Collection Protocol

Overview

Goal: capture viable nasal mast cells in sufficient quantity for ex vivo degranulation assay. The collection step is the single biggest unknown in the entire protocol — cell yield determines whether the assay is feasible at all.

Materials

• NasalCrom nasal spray (cromolyn sodium, BAK-free formulation — check label, benzalkonium chloride is a mast cell liberator)
• Nasal brush (preferred) or swab — cytobrush or similar cytology brush gives better yield than cotton swab
• PBS buffer, cold (4°C)
• Microcentrifuge tubes, 1.5mL
• Ice or cold block to keep tubes on during collection

Step-by-Step

1. Stabilizer Pre-Spray

One spray NasalCrom per nostril. Wait 90-120 seconds.

Purpose:

• Suppresses mechanical degranulation from collection stress — cells arrive in buffer without having already fired from handling
• Partially normalizes activation state from environmental exposure that day (allergens, cold air, recent food)
• Becomes part of the assay definition — all scores are “reactivity above the cromolyn floor,” which is reproducible and clinically meaningful

Note on BAK: Some NasalCrom formulations contain benzalkonium chloride as preservative. BAK directly triggers mast cell degranulation. Using a BAK-containing spray defeats the purpose entirely. Generic cromolyn sodium nasal sprays often omit BAK — read the inactive ingredients. If BAK-free commercial spray is unavailable, pharmaceutical-grade cromolyn sodium dissolved in saline is an alternative.

2. Collection

Insert nasal brush gently into nostril. Gentle rotating motion against the lateral nasal wall mucosa — this is where mast cell density is highest. 3-5 full rotations. Remove carefully.

Immediately transfer brush/swab to microcentrifuge tube containing 300µL cold PBS. Swirl and press brush against tube wall to release cells. Remove brush.

Repeat from second nostril into same tube for higher yield if needed.

Keep tube on ice from this point.

Yield optimization if counts are low:

• Nasal brush over cotton swab — significantly better cell capture
• Pre-spray with plain PBS before cromolyn spray to hydrate mucosa — loosens epithelial cells
• More vigorous brush rotation (within comfort)
• Both nostrils into same tube

3. Wash Step

Spin at 300g, 7 minutes, 4°C.

Pour off supernatant carefully — this removes:

• Background histamine from diet, environment, baseline low-level activation
• Cromolyn sodium from the pre-spray (removes stabilizer before assay)
• Debris and mucus
• Any free mediators already in solution

Resuspend pellet in 200µL fresh cold PBS. Pipette gently 5-6 times to homogenize. Do not vortex.

This step concentrates cells (dilute nasal wash → controlled small volume) and cleans baseline, making histamine detection post-degranulation meaningful.

4. Quality Check Before Assay

Take 10µL aliquot for viability check (trypan blue exclusion). Take 10µL aliquot for cell identification (toluidine blue stain).

If toluidine blue confirms mast cells present and trypan blue shows >70% viability → proceed to assay.

If cell count is low but viability is good → proceed but note yield, interpret results cautiously.

If viability is poor → collection or buffer problem. Troubleshoot before proceeding:

• Was tube kept cold throughout?
• Was PBS pH correct (~7.4)?
• Was transfer time too long?

Timing

Collection to assay should be completed within 30 minutes of collection. Mast cell viability ex vivo in buffer degrades over time. The viability window characterization experiment will establish the actual outer limit, but 30 minutes is a conservative safe target.

Centrifuge Options

Lab microcentrifuge (Splatspace): Set to 300g, 7 min, 4°C. Most reliable for feasibility experiments.

Drill-mounted rotor: See Hand Centrifuge for 3D printed rotor design. Run at lowest speed setting, calibrate RPM first.

Whirligig (kit version): See Hand Centrifuge. Paracord spinner, 7 minutes, achieves sufficient G-force for cell pelleting.