Physiologic Lung³Gel
Culturing microbial samples in a healthy human airway mucus model, in syringes, and preparing them for sequencing and metabolomics.
Product
Reproducible and high-throughput in vitro models are essential for studying complex microbial communities. This kit is designed for the in vitro modelling of healthy human airway mucus.
The protocol describes how to culture microbial samples in Physiologic Lung³Gel in a syringe format, and how to prepare the samples for downstream analyses: cell viability, flow cytometry, metabolomics and sequencing.
Before you begin
- The steps below are written for single-strain samples. The same protocol has also been used for co-cultures, microbial consortia and complex microbial samples.
- The gel in this kit contains porcine stomach type III mucin at 19.0 mg/mL with 4.78 mg/mL NaCl. The composition can be tuned, and custom compositions are available.
- Sterilize all instruments and consumables, and work under aseptic conditions, ideally in a Class II biological safety cabinet. Prepare a sterile 0.9% (w/v) NaCl solution.
Procedure
APreparing the microbial suspension~15 min
- Grow the species of interest, for example Pseudomonas aeruginosa, to logarithmic phase in the appropriate medium.
- Centrifuge to pellet the cells and discard the supernatant.
- Resuspend the pellet in LB or another appropriate culture medium.
- Measure the optical density at 600 nm and adjust the cell concentration as needed.
Critical If the gel was produced in a culture medium, resuspend the pellet in 0.9% (w/v) NaCl rather than in medium.
BCulturing in Physiologic Lung³Gel~5 min
- Discard the first 2 to 3 drops by gently pressing the syringe plunger.
- Using the double syringe mixing method, combine the suspension from stage A with the gel at a 1:1 volume ratio, mixing 6 to 7 times.
- Dispense the contents into a 15 mL tube, then distribute equal volumes into microcentrifuge tubes with a positive displacement pipette.
- Without a syringe connector: dispense the gel into a sterile 15 mL tube, transfer 500 µL into each microcentrifuge tube, add an equal volume of the suspension and homogenize by gentle pipetting.
- Incubate at 37 °C for up to 72 hours, aerobic or anaerobic, according to the experimental design.
Note 24-well plates can be used instead of microcentrifuge tubes.
CDissolving the gel~5 min per syringe
- Add Bac³Gel dissolution medium (200 mM sodium citrate) at a 1:1 ratio to each sample.
- Mix thoroughly until the gel has fully dissolved. The homogenized samples are ready for downstream assays.
Downstream analysis
Sequencing
Centrifuge at 14,000 × g for 5 minutes, then use the pellet for DNA extraction.
Metabolomics
Centrifuge at 14,000 × g for 10 minutes, then collect and lyophilize the supernatant.
Metabolic activity or optical density
Centrifuge at 14,000 × g for 5 minutes, discard the supernatant and resuspend in 0.9% (w/v) NaCl.
Other readouts
Also suited to cell viability assessment, flow cytometry and standard microbiological characterization assays.
Troubleshooting
For research and development use only. Not for diagnostic or clinical use.