Product manual
Product use, preparation, operating conditions and storage.
Cell-Life DyeReagent kit
Cell-Life Dye 405/540
Cat. No.QH-66-6004
Stain viability before fixation to distinguish intact and membrane-compromised populations during subsequent surface or intracellular analysis.
Packs & quotePrepare cells and viability controls
Stain before fixation, protected from light
Wash and perform downstream staining
Excitation405 nm
Emission540 nm
TargetAccessible cellular protein amines and differences in membrane integrity
Preparation and storage
- Storage
- Store solid dye at −20°C, dry and protected from light; stable for at least one year from receipt. Aliquot DMSO stocks and store at −20°C with desiccant, protected from light.
Fixable viability staining
The dye covalently reacts with accessible protein amines. Intact cells mainly show lower surface labeling; compromised cells expose more intracellular proteins and stain more strongly. Stain before fixation and permeabilization, then validate separation through downstream processing.
Flow-cytometric viability exclusion
Stain viability before multicolor surface or intracellular staining to reduce artifacts from nonspecific antibody binding to dead cells.
Post-fixation analysis
For validated fixation and permeabilization workflows, with viability controls processed identically to the complete assay.
Application data
Jurkat cell staining example
Unfixed Jurkat cells; at 1 µL/mL, the percentage of events in gate P4 changes from 0.08% in the blank to 12.18%.
- Cell line: Jurkat
- Sample state: unfixed
- Cat. No.: QH-66-6004 · 405/540
Specifications
Product information
- Product name
- Cell-Life Dye 405/540
- Cat. No.
- QH-66-6004
- Product form
- Cell-Life Dye
- Target
- Accessible cellular protein amines and differences in membrane integrity
- Pack sizes
- 100T / 500T
Preparation and properties
- Excitation wavelength
- 405 nm
- Emission wavelength
- 540 nm
- Storage
- Store solid dye at −20°C, dry and protected from light; stable for at least one year from receipt. Aliquot DMSO stocks and store at −20°C with desiccant, protected from light.
- Reconstitution of a 100T vial
- 100 µL anhydrous DMSO
- Staining order
- Stain before fixation and permeabilization
Spectral information and detection
Product parameters
Use parameters for the exact catalog number when preparing reagents, setting reagent input or calculating results. Request a lot-specific CoA for test results.
Protocol
Protein-free staining medium
Use a suitable BSA- and serum-free buffer during viability staining; proteins and free amines can consume the dye. Wash before proceeding to protein-containing steps.
Reconstitution and amount
Reconstitute a 100T dye vial with 100 µL anhydrous DMSO. Titrate the working amount for cell count, cell type and the applicable pack.
Prepare cells and viability controls
Collect and wash gently, record cell count and prepare known viable and membrane-compromised controls.
Stain before fixation, protected from light
The Jurkat reference procedure uses 1×10⁶ cells/mL and 1 µL dye per mL for 30 minutes at room temperature or on ice, protected from light. Treat this as a starting assessment and titrate other cells.
Wash and perform downstream staining
Wash out free dye before surface staining, fixation and suitable permeabilization. Process complete controls identically.
Set the gates
Exclude debris and doublets, then set viability gates, compensation or unmixing with unstained, live/dead and single-stain controls.
Request product documentation
Select a document and enter your phone and email. Include the lot number for a CoA.
Cell-Life Dye 405/540QH-66-6004
Spectra and measurement conditions
Spectra, measurement conditions and detection settings for the exact catalog number.
Certificate of analysis (CoA)
Lot-specific test results. Include the lot number from the vial.
Safety data sheet (SDS)
Handling, storage and emergency response information.
Quality and lot information
Live and dead cells
Define negative and positive populations with known intact and compromised samples rather than moving the viability gate to fit each experimental distribution.
Fixation and permeabilization controls
Compare separation before and after downstream processing to verify compatibility of the complete workflow.
Multicolor single stains
Use cells stained with this dye or a validated compatible compensation substrate for compensation or unmixing.
References
Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry
Perfetto SP, Chattopadhyay PK, Lamoreaux L, et al. · Journal of Immunological Methods · 313(1–2):199–208 · 2006
Examines titration, live/dead separation and post-fixation detection with amine-reactive viability dyes, informing control design for fixable staining.
Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)
Cossarizza A, Chang HD, Radbruch A, et al. · European Journal of Immunology · 51(12):2708–3145 · 2021
Provides methodological guidance on detection settings, controls and experimental records for downstream conjugate evaluation.
Frequently asked questions
Can cells be fixed before staining?
Stain first to assess original viability. Fixation or permeabilization changes membrane integrity, so later staining cannot reconstruct the original state.
Why do live cells show weak signal?
Intact cells also have reactive surface amines. Assess live/dead separation together with dose, washing and handling.
Does fixable mean compatible with every protocol?
Fixative, permeabilization chemistry and storage time can affect signal. Validate the complete workflow.