I am looking for standard Phenotype shift protocol of RAW-264.7 on induction with LPS and IL-4 and their fluorescence imaging.

Here’s a concise, standardizable RAW 264.7 polarization and IF imaging workflow.

Culture

• Cells: RAW 264.7 (mouse macrophage line).
• Medium: DMEM high glucose + 10% FBS + 1% Pen/Strep. 37 °C, 5% CO₂.
• Plate for imaging: 12-mm glass coverslips in 24-well plates at 1–2×10⁵ cells/well (≈5×10⁴ cells/cm²). Let adhere 6–12 h.

Induction

M1-like (pro-inflammatory)

• Reagents: LPS (E. coli O111:B4) 100 ng/mL. Optional: IFN-γ 10–20 ng/mL to strengthen M1.
• Duration: 18–24 h (up to 48 h if responses are weak).

M2-like (alternatively activated)

• Reagents: IL-4 20 ng/mL. Optional: IL-13 10–20 ng/mL for synergy.
• Duration: 24–48 h.

Controls

• Untreated (vehicle).
• Cytotoxicity control: staurosporine 0.5–1 µM, 4 h (optional).

Immunofluorescence (end-point)

1. Wash 2× PBS. Fix 4% PFA, 10 min RT. Quench 50 mM NH₄Cl, 5 min.
2. Permeabilize 0.1% Triton X-100 in PBS, 5 min (skip for surface markers).
3. Block 5% BSA or 5% donkey serum in PBS, 30 min.
4. Primary Abs, 1–2 h RT or overnight 4 °C:

• M1 markers: iNOS (NOS2), CD86, p-p65, TNF-α.
• M2 markers: Arg1, CD206 (MRC1), YM1/Chi3l3 (sometimes low in RAWs), p-STAT6.

1. Wash 3× PBS. Secondary Abs (Alexa Fluor 488/555/647), 1 h RT, dark.
2. Counterstain: DAPI (nuclei). Optional: Phalloidin (F-actin) for morphology.
3. Mount with anti-fade. Image on epifluorescence or confocal (40×–63× oil). Keep exposure constant across groups.

Live-cell fluorescent probes (optional, before fixation)

• ROS: DCFDA 10 µM, 30 min (↑ in M1).
• NO: DAF-FM DA 5 µM, 30 min (↑ in M1).
• Mito potential: TMRM/JC-1 per kit (often ↓ in M1).
• Wash, image live, or proceed to fixation.

Expected readouts

• M1/LPS(±IFN-γ): Higher iNOS/CD86, stronger DAF-FM/ROS, spread/amoeboid morphology.
• M2/IL-4(±IL-13): Higher Arg1/CD206, elongated/spindle-like morphology, lower ROS/NO.

Validation (recommended)

• qPCR: Nos2, Tnf, Il6 (M1); Arg1, Mrc1, Il10 (M2).
• ELISA (supernatant): TNF-α, IL-6↑ in M1; IL-10↑ in M2.
• Flow (alternative to IF): surface CD86 (M1), CD206 (M2).

Timing summary

• Day 0 plate; Day 1 induce; Day 2 image/fix and stain.

Troubleshooting

• Weak M1: add IFN-γ, extend to 24–36 h, verify LPS potency, keep FBS ≤10%.
• Weak M2: co-stimulate with IL-13, extend to 48 h; note RAW 264.7 can show variable IL-4 responsiveness by lot.
• High background IF: increase blocking, include isotype controls, keep identical exposure.
• Cytotoxicity: titrate LPS to 10–50 ng/mL or shorten exposure.

Reagent notes

• Use endotoxin-free plastics and buffers.
• Typical working ranges: LPS 10–200 ng/mL; IFN-γ 10–20 ng/mL; IL-4 10–30 ng/mL.