Obtaining Cytotoxic Amyloids Following Bacterial Infection of Rat Pulmonary Endothelial Cells

Abstract

Source: Balczon, R., et al. Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa. J. Vis. Exp. (2018)

This video demonstrates the procedure for generating cytotoxic supernatant from pulmonary endothelial cells following infection with Pseudomonas aeruginosa. It outlines how bacterial effector proteins disrupt host cell structure and induce the release of cytotoxic amyloid oligomers into the supernatant.

Protocol

1. Generation of Cytotoxic Supernatants:

Primary cultures of rat pulmonary microvascular endothelial cells (PMVECs) were obtained from the Cell Culture Core Facility at the University of South Alabama’s Center for Lung Biology.

1. For generation of cytotoxic supernatants, plate 2 x 10⁶ cells into individual 150 cm dishes and grow for 3 - 4 days until confluence is achieved. Use two plates per experiment.
2. To produce supernatant, wash one of the two 150 cm dishes of PMVECs with phosphate-buffered saline, trypsinize, and then count the cells. Wash the other dish of PMVECs with Hank’s Balanced Salt Solution (HBSS) and then infect with either strain of Pseudomonas aeruginosa at a multiplicity of infection (MOI) of 20:1. This is achieved as follows:
   1. For P. aeruginosa, OD₅₄₀ 0.25 = 2 x 10⁸ CFUs/mL. To prepare this dilution, add 1 mL HBSS to a 1 mL disposable cuvette. Scrape enough bacteria off of the plate until an OD₅₄₀ of 0.25 is achieved when measured using a spectrophotometer. The next step will provide a calculation of how much bacteria will be needed.
   2. Once the number of PMVECs in the culture dish is determined, determine the appropriate number of bacteria to be added to the second, non-trypsinized culture dish containing the PMVECs. For example, if it is determined that the culture dish of PMVECs contains 1.3 x 10⁷ cells, then prepare 1.3 x 10⁷ cells x 20 bacteria/cell x 1 mL/2 x 10⁸ CFUs = 1.3 mL bacteria.
   3. Dilute the 1.3 mL of bacteria into HBSS to a final volume of 20 mL, so that the entire surface of a 150 cm dish can be covered with fluid, then add the diluted bacteria to the plate of PMVECs.
   4. Incubate the PMVECs inoculated with the bacteria at 37 °C in a 5% CO₂ incubator for 4 - 5 h, until gaps can be seen forming in the cell monolayer when the plate is observed microscopically (See Figure 1 for proper level of gap formation).
   5. Collect the supernatant, then centrifuge for 10 min at 2,000 x g in a tabletop centrifuge to remove cellular debris.
      Note: Any table top centrifuge capable of generating sufficient g force to pellet unlysed cells and large cell fragments can be used for this step.
   6. Pour the supernatant into a syringe that has a 0.2 µm filter attached to its end, then pass the supernatant through the filter to remove bacteria.
   7. Use an aliquot of the sterile supernatant to test cytotoxicity and freeze the rest of the supernatant at -80 °C for future use.

Results

Figure 1. Effects of P. aeruginosa inoculation on PMVEC morphology. A confluent monolayer ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HBSS	Gibco	14025-092
PBS	Gibco	10010-023
0.22 micron syringe filters	Millipore	SLGP033RS
DMEM	Gibco	11965-092
Countess Automated Cell Counter	Invitrogen	C10277
Microcetrifugation concentrators- 10,000 MW cut-off	Millipore	UFC801008
Strains PA103 and ΔPcrV			These strains of P. aeruginosa were obtained from Dr. Dara Frank, University of Wisconsin Medical College, Milwaukee, WI
Agar	Fisher	BP1423-500
Fetal bovine serum	Hyclone	SH30898.03IH
PenStrep	Gibco	15070063