IF/ICC).
8. Stain the cells/tissues with the working solution for 20 minutes at 4°C or room temperature in the dark.
   • Protect from light prior to imaging.
   • No wash step is needed after staining.
9. Mount the slides with an antifade medium.
10. Image slides with a filter ideal for ex/em max of 495 nm/519 nm (Alexa Fluor® 488 or FITC channel.

Antigen Details

Biology Area

Apoptosis/Tumor Suppressors/Cell Death, Cell Biology, Cell Cycle/DNA Replication, Cell Proliferation and Viability

Gene ID

NA

Documentation

• Technical Data Sheet (pdf)
• Certificate of Analysis
• Safety Data Sheet

Reviews

Related Pages & Pathways

Related FAQs

Certificate of Analysis