Jun 03, 2024
HTBE ALI System Infection & Sample Collections
This protocol is a draft, published without a DOI.
- 1mount sinai icahn school of medicine
Protocol Citation: Briana L McGovern 2024. HTBE ALI System Infection & Sample Collections. protocols.io https://protocols.io/view/htbe-ali-system-infection-amp-sample-collections-dezy3f7w
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: June 03, 2024
Last Modified: June 03, 2024
Protocol Integer ID: 101144
Abstract
HTBE ALI System
Background on the HTBE ALI System
Background on the HTBE ALI System
The air liquid interface (ALI) system provides a life-like experimental set up for human tracheal/bronchial epithelial cells. Cells are cultured (by Mattek) in a small circular insert. This insert is then gently placed, or optionally suspended, in a 6-well/12-well/24-well plate depending on the area of the insert.
The liquid interface:
Media in the wells of the plate is referred to as basal media. This basal media should be warmed before being plated. Basal media should be replaced every 2-3 days as the nutrients are depleted over time.
Basal media is what will be manipulated in these HTBE ALI experiments. You can introduce drugs, antibodies, interleukins, etc into this basal media to treat the HTBE cells.
The air interface:
The actual area inside of the insert is known as the apical side, and is the air interface of the system. This apical surface generates mucus, much like the mucus of the airways. This mucus must be washed off of the surface every 2-3 days using TEER buffer or DPBS. You will also infect the cells using the TEER/DPBS.
Apical washes will be collected and tittered at various time points of interest.
Schematic and actual photo of an ALI system using a suspension insert (NOT the Mattek system - a murine ALI system cultured by a colleague here at Mount Sinai)
Plating the inserts
Plating the inserts
Inserts will arrive embedded in an agar/gel to prevent movement during shipping.
Once you receive the cells you should immediately move them to the incubator and prepare the plates that you wish to keep them in.
The following is for a 12-well plate:
Warm the provided media and add 0.5-1ml to each well.
Disinfect a pair of tweezers using ethanol and gently lift an insert out of the gel/agar. Avoid having excess gel stuck to the insert. If gel is stuck on the insert gently remove it.
Move the insert to the well on the new plate*.
Repeat until all inserts are set in the new plates.
Incubate the plates overnight for acclimation.
*Mattek offers suspension set ups that float the insert in the well rather than have the insert placed in the well. We have not personally purchased with these pieces.
Treatment via Basal Media
Treatment via Basal Media
Using the provided media, set up your treatments.
I usually set up the treatments in 10ml/15ml tubes since you would want to make enough volume for triplicates.
An example of how I set up a treatment group of serial dilutions. For this experiment we plated <1ml per well so if you'd like to use a larger volume you can scale up.
ex) treating inserts on a 12-well plate with 3 concentrations (1:3 serial dilutions) of a drug
Conditions should be tested in triplicate at minimum and there should be both a DMSO (no treatment) control and a treatment control (for SARS-CoV-2 it would be Nirmatrelvir).
An example of how I layout the treatments on the plates. Triplicates
Infection
Infection