LPS-Induced Cellular Inflammation Model: Experimental Procedure

I. Principle

Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, binds to the TLR4 receptor and its co-receptors (e.g., CD14, MD-2) on macrophages, activating NF-κB, MAPK, and other pathways and inducing production/secretion of inflammatory cytokines such as TNF-α, IL-6, and IL-1β.

II. Materials and Reagents

1.Cells

Mouse macrophage line RAW264.7.

2.Culture/handling reagents

High-glucose DMEM; FBS; penicillin–streptomycin; trypsin or enzyme-free dissociation buffer (note: RAW264.7 can often be collected by gentle pipetting or scraping; avoid prolonged trypsinization to protect surface receptors); PBS for washes.

3.Modeling/detection reagents

LPS (commonly E. coli–derived; prepare stock, aliquot, store at −20 °C); CCK-8 for cell viability; ELISA kits for TNF-α, IL-6; optional assay kits for NO, PGE₂, etc.

4.Instruments/consumables

CO₂ incubator (37 °C, 5% CO₂, humidified); biosafety cabinet; inverted microscope; microcentrifuge; microplate reader (e.g., 450 nm); flasks, dishes, 96-well plates; pipettes and sterile tips, tubes.

III. Procedure

1.Thawing

Retrieve RAW264.7 vial from liquid nitrogen, rapidly thaw in a 37 °C water bath with gentle agitation. Transfer to the cabinet, dilute with ~5 mL DMEM + 10% FBS in a tube, spin 1000 rpm ~5 min, discard supernatant, resuspend in fresh DMEM + 10% FBS, seed into a flask, and culture at 37 °C, 5% CO₂ to recover.

2.Passaging

At ~80–90% density, remove medium, rinse 1–2× with PBS. Detach using a brief exposure to trypsin or enzyme-free dissociation buffer (stop promptly with medium when cells round/loosen), or use pipetting/scraping. Make a single-cell suspension and split 1:3–1:4 into new flasks; continue culture to maintain good status.

3.Seeding

Collect log-phase, healthy cells and count. Adjust to ~5×10⁴–1×10⁵ cells/mL in DMEM + 10% FBS. Seed 100 µL/well into 96-well plates (≈5×10³–1×10⁴ cells/well) and incubate overnight for attachment and recovery.

4.LPS induction

After attachment, replace with serum-free or low-serum medium (e.g., 1% FBS) for 2–4 h to synchronize. Groups: Blank/control (no LPS, equal volume medium); LPS groups (e.g., 0.1, 1, 10 µg/mL), 3–5 replicates each. Pre-prepare LPS working media at target concentrations; add gently to wells, avoiding localized high concentrations. Incubate 12–24 h (optimize by pilot tests).

5.Readouts

(1) Cell viability (CCK-8): At chosen timepoints (e.g., 12 h or 24 h post-LPS), add ~10 µL CCK-8 per well, mix gently, incubate 1–4 h, read OD450. Subtract blank (no cells, medium + CCK-8) and compare groups.

(2) Cytokine secretion (ELISA): Collect supernatants at set timepoints; dilute if needed per kit. Follow kit steps (loading, incubation, washing, development, stop), read at specified wavelengths, and calculate TNF-α, IL-6 concentrations from standard curves to assess inflammatory response.

IV. FAQ

Q1: Extensive cell death/detachment after LPS?

A: Likely excessive LPS dose, prolonged exposure, or high sensitivity. Lower the dose, shorten exposure, and pilot to identify conditions that elevate cytokines while preserving viability. Also check seeding density, medium freshness, cell passage/age, and potential stresses (pH, osmolarity).

Q2: Must induction be serum-free?

A: Many protocols use low serum (0–1% FBS) for 2–4 h pre-induction to reduce serum confounders. Whether to use serum-free during LPS exposure depends on tolerance and goals: serum-free may yield a “cleaner” response but can impair survival; low serum better preserves viability. Compare conditions in pilots (cytokines + viability) before finalizing.

Aladdin: https://www.aladdinsci.com/