Helix NP™ Blue

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Other Names
HelixBlue
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A_HelixNP_Blue_FC_1_051617
One-day-old C57BL/6 mouse splenocytes were stained with Helix NP™ Blue (filled histogram) at 5nM. Cells alone, without Helix NP™ Blue staining, are also shown (open histogram).
  • A_HelixNP_Blue_FC_1_051617
    One-day-old C57BL/6 mouse splenocytes were stained with Helix NP™ Blue (filled histogram) at 5nM. Cells alone, without Helix NP™ Blue staining, are also shown (open histogram).
  • B_HelixNP_Blue_ICC_3_051617
    HeLa cells were fixed with 1% paraformaldehyde (PFA) for 10 minutes, permeabilized with 0.5% Triton X-100 for 10 minutes, and blocked with 5% FBS for 30 minutes. Then the cells were intracellularly stained with 5 µg/mL of Alexa Fluor® 647 anti-Cytokeratin (pan reactive) (clone C-11) antibody (red) in blocking buffer overnight followed by 25 µM of Helix NP™ Blue (green) and Flash Phalloidin™ Red 594 (blue) staining for 15 minutes at room temperature. The image was captured with a 60X objective using the Alexa Fluor® 488 filter.
  • C_HelixNP_Blue_IHCF_4_051617
    Mouse frozen cerebellum tissue was fixed with 4% paraformaldehyde (PFA) for 10 minutes, permeabilized with 0.5% Triton X-100 for 10 minutes, and blocked with 5% FBS for one hour. Then the tissue was intracellularly stained with 5 µM of Helix NP™ Blue (blue) 15 minutes at room temperature and co-stained with Flash Phalloidin™ Red (red). The image was captured with a 10X objective using the Alexa Fluor® 488 filter.
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Description

Helix NP™ Blue is a cell-impermeant nucleic acid stain suitable for use as a viability dye in a live cell sample and assessing DNA content and cell cycle following cell fixation and permeabilization.  It can also be used as a nuclear counterstain in immunocyto- and immunohistochemistry. It is a blue fluorescent dye with an excitation/emission max of 430 nm/470 nm that can be the Pacific Blue™ / Brilliant Violet 421™ channels in flow cytometry. For use in viability assays, the optimal concentration can range from 5nM - 50nM. For use in cell cycle, the optimal concentration can range from 0.2nM - 20nM.  To ensure the brightest signal when used in counterstaining the nuclei of fixed and permeabilized cell specimens, the spectra of Helix NP™ Blue should be matched to the ideal filter specifications on the microscope. The optimal concentration for this application can range from IHC-F: 2.5 μM - 50 μM and IF/ICC: 2.5μM - 25 μM.

Product Details
Cat # Size Price Quantity Avail. Save
425305 250 µl $180.00
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Technical data sheet

Product Details

Preparation
Helix NP™ Blue is supplied at 250 µl per vial.
Concentration
5.0 mM
Storage & Handling
Upon receipt, store at -20°C.
Application

FC - Quality tested
ICC, IHC-F - Validated

Recommended Usage

For flow cytometric viability staining, the suggested use of this reagent is 5-50nM. For flow cytometric fixed cell cycle analysis, the suggested use of this reagent is 0.2-20nM. For immunocytochemistry, the suggested use of this reagent is 2.5-25 µM. For immunohistochemical staining on frozen tissue sections, the suggested use of this reagent is 2.5-50 μM.

Application Notes

Protocol for viability staining using Helix NP™ Blue: 

  1. Isolate cells following protocol of choice.
  2. Dilute Helix NP™ Blue to required concentration. We recommend titrating the reagent to determine optimal concentration for cells of interest. We have observed good results in flow cytometry at 5-50 nM.
  3. Following the addition of Helix NP™ Blue to cells, do not wash, cells are ready for acquisition.
  4. Analyze cells on a cytometer equipped with the 405nm violet laser.


Protocol for fixed cell cycle analysis using Helix NP™ Blue: 

  1. Isolate cells following protocol of choice.
  2. Wash cells twice with phosphate buffered saline (PBS). Using 70% ethanol (EtOH) chilled to -20°C, slowly add to desired cells while vortexing. Following the addition of 70% EtOH, continue vortexing for an additional 30 seconds. Incubate fixed cells in -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).
  3. Dilute Helix NP™ Blue to required concentration. We recommend titrating the reagent to determine optimal concentration for cells of interest. We have observed good results in the 0.2-20nM range.
  4. Following the addition of Helix NP™ Blue to cells, do not wash. Cells are ready for acquisition.
  5. Analyze cells on a cytometer equipped with the 405nm laser.


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

  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 prior to the addition of the Helix NP™ Blue counterstaining.
  7. Prepare the working solution.
    • In initial experiment, it may be the best to try a range of dye concentration to determine optimal concentration for cells/tissues of interest in order to achieve the best image (suggesting range: 2.5 μM -50 μM for IHC-F and 2.5μM -25 μM for IF/ICC).
  8. Stain the cells/tissues with working solution for 20 minutes at 4°C or room temperature in dark.
    • Make sure to protect from light prior to imaging.
    • No wash step needed after staining.
  9. Mount the slides with an antifade medium.
  10. Image slides with a filter ideal for ex/em max of 430nm/470nm (either Alexa 488 or BV510 filter)

Antigen Details

Gene ID
NA

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Go To Top Version: 1    Revision Date: 05/17/2017

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