Cowell1 project protocol

Host response to Staphylococcus aureus infection in females of 13 inbred strains of mice   (2012)

Cowell LG, Fowler VG
With: Johnson NV, Ahn SH, Deshmukh H, Levin MK, Nelson CL, Scott WK, Allen A




Cowell1 Protocol

Project protocol - Contents

Workflow and sampling

Step
Procedure
Data collected
1
 Cohort 1: Injected with S. aureus for survival study (max 104 h)
Survival time ( in hours)
2
 Cohort 2: Injected with S. aureus for determining bacterial load (at 24h)
colony forming units (kidney and peritoneal fluid)
3 Cohort 3: Injected with S. aureus for determining serum IL-6 levels (at 24h; compared to uninjected controls) IL-6 levels

Equipment and supplies

  • Syringes and needles
  • Dissection kit
  • Trypic Soy Agar plates

Reagents and solutions

  • Phosphate buffered saline (PBS)
  • Bicinchoninic acid method kit (Pierce)
  • ELISA kit (Due Kit, Invitrogen)

Procedure: Bacterial preparation and injection

    1. Sanger 476 strain of Staphylococcus aureus is used.
    2. Mice are injected intraperitoneally with 107 colony forming units per gram (CFU/g) of S. aureus.

Procedure: Survival study

    1. S. aureus injected mice are monitored every 8h for 5 days.
    2. Mice are euthanized using CO2 asphyxiation if they appear moribund.
    3. Survival times are recorded in hours (max 104 h).

Procedure: Bacterial load

    1. S. aureus injected mice are euthanized 24h post-infection.
    2. Kidneys are collected and homogenized in PBS; diluted serially with PBS 10-fold.
    3. Peritoneal fluid is collected; diluted serially with PBS 10-fold.
    4. Serial dilutions (50 µL) are plated on Tryptic Soy Agar plates and incubated overnight at 37°C.
    5. Bacterial colonies are counted (CFU/g for kidney; CFU/mL for peritoneal fluid).

Procedure: Serum IL-6 levels

    1. S. aureus injected mice are euthanized 24h post-infection.
    2. Blood is obtained by intracardiac puncture.
    3. Serum is separated by centrifugation.
    4. Total protein is determined using the bicinchoninic acid method.
    5. Serum is diluted to ensure equal amounts of total protein.
    6. IL-6 levels are estimated using an enzyme-linked immunosorbent assay (ELISA).
    7. Fold-change is determined compared to uninfected mice of the same strain.

Data collected by investigator

  • Survival (hours post-infection; max=104 h)
  • Bacterial load: kidneys
  • Bacterial load: peritoneal fluid
  • Serum IL-6 levels (fold-change compared to uninfected controls)