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Technical Specification: Cell Staining Reagents

1. Product Range

Solarbio cell staining reagents cover essential fluorescent probes for nuclear counterstaining, DNA content analysis, viability discrimination, and mitochondrial health assessment. These probes are validated for use in fluorescence microscopy, confocal microscopy, flow cytometry, and high-content imaging platforms.

Product SKU Ex/Em (nm) Application Format
DAPI (4′,6-diamidino-2-phenylindole) D1060 358/461 Nuclear counterstain, DNA content 5 mg/mL in DMSO, 1 mL
Hoechst 33258 H1398 352/461 Live cell nuclear stain 10 mg/mL in DMSO, 1 mL
Hoechst 33342 H1399 350/461 Live cell nuclear stain (membrane-permeant) 10 mg/mL in DMSO, 1 mL
PI (Propidium Iodide) P8080 535/615 Dead cell stain, DNA content (cell cycle) 1 mg/mL in PBS, 1 mL
JC-1 Mitochondrial Membrane Potential Assay M8650 514/529 (monomer), 585/590 (aggregate) Apoptosis, mitochondrial health 200× in DMSO, 0.5 mL
Calcein-AM CA1330 495/515 Viable cell stain (esterase activity) 1 mM in DMSO, 100 μL
FITC-Phalloidin CA1610 495/520 F-actin cytoskeleton labeling 50 μg, lyophilized

2. Spectral Properties and Working Concentrations

2.1 Nuclear Stains

Stain Stock Solution Working Concentration Excitation Source Staining Pattern
DAPI 5 mg/mL in DMSO (store -20°C, dark) 0.5–1 μg/mL 358 nm (UV, 405 nm laser) Nucleus; AT-rich regions brighter
Hoechst 33342 10 mg/mL in DMSO (store -20°C, dark) 1–10 μg/mL 350 nm (UV, 355 nm laser) Nucleus; live cell permeable (30 min labeling)
Hoechst 33258 10 mg/mL in DMSO 1–10 μg/mL 352 nm Nucleus; less permeable than 33342
PI (viability) 1 mg/mL in PBS (store 2–8°C, dark) 1–5 μg/mL 535 nm (488 nm laser, >585 nm emission) Nucleus; only permeabilized/dead cells

2.2 Functional Probes

Probe Stock Working Dilution Incubation Application Notes
JC-1 200× in DMSO (store -20°C, dark) 1× in culture medium 15–30 min at 37°C Red/green ratio measures ΔΨm depolarization
Calcein-AM 1 mM in DMSO (store -20°C, dark) 0.5–5 μM 15–30 min at 37°C Non-fluorescent until cleaved by intracellular esterases
FITC-Phalloidin Reconstitute in 1.5 mL methanol (store -20°C) 1:40–1:200 20–40 min at RT (dark) Requires fixation (4% PFA) and permeabilization

3. Dye Mechanism Details

DAPI

DAPI (C₁₆H₁₅N₅·2HCl, MW 350.25) binds non-fluorescently to the minor groove of dsDNA, with strong preference for AT-rich sequences (binding constant ~10⁶ M⁻¹). Upon binding, its fluorescence quantum yield increases approximately 20-fold.

DAPI (free, low fluorescence)  +  dsDNA  →  DAPI-DNA complex (20× brighter)
                                            λmax ex: 358 nm, λmax em: 461 nm

JC-1

JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) is a cationic carbocyanine dye that accumulates in the mitochondrial matrix in proportion to the mitochondrial membrane potential (ΔΨm). At low concentrations (depolarized mitochondria), JC-1 exists as green-fluorescing monomers (Ex/Em 514/529 nm). At high concentrations (polarized mitochondria, ΔΨm > -140 mV), JC-1 forms J-aggregates with red fluorescence (Ex/Em 585/590 nm). The ratio of red to green fluorescence provides a sensitive, ratiometric measure of ΔΨm.

JC-1 (monomer, green)  ←→  JC-1 (J-aggregate, red)
  (depolarized ΔΨm)            (polarized ΔΨm > -140 mV)

Ratio: A₅₉₀ / A₅₂₉ — decrease indicates mitochondrial depolarization

4. Multiplex Compatibility

Primary Probe Compatible Counterstain Notes
FITC-phalloidin (green) DAPI (blue) Minimal spectral overlap
Calcein-AM (green) PI (red) Standard live/dead assay
JC-1 (green/red) None needed Ratiometric; avoid co-staining with overlapping spectra
FITC-labeled antibodies DAPI IF + nuclear counterstain
GFP-expressing cells PI or DAPI Cell cycle + reporter analysis
Alexa Fluor 555/594 DAPI Fixed cell imaging

Nuclear Staining (Fixed Cells)

Step Detail
Fixation 4% paraformaldehyde, 15 min at RT
Permeabilization 0.1–0.5% Triton X-100 in PBS, 10 min at RT
Wash 3× PBS, 5 min each
DAPI staining Add 300 nM DAPI (0.5 μg/mL) in PBS, 5 min at RT
Wash 1× PBS, 5 min
Mount Anti-fade mounting medium; seal coverslip
Image DAPI filterset (Ex 340–380 nm, Em 435–485 nm)

JC-1 Mitochondrial Membrane Potential Assay

Step Detail
Seed cells 1×10⁵ cells/mL, 200 μL culture medium (96-well or chamber slide)
Treatment Add experimental agents (e.g., FCCP 10 μM as positive control, 30 min)
Prepare JC-1 Dilute 200× stock to 1× in pre-warmed culture medium (37°C)
Load cells Replace medium with 100 μL 1× JC-1; incubate 15–30 min at 37°C, 5% CO₂
Wash 2× with warm PBS or culture medium
Read Fluorescence plate reader: Ex 485 nm, Em 535 nm (monomer); Ex 560 nm, Em 595 nm (aggregates)
Calculate Ratio = F₅₉₀ / F₅₂₉

Live/Dead Cell Viability Assay

Step Detail
Prepare staining solution Calcein-AM (1:1000 = 1 μM final) + PI (1:1000 = 1 μg/mL) in PBS
Stain cells Add solution to cells; incubate 15–30 min at RT, dark
Wash 1× with PBS
Image Calcein-AM (Ex 495 nm, FITC filter); PI (Ex 535 nm, TRITC filter)
Interpretation Live cells: green fluorescence only; dead cells: red fluorescence only

6. Troubleshooting

Issue Likely Cause Solution
High background, all nuclei stain weakly Stain concentration too high Reduce probe concentration 2–5×
Patchy nuclear staining Fixation or wash incomplete Increase permeabilization time; ensure thorough washing
JC-1 does not show red aggregates Mitochondria depolarized; wrong culture conditions Use FCCP positive control; verify cells are healthy; warm all solutions to 37°C
Calcein-AM leaks out of cells Over-staining or prolonged incubation Reduce loading time; wash thoroughly and image immediately
PI stains all cells including control Cells damaged during harvesting Gentle dissociation; include live cell gate in flow cytometry

7. Cross-References

For product procurement: solarbio.store