Phosphate buffered saline solution pbs

Two commercially available head and neck squamous cell carcinoma lines UM-SCC-1 and SCC-9 were used because of the lack of established, readily available conjunctival SCC lines. All lines were routinely screened for mycoplasma and mouse pathogens and cleared for murine experiments. UM-SCC-1 was kept in Dulbecco's modified Eagle medium (Gibco, Grand Island, NY, USA), 10% fetal bovine serum (Omega Scientific, Tarzana, CA, USA), 1% non-essential amino acids (Gibco), and 1% penicillin/streptomycin (Gibco). SCC-9 was kept in DME F/12 media (Gibco), fetal bovine serum (Omega Scientific), 400 ng/mL hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin/streptomycin (Gibco). On the injection day, cells were collected with trypsin (Gibco), washed with phosphate-buffered saline solution (PBS) (Gibco), counted, and suspended in PBS. Suspended cells were placed on ice and resuspended before each injection.

The overnight culture of E. coli was 10-fold diluted in LB broth and was used for acid treatment. Acid treatment was carried out in LB containing 5.5% (vol/vol) of 90% lactic acid (Sigma-Aldrich, Oakville, ON, CA) with the pH adjusted to around 2.9, followed by sterilization using 0.02-μm filtration units (Nalgene, VWR, Edmonton, AB, CA). Aliquots of 100 μL of cell suspension in LB broth of each strain were added to 0.9 mL acidified LB broth and LB broth without the addition of lactic acid or adjustment of pH (control), followed by incubation in ice water for 1 h. At the end of the treatment, 100 μL of treated and control samples were transferred into 9.9 mL of phosphate-buffered saline solution (PBS) (Fisher Scientific, Ottawa, ON, Canada) to neutralize the solution and stop the treatment. After neutralization, the pH of the cell suspension in PBS ranged from 6.5 to 6.8 as measured by a pH meter (Fisherbrand accumet AP115, Fisher Scientific). Cell suspensions in PBS solution were serial-diluted in PBS, and then 1 mL of appropriate dilutions was plated onto Aerobic 3M Petrifilm Aerobic Count Plates (3M Corp., St. Paul, MN, USA). Plates were incubated at 35°C for 18 h. The colonies were then counted following the manufacturer’s instructions and regarded as surviving E. coli.

All solutions were made using filtered, sterile, deionized water (Direct-Q 3 Millipore, Billerica, MA, USA). All glassware was cleaned with 70 vol% isopropyl alcohol (Supelco, Burlington, MA, USA). 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Perfluorobutane gas (n-C₄F₁₀, PFB) was purchased from FluroMed (Round Rock, TX, USA). Polyoxyethylene-40 stearate (PEG40S) and chloroform were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sterile saline solution and phosphate-buffered saline solution (PBS) were purchased from Fisher Scientific (Pittsburg, PA, USA). The purity of all the reagents was ≥99%, and they were used as received without further purification.