Nov 10, 2023
Elution of nasal lining fluid collected via nasosorption
- 1Telethon Kids Institute;
- 2University of Western Australia
Protocol Citation: Samuel Montgomery 2023. Elution of nasal lining fluid collected via nasosorption. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn3nn6l5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 10, 2023
Last Modified: November 10, 2023
Protocol Integer ID: 90751
Keywords: nasosorption, nasal lining fluid
Abstract
This procedure outlines the materials and processes for eluting nasal lining fluid collected using a Mucosal Diagnostics FX-i nasosorption device. This protocol has been successfully used to elute nasal lining fluid for both DNA extraction for 16s rRNA sequencing & metagenomics, and for proteomics using the SomaScan assay.
Guidelines
This protocol should be performed in a sterile environment to reduce cross contamination between nasosorption devices, to enable metagnomic DNA extraction and analyses
Materials
- Phosphate Buffered Saline (PBS) (#10010023)
- Tween 20 (Sigma, #P9416-50ML)
- Corning SpinX columns (Sigma, #CLS9301)
- 2mL Corning microfuge tubes (Sigma, #CLS3213)
- Sterile forceps (two pairs per swab)
- 1.6mL sterile microfuge tubes
Making elution buffer
Making elution buffer
Add 0.05 % volume of Tween20 to PBS in a sterile environment to reduce cross-contamination
Store at 4 °C until required
Elution of nasal lining fluid
Elution of nasal lining fluid
30m 30s
Thaw nasosorption device in cryotube on ice
Add 300 µL of elution buffer to a labelled 2mL microcentrifuge tube and place on ice
Remove the synthetic absorptive matrix (SAM) from the nasosorption device using sterile forceps by tearing it from the handle and place in the microcentrifuge tube containing elution buffer
Vortex the microcentrifuge tube for 00:00:30
30s
Using a new pair of sterile forceps, remove the SAM from the elution buffer, and place into a spinX column on a new 2mL microcentrifuge tube
Add the elution buffer from the first tube onto the SAM in the spinX column
Centrifuge the SAM and spinX column at 16000 x g for 00:30:00 at 4 °C
30m
Discard the spinX column and SAM
Aliquot the eluate into labelled microtubes, and measure total protein concentration using the assay of your choice (Pierce BCA assay, Qubit protein assay etc)