Live-Cell Time-Lapse Single-Cell Tracking of Random and Wound-Directed Migration in RFP-Expressing MDCK Epithelial Monolayers

Conditions: vehicle (DMSO) and gefitinib (1, 10 µM). Each condition is imaged in n = 3 wells per biological replicate, with N = 3 independent biological replicates. For each well, >=50 individual cells in the migrating front and >=50 in the bulk are tracked. Imaging is at 10-min intervals for 18 h on a stage-top incubator (37 C, 5% CO2). Field positions are fixed per well; treatment assignment to wells is randomized; track curation is performed on coded, blinded movies. Drift is corrected using fiducial features.

BSL-1 for MDCK. Gefitinib is a pharmacologically active kinase inhibitor — handle with gloves, avoid inhalation/skin contact, and dispose as chemical waste. Mitomycin C is a mutagen/carcinogen — biosafety cabinet, double gloves, dedicated waste, no drain disposal. HEPES medium and DMSO standard handling. Laser/LED illumination: use appropriate enclosure/interlocks. Standard PPE: lab coat, nitrile gloves, eye protection. Decontaminate surfaces with 70% ethanol; bleach biological waste.

Vehicle control: DMSO matched to the highest gefitinib carrier isolates solvent effects. Random-migration control: tracking cells in the bulk monolayer (away from the wound) provides non-directed baseline motility for each condition. Proliferation control: paired wells with/without mitomycin C confirm whether division contributes to apparent track displacement. Phototoxicity control: a no-drug well imaged with identical illumination verifies that exposure alone does not slow cells. A fixed-field, drift-correction fiducial controls for stage drift artifacts.

Vehicle front cells should show directed motility with speeds ~0.3-0.6 µm/min and a directionality ratio of ~0.5-0.7 toward the wound, with MSD growing super-diffusively early. Gefitinib at 10 µM should reduce speed (e.g., to ~0.15-0.3 µm/min), lower the directionality ratio toward random (~0.3), and flatten MSD, with a smaller effect at 1 µM. Bulk cells should be slower and less directed than front cells in all conditions. >50 high-quality tracks per condition are expected after curation.

• MDCK cells stably expressing H2B-RFP (nuclear marker), passage 10-30
• DMEM, high glucose, with 10% FBS, 1% pen-strep; DMEM/1% FBS + 25 mM HEPES for imaging
• Gefitinib, 10 mM stock in DMSO; working 1 and 10 µM
• DMSO vehicle, matched (<0.1% final)
• Mitomycin C, 10 µg/mL (optional proliferation block during tracking)
• Ibidi 2-well culture-insert (defined 500 µm gap) in glass-bottom 35 mm dishes or 24-well glass-bottom plate
• Fibronectin coating, 10 µg/mL in PBS
• Hoechst 33342 (optional, 1 µg/mL) for cross-check of nuclei
• Dulbecco's PBS, Trypsin-EDTA
• Mineral oil or sealed lid to limit evaporation

To capture phase + RFP time-lapse images of a wounded H2B-RFP MDCK monolayer at 10-min intervals over 18 h and extract per-cell migration trajectories, computing instantaneous speed, directional persistence (directionality ratio), and mean-squared displacement, in order to resolve how EGFR inhibition with gefitinib changes single-cell motility parameters that bulk wound-closure assays cannot distinguish.

1. Coat glass-bottom dishes with 10 µg/mL fibronectin (1 h, 37 C); place an Ibidi 2-well insert; seed 3.5 x 10^4 H2B-RFP MDCK per chamber in DMEM/10% FBS; grow to confluence (24 h).
2. Optionally apply mitomycin C (10 µg/mL, 2 h) to suppress division so tracks reflect migration; wash 2x PBS.
3. Remove the insert to create a 500 µm cell-free gap; wash gently; add DMEM/1% FBS + 25 mM HEPES with vehicle or gefitinib (1/10 µM).
4. Mount on a stage-top incubator (37 C, 5% CO2, humidified) on a widefield or spinning-disk microscope; set up phase + RFP channels at 10x or 20x.
5. Acquire multipoint time-lapse: 1 frame per 10 min for 18 h, with autofocus and minimal RFP exposure to limit phototoxicity.
6. At the end, confirm cell viability (morphology, continued division if not blocked) and check for focus drift.
7. Export 16-bit image stacks per position for nuclear segmentation and tracking.

Independent variable: gefitinib concentration (0, 1, 10 µM). Dependent variables: per-cell mean speed (µm/min), directionality ratio (net/total displacement), mean-squared displacement, and forward migration index toward the wound. Controlled variables: imaging interval (10 min) and duration (18 h), temperature/CO2/humidity, illumination/exposure, gap width (500 µm), fibronectin coating, serum (1%), passage range, number of cells tracked per condition.

If wound-directed MDCK migration depends on EGFR-driven motility and leader-cell guidance, then gefitinib (10 µM) will reduce per-cell speed and directional persistence (lower directionality ratio and MSD) relative to DMSO vehicle, even at concentrations that only partially slow bulk gap closure, revealing a motility-quality defect distinct from a pure rate change.

Nuclei are segmented from the H2B-RFP channel and linked into trajectories using TrackMate (Fiji) or Trackpy; tracks shorter than a threshold or with gaps are discarded. Per-track speed, directionality ratio, forward migration index, and MSD are computed (e.g., with the DiPer/Chemotaxis tool). Drift is subtracted using background features. Rose plots and trajectory overlays visualize directionality. All movies are coded before track curation to blind the analyst.

Tracks break / nuclei lost: increase frame rate or RFP exposure slightly, and improve segmentation thresholds; ensure H2B-RFP signal is bright and not bleached. Focus drift over 18 h: enable hardware autofocus and pre-equilibrate the stage incubator >=1 h before imaging. Phototoxicity (cells round up/stop): reduce excitation intensity/exposure and lengthen the interval; verify with a no-illumination control. Evaporation/medium concentration changes: use a sealed lid, humidified chamber, or mineral-oil overlay. Apparent motility from division not migration: include the mitomycin C arm so tracks reflect translocation, not mitotic splitting.

Per-cell parameters are summarized to a per-well median, then to a per-biological-replicate mean (N = 3) to respect nesting and avoid pseudoreplication. Speed and directionality are compared across concentrations by one-way ANOVA with Dunnett's test vs vehicle (alpha = 0.05); non-normal metrics use Kruskal-Wallis with Dunn's correction. A linear mixed-effects model (cell within well within replicate) is used as a sensitivity analysis. Power analysis (alpha 0.05, power 0.8) with >50 cells/condition x 3 replicates detects a 30% speed difference.