(e)

0.5 mL of Primocin™Invivogen; (Note that this is an

antimicrobial agent).

(f)

0.5 mL of Fungin™Invivogen; (Note that this is antifun-

gal reagent).

3.1.3

1% Sodium

Dodecyl Sulfate

1. Weigh 5 g SDS and add to a 500-mL Duran bottle. Avoid

inhalation using a fume mask or weigh the powder under a

fume hood.

2. Measure 500 mL of deionized H2O and add to the Duran

bottle.

3. Add a magnetic stir bar and place on a magnetic stirring plate to

ensure mixing of the solution; use a low-to-medium speed and

gently heat up the solution (60 ^C) using a hot plate.

4. Verify and adjust the pH to 7.4 (see Note 3).

5. Filter the solution inside the BSC and store at room tempera-

ture (RT).

3.1.4

1% Triton X-100

1. Measure 500 mL of deionized H2O and add to a 1000-mL

Duran bottle.

2. Use a plastic pipette to add 5 mL of Triton X-100 to the Duran

bottle.

3. Add a magnetic stir bar and place on a magnetic stirring plate to

mix the solution; use a low-to-medium speed and gently heat

up the solution (60 ^C) using a hot plate.

4. Verify and adjust the pH to 7.4 (see Note 3).

5. Filter the solution in the BSC and store the solution at RT.

3.2

Graft

Procurement and

Preparation

Use sterile surgical instruments (see Note 4).

1. At the Operating Room (OR):

(a)

Procure the trachea aseptically in a sterile fashion follow-

ing standard surgical procedures (see Note 5).

(b)

Scrape the tracheal luminal surface with a sterile metal

spatula to remove residual mucus.

(c)

Submerge the trachea in a sterile hybridization bottle with

100 mL DMEM + Antibiotics, then transport it to the

laboratory.

2. At the laboratory:

(a)

Place

the bottle

with the

trachea in

a

rocker at

low-medium speed for 1 h at RT.

(b)

Inside the BSC, transfer the trachea to another sterile

bottle with fresh DMEM + Antibiotics after scraping the

lumen with another sterile spatula.

(c)

Repeat steps (a and b) twice to complete a 3-h wash

period.

172

Alba E. Marin-Araujo et al.