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.

Condensate dynamics contribute most when studying rapid molecular exchange, fusion/fission events, and phase transitions in live-cell environments using time-lapse fluorescence or FRAP imaging.

They are especially informative in contexts where biochemical reactions are spatially confined (e.g., transcriptional hubs, stress granules, signaling clusters) and where changes in viscosity or material state reflect function.

Microscopy excels when paired with quantitative metrics (recovery curves, droplet coalescence kinetics, morphological changes) to link physical properties with biological outcomes.

In condensates, molecules show restricted yet dynamic mobility that reflects liquid-like phase behavior, distinct from diffusion in other compartments:

• Cytoplasm: Diffusion is largely unrestricted and FRAP recovery is fast and complete (milliseconds–seconds) because molecules move freely in solution.
• Membranes: Lateral diffusion is 2-D confined; FRAP recovery depends on membrane viscosity and often reaches full recovery but more slowly.
• Organelles (e.g., nucleus, mitochondria): Movement may be hindered by crowding or compartment boundaries, producing slower but still complete recovery.
• Condensates: Molecules display partial and slower recovery. A rapid initial phase reflects exchange of mobile molecules, followed by an incomplete plateau indicating an immobile or stably bound fraction. This biphasic curve and high viscosity reveal liquid-to-gel or solid-like states and weak multivalent interactions that define condensate material properties.

Reconstitute NPC1 in nanodiscs and compare ±cholesterol/ligands by cryo-EM, then map conformational shifts with HDX-MS or crosslink-MS plus MD simulations.

In cells, build an intramolecular NPC1 FRET biosensor targeted to lysosomes to track state changes under cholesterol flux perturbations (e.g., NPC2 addition, U18666A) and correlate with FRAP/trafficking readouts.

Validate structure–function by cholesterol transport assays and, if needed, smFRET or site-directed spin labeling/EPR on purified NPC1.

Applications

• The combined techniques of liquid–liquid phase separation imaging, Fluorescence Recovery After Photobleaching (FRAP) and X‑Ray Photon Correlation Spectroscopy (XPCS) enable quantification of molecular mobility, exchange kinetics, and material states (liquid, gel, solid) of biomolecular condensates in vitro and in cells.
• They can be applied to study phase transitions, ageing or maturation of condensates (e.g., toward solid or pathological states) in disease contexts such as neurodegeneration.

Limits

• XPCS requires access to high-coherence X-ray sources (synchrotrons / XFELs) and careful sample handling to avoid radiation damage, limiting its general availability.
• FRAP and microscopy report on relatively large-scale/slow dynamics and may not resolve nanoscale motions or heterogeneous sub-populations within condensates; additionally, artefacts (bleaching, phototoxicity, surface effects) can distort interpretation.

Yes. Different biocondensates show distinct FRET efficiency profiles reflecting their molecular packing density and interaction strength.

High, stable FRET indicates tight, static interactions (gel-like or solid condensates), while low or fluctuating FRET suggests dynamic, liquid-like states.

Thus, FRET can classify condensates along a liquid-to-solid continuum and monitor phase transitions in real time.

Phase separation in living cells is typically visualized using fluorescence microscopy techniques such as confocal, lattice light-sheet, or super-resolution (e.g., STED, SIM) imaging with fluorescently tagged proteins. These allow observation of droplet-like condensates, fusion events, and dynamic exchange.

Dynamic properties are probed by FRAP (fluorescence recovery after photobleaching) to measure molecular mobility within condensates, and FCS (fluorescence correlation spectroscopy) or XPCS (X-ray photon correlation spectroscopy) for nanoscale dynamics.

For extracellular condensates, detection is more challenging. Emerging tools include cryo-electron tomography, atomic force microscopy (AFM), and phase-sensitive light scattering or Raman imaging to detect biomolecular clustering in extracellular fluids or matrices. Extracellular vesicle–like condensates and secreted protein droplets are also being explored using label-free imaging (OCT, DIC) and microfluidic confinement assays.

Use organelle-specific fluorescent markers (e.g., DAPI or H2B-GFP for nucleus, calnexin or ER-Tracker for ER) and colocalization imaging to compare signal overlap.

Confirm localization with confocal z-stacks or super-resolution microscopy, optionally supported by fractionation or immunostaining.