9. Gibco™Gentamicin (50 mg/mL).

10. Primocin™Invivogen—Antimicrobial Agent.

11. Fungin™Invivogen—Antifungal Reagent.

12. Sodium dodecyl sulfate.

13. Triton X-100.

14. Filtered deionized H2O.

15. PBS without Ca²⁺/Mg²⁺.

3

Methods

3.1

Preparation of

Antibiotics, Media, and

Detergents (See Notes

1 and 2)

3.1.1

Cocktail of

Antibiotics

Prepare under sterile conditions inside the BSC using cell culture

H2O:

1. Reconstitute one vial of Imipenem/Cilastatin 500 mg/

500 mg with 8 mL of H2O, i.e., 67.5 mg/mL.

2. Reconstitute two vials of Ceftazidime 1 g by adding 5.2 mL of

H2O to each vial, i.e., 192 mg/mL.

3. Reconstitute one vial of Colistimethate 150 mg with 6 mL of

H2O, i.e., 25 mg/mL.

4. Measure out 340 mL of H2O and add:

(a)

8 mL of Imipenem/Cilastatin.

(b)

8 mL of Ceftazidime.

(c)

4 mL of Colistimethate.

(d)

40 mL of Fluconazole 400 mg/200 mL, i.e., 2 mg/mL.

5. Discard unused portions of Ceftazidime, Colistimethate, and

Fluconazole.

6. Prepare 10 mL aliquots of the cocktail solution into 15-mL

conical centrifuge tubes.

7. Store them at 20 ^C. Aliquots are stable at 20 ^C for up to

6 months.

3.1.2

DMEM +

Antibiotics

Inside the BSC under sterile conditions:

1. Measure 430 mL of Dulbecco’s Modified Eagle’s Medium

High Glucose L-Glutamine (DMEM).

2. Add:

(a)

50 mL of fetal bovine serum.

(b)

10 mL of cocktail of antibiotics.

(c)

10 mL of Gibco™Antibiotic-Antimycotic (100).

(d)

0.5 mL of Gibco™Gentamicin (50 mg/mL).

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