Stain live with a fixation-compatible probe (CellROX Green or Deep Red for general ROS; BODIPY 581/591 C11 for lipid peroxidation), then fix quickly with 3.7–4% PFA, wash, and perform gentle permeabilization (saponin recommended) for antibody staining. Validate the exact fix/perm conditions and analysis time window for your cell type — signal decays with time and harsh detergents.

Recommended probes (practical picks)

CellROX Green — robust for general intracellular ROS and reasonably stable after PFA fixation (analyze within vendor window; usually up to ~24 h, but shorter is safer).

CellROX Deep Red — good if you need a far-red channel (analyze sooner).

BODIPY 581/591 C11 — best for lipid peroxidation (spectral shift readout).

Avoid relying on CM-H₂DCFDA, DCFH-DA, MitoSOX, DHE for fixed readouts — treat them as live-only unless rigorously validated.

General principles (must follow)

1. Always stain live — the fluorescent probe has to be oxidized in metabolically active cells. Fixing before oxidation gives zero meaningful signal.

2. Minimize fix time and aldehyde concentration consistent with antigen preservation (3.7–4% PFA, 10–15 min, RT, is a good starting point).

3. Use gentle permeabilization (0.05–0.1% saponin or very brief 0.05% Triton X-100) if intracellular antibodies are required — test as this is the main step that can quench signal.

4. Acquire as soon as practical; signals often degrade over hours. If you must store, keep dark at 4°C and test the decay curve.

---

Copy-pasteable protocol A — Flow cytometry (single-tube workflow)

Reagents: CellROX Green (stock per vendor), PBS, 4% PFA, 0.05% saponin in PBS (perm buffer), antibody buffers.

1. Plate or collect cells; include controls (see validation layout below).

2. Prepare CellROX working solution (start with vendor concentration; typical final ≈ 2.5 µM for CellROX Green — optimize).

3. Incubate cells live with probe at 37°C, 30 min, protected from light.

4. Wash 2× with warm PBS (300–500 g, 5 min).

5. (Optional) Surface antibody staining before fixation if needed — use fixable antibody clones/dyes.

6. Fix: add 4% PFA, room temp, 10 min.

7. Wash 2× PBS.

8. Permeabilize for intracellular staining: 0.05–0.1% saponin in PBS (10 min, RT) — add intracellular antibodies in saponin buffer, incubate per antibody spec (usually 30–60 min). Avoid long exposures.

9. Wash with saponin buffer then PBS, keep on ice/dark.

10. Acquire on flow cytometer as soon as possible. Record fluorescence mean/median and % positive. Include single-color and unstained controls for compensation.

Copy-pasteable protocol B — Confocal/epifluorescence microscopy (adherent cells)

1. Treat cells on coverslips/dishes. Prepare controls.

2. Prepare CellROX (or BODIPY C11) per vendor; incubate live at 37°C for 20–30 min in the dark. For BODIPY C11, use imaging media without phenol red.

3. Wash gently with warm PBS (2×).

4. Fix with 3.7%–4% PFA for 10 min at RT.

5. Wash 3× PBS.

6. Permeabilize (if required) with 0.05% saponin or 0.05% Triton X-100 for short times (2–5 min) — test both on pilot slides.

7. Block and incubate with antibodies in saponin buffer (so ROS probe stays embedded).

8. Mount with anti-fade mounting medium, image quickly (acquire Z stacks if needed). Keep laser power consistent and avoid overexposure.

---

Validation experiment (plate layout + conditions) — run this first (8 wells per condition if plate)

Goal: know whether your fix/perm protocol preserves ROS signal and how fast it decays.

Plate layout (6× conditions × duplicates recommended):

1. Unstained (negative)

2. Probe only (live) — stain, wash, immediate acquire (positive live control)

3. Probe → PFA (10 min) → immediate acquire

4. Probe → PFA → saponin perm → immediate acquire

5. Probe → PFA → Triton 0.05% perm → immediate acquire

6. Probe → PFA → saponin perm → intracellular Abs → immediate acquire

Add these variations: include a positive inducer (e.g., 200 µM H₂O₂ or 50 µM menadione for 30 min prior to probe), and an antioxidant control (5–10 mM NAC pre-treatment). For kinetics, pick 0, 1, 4, 24 h post-fix acquisition to see signal decay.

Readouts: MFI and % positive (flow) or mean pixel intensity (microscopy). Acceptable outcome = clear separation between positive inducer and antioxidant, and reasonable signal retention after your fix/perm.

---

Typical starting concentrations / times (optimize!)

CellROX Green: start at 2–5 µM, 30 min, 37°C.

CellROX Deep Red: similar to Green but analyze sooner.

BODIPY 581/591 C11: 2–5 µM, 20–30 min; image in both channels (red→green shift indicates lipid peroxidation).

H₂O₂ positive control: 100–500 µM, 15–30 min (optimize for toxicity).

NAC antioxidant: 5–10 mM pre-incubation 30–60 min.

---

Notes about permeabilization reagents

Saponin (0.05–0.1%) — mild, reversible; usually best for keeping dyes intact.

Triton X-100 (≥0.05%) — stronger; can extract cytoplasmic contents and quench signal — use only briefly if needed.

Methanol or acetone fixation/permeabilization — generally bad for retention of oxidation-sensitive fluorescent adducts; avoid unless necessary.

Co-staining tips

Do surface staining before fixation where possible.

If you need both ROS and many intracellular markers, consider splitting sample: one aliquot for ROS + minimal downstream markers; second aliquot for full intracellular panel (no ROS). Then compare populations by gating strategies.

When using secondary antibodies, ensure their fluorochromes don’t overlap with your ROS probe channel.

Troubleshooting checklist

No ROS signal: probably stained after fixation — re-run live staining; confirm positive control induces signal.

Signal lost after perm: try saponin or reduce Triton concentration/time; try staining antibodies on separate aliquot.

High background: lower probe concentration/time; include unstained autofluorescence control; gate carefully.

MitoSOX/DHE weird localization: these are mitochondrial/superoxide-specific and sensitive to fixation — interpret with caution.

Alternatives / advanced

Genetically encoded sensors (HyPer, roGFP) — excellent for live measurements and kinetics but require transfection/stable expression; fixation will freeze the oxidation state but interpretation and antibody co-staining are more complex.

Immunodetection of oxidative damage products (8-oxo-dG, 4-HNE, nitrotyrosine) — these are fixed-stable endpoints (immunostaining or ELISA) and useful if you only need evidence of oxidative damage rather than instantaneous ROS levels.

Thank you for your kind response Sir. I hope it will be helpful for my experiment.