Feasibility Experiment

Goal

Answer one question: Can you capture viable nasal mast cells on a swab, transfer them to buffer, and observe degranulation in response to a controlled liberator?

Everything else is downstream of this. Do not proceed to detection chemistry optimization, liberator calibration, or product design until this is confirmed.

Location

Splatspace biolab, Durham NC. Available equipment: microcentrifuge, NanoDrop, fume hood, PCR machine, biohacking tools.

What You Need

Reagents

• NasalCrom (cromolyn sodium nasal spray) — OTC, CVS. Verify BAK-free formulation. Benzalkonium chloride is a mast cell liberator — counterproductive. Check label.
• PBS tablets — dissolve in distilled water. Available from lab suppliers, some aquarium suppliers, brewing suppliers.
• Toluidine blue stain — mast cells stain metachromatically (purple against blue background). Research supplier or lab supplier.
• Trypan blue — viability stain. Dead cells take it up, live cells exclude it.
• Citric acid (food grade) — first liberator. Cheap, accessible, known mast cell liberator via osmotic/pH stress. Has existing precedent in airway provocation testing. Note: eliminates pH as a readout — choose histamine or visual degranulation as detection method instead.
• Distilled water

Equipment

• Light microscope (available at Splatspace or cheap USB microscope)
• Nasal brush (preferred over swab for cell yield — cytobrush or similar)
• Microcentrifuge tubes
• Pipettes
• Depression slides or microwells

Optional for This Experiment

• Brewing histamine test kit (OPA-based colorimetric) — proxy readout if visual counting is insufficient
• USB microscope + laptop for vision model quantification

Protocol

1. Pre-Collection Stabilization

One spray NasalCrom (BAK-free) per nostril. Wait 2 minutes.

Purpose: suppresses mechanical degranulation stress during collection. Cells arrive in buffer without having already fired from handling. Also partially normalizes activation state from environmental exposure that day.

2. Collection

Nasal brush (or swab if brush unavailable) — gentle circular motion against nasal mucosa. Transfer immediately to microcentrifuge tube containing 500µL PBS. Cap.

3. Wash Step (Critical)

Spin at 300g, 7 minutes, 4°C if available.

Pour off supernatant — this removes:

• Background histamine from environment, diet, baseline activation
• Cromolyn sodium (removes stabilizer before assay so it doesn’t suppress liberator response)
• Any debris

Resuspend pellet in 200µL fresh PBS. This concentrates cells and cleans baseline.

4. Viability Check

Take 10µL aliquot. Add trypan blue. Count live (clear) vs dead (blue) cells under microscope.

If cell count is very low: experiment may still proceed but note yield. If cells are predominantly dead: collection or buffer problem, troubleshoot before proceeding.

5. Toluidine Blue Baseline

Take 10µL aliquot. Stain with toluidine blue. Confirm mast cells present (purple metachromatic granules). Count intact (granulated) vs degranulated cells. Record as baseline ratio.

6. Liberator Challenge

Add citric acid solution to remaining sample. Start at low concentration (1mM). Add incrementally every 60 seconds.

Take 10µL aliquot at each step. Stain with toluidine blue. Count degranulated vs intact.

7. Readout

Plot degranulation percentage over time/concentration. Looking for:

• Any increase in degranulated cell ratio above baseline → assay concept is viable
• Dose-response relationship → titration protocol is feasible

Success Criteria

Minimum: Cell capture confirmed (toluidine blue shows mast cells present) AND any measurable increase in degranulation in response to liberator above baseline.

Good: Dose-response relationship visible across concentration steps.

Excellent: Cell count sufficient for histamine colorimetric readout as secondary confirmation.

If Cell Yield Is Low

Try:

• Nasal brush instead of swab
• Pre-spray with PBS before cromolyn to hydrate mucosa
• Longer, more vigorous brush stroke
• Second brush from opposite nostril, combine in same tube

Cell yield is the single biggest unknown and potential fatal flaw. If yield is consistently too low to generate signal, the nasal route may not be viable and skin biopsy or alternative collection would need to be explored.

Next Steps If Successful

1. Characterize viability window — how long do cells remain responsive after collection
2. Calibrate liberator dose-response with compound 48/80 (requires fume hood — Splatspace)
3. Develop drop titration protocol
4. Test histamine colorimetric readout post-wash
5. Prototype vision model cell counting