Indoor Lighting Spectra Influences on Circadian Gene Expression in Human Cells

Problem Statement

This experiment tests whether exposure to different indoor lighting spectra, under consistent laboratory conditions, causes acute changes in circadian gene expression in cultured human cells. Only direct gene expression changes are measured. No behavioral, sleep, cognitive, or clinical conclusions are drawn.

Hidden Assumption Being Tested

Common indoor lighting spectra do not significantly alter circadian gene expression when exposure occurs over short durations under otherwise stable laboratory conditions.

Lighting Spectra Tested

• Blue-enriched LED (peak ~460 nm, FWHM ~20 nm)
• Warm white LED (CCT ~3000 K)
• Cool white LED (CCT ~6500 K)
• Dark control (no incident light; plates wrapped in opaque foil)

Exposure Conditions

• Exposure duration: 4 hours continuous illumination
• Incubation conditions: 37 °C, 5% CO₂
• Light intensity: ~100 lux at the cell monolayer for all illuminated conditions
• Exposure begins immediately after circadian synchronization

Cell System and Synchronization

• Cell line: Human fibroblasts (e.g., Hs68) or equivalent
• Confluence at exposure: 70–80%
• Synchronization protocol:
  • 2-hour serum shock using 50% serum
  • Replace with standard growth media for 24 hours before exposure

Gene Expression Measurement

• Total RNA extracted immediately after 4-hour exposure
• cDNA synthesized using standard reverse transcription
• Quantitative PCR performed for:
• PER2 (core circadian gene)
• BMAL1 (core circadian gene)
• GAPDH (housekeeping normalization control)
• Data analysis performed using ΔΔCt method relative to dark control

Binary Disruption Threshold

For each lighting condition (blue, warm white, cool white):

• Disruption: Expression of PER2 or BMAL1 changes by ≥1.5-fold (up or down; |log₂ fold-change| ≥0.58) relative to dark control
• No disruption: Both genes change by <1.5-fold relative to dark control

Controls and Technical Requirements

• Minimum three biological replicates per condition
• All non-light variables held constant (media batch, plasticware, temperature, CO₂)
• Light sources calibrated before each experiment for spectral output and intensity
• No unintended light exposure before, during, or after synchronization

Boundaries

• Only in vitro gene expression is measured
• No claims about sleep, behavior, cognition, or health
• No extrapolation to humans, animals, or long-term exposure
• No live animal or human testing

Why This Matters

This experiment directly tests whether widely used indoor lighting spectra interact with circadian molecular machinery at the cellular level. Any detected disruption challenges the assumption that indoor lighting is biologically neutral over short exposure windows, without invoking downstream or clinical interpretations.