Quantification of Immune Mediator Levels in Human Mucosal Lining Fluid

Protokoll

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Experimental Setup

1. Use sheets of filter paper (fibrous hydroxylated-polyester sheets, see the Table of Materials).
   1. Cut out strips 3 x 15 mm in size for a 1-month-old child and in an L-shape for older children and adults (5 x 20 x 10 mm (short arm of the L)).
2. Insert one piece of filter paper into each nostril of an infant using forceps. Place the filter paper at the anterior part of the inferior turbinate (approximately 1 cm inside the nostril for neonates and 1.5 cm for all other ages).
   1. Place neonates on a bed and give them sugar water for comfort.
      NOTE: For children of all other ages, the sampling is performed most easily with the subject seated on a chair.
   2. For older children, insert the long arm of the L-shaped filter paper into each nostril.
3. Apply a nose clip around the nostrils to minimize discomfort and avoid the accidental loss of the filter paper.
4. Remove the filter papers after 2 min of absorption.
5. Place the filter papers in labeled tubes and freeze them immediately at -80 °C.
6. Make a note of any symptoms of airway infection shown by the child on the day of sampling.
7. Record any sneezing, persistent crying, or epistaxis that occurs during the 2 min of sampling.

2. Quantification of Airway Immune-mediators

1. Take up to 10 random samples from the freezer. Record the identification numbers. Keep the samples on ice during the whole working process.
2. After thawing on ice, immerse the filter papers (per subject) from both nostrils in 300 µL of freshly prepared buffer (see the Table of Materials) containing one complete protease inhibitor tablet (see the Table of Materials) per 25 mL of buffer.
   1. Adjust the volume of the buffer as per the size of the filter paper.
      1. Use 300 µL of buffer for filter papers 3 x 15 mm in size.
      2. If only one filter paper is available, use half the buffer volume (150 µL).
3. Transfer the samples to a plate shaker (400 rpm) for 5 min. Use a timer.
4. Transfer the moist filter papers and assay buffer into the cup of a cellulose acetate tube filter (0.22 µm pore size) placed within a microcentrifuge tube (see  Table of Materials).
5. Centrifuge at 16,000 x g for 5 min in a cooled centrifuge (at 4 °C) to obtain filtered nasal extract in the tube.
6. Remove the cup and keep the tubes on ice while aliquoting the nasal extract into the wells of low-protein binding storage plates (see  Table of Materials).
7. Store at -80 °C until analysis.
8. Determine the concentrations of cytokines and chemokines in the nasal extracts using a high-sensitivity, electrochemiluminescence-based multiplexed array system (see the Table of Materials).   
   c: A human 10-plex T_H1/T_H2 cytokine assay, 9-plex chemokine assay, and single-plex interleukin-17A (IL-17A), transforming growth factor-beta1 (TGF-β1), and thymic stromal lymphopoietin (TSLP) were performed here.
   1. For the assays, incubate the nasal extracts overnight at 4 °C. Conduct the measurements as per the standard manufacturer protocol (see the Table of Materials).  
      NOTE: The immunoassays (see the Table of Materials) typically have a high dynamic range for measurement ranging between 1 & 10,000 pg/mL, but for some assays, it can be 100,000 pg/mL. This means that all samples can be run at the same dilution, thus limiting the influence of dissimilar dilutions in healthy and diseased subjects, as is the case in other similar immunoassays. The lower limit of detection for all cytokines was 1 pg/mL or less, and for chemokines, it ranged between 1 & 50 pg/mL. TSLP was not detectable in 98% of samples at 1 month of age.

Materialien

Name	Company	Catalog Number	Comments
Fibrous hydroxylatedpolyester sheets	Accuwik Ultra	SPR0730	Filter paper
Milliplex Assay Buffer	Millipore	L-AB	Buffer
low-protein binding storage plates	Thermo Scientific	CLS8161	Plates
Protease Inhibitor	Roche	11873580001	Complete EDTA-free Protease Inhibitor Cocktail
Reader of multi-spot plates	Mesoscale	NA	Sector imager 6000
Assays	Mesoscale		Human 10-plex TH1/TH2 cytokine assay and 9-plex chemokine assay, and singleplex IL-17A, TGF-β1and TSLP. A description can be found online on www.mesoscale.com