AFP solution
Fungal solution (10⁵ CFU/mL)
Positive control drugs
96-well plates
LB medium or MHB (Mueller-Hinton Broth) medium
Pipettes and tips
Spectrophotometer
Hemocytometer
PBS (Phosphate-Buffered Saline)
Microplate reader
Sterile water
Bacterial Fluid Resuscitation
Inoculate the target strain in liquid medium (LB or MHB), gently blow and mix.
Incubate the culture at 37°C for 12-18 hours to obtain high-density bacterial solution.
Bacterial Solution Dilution
Remove the culture, centrifuge at 12,000 rpm, and aspirate the supernatant.
Add 1 ml PBS solution, resuspend, centrifuge again, and aspirate the supernatant.
Resuspend with PBS and dilute to 10⁵ CFU/mL using 1640 medium.
Drug Dilution
Take 10 μL of the drug and add 90 μL of 1640 medium for a 10x dilution.
Use fluconazole as a control and prepare a 128 μg/μL solution.
Concentration Determination
Add the drug solution to the first row of a 96-well plate in triplicate for each drug.
Incubate at 25°C for 2-3 days, then observe turbidity or measure OD600.
Pipette tips
Fungal solution (10⁵ CFU/mL)
Positive control drugs
96-well plates
Mouse blood (pre-collected)
Centrifuge
Microplate reader
Plasma Dilution and Treatment
Take 100 μL of plasma, add 900 μL of saline, and mix gently to dilute the plasma.
Centrifuge at 2,000 rpm for 5 minutes, discard the supernatant, and repeat 3 times.
Preparation of 2% Plasma Suspension
Resuspend the plasma pellet in normal saline to create a 2% plasma suspension.
96-well Plate Preparation
Add 100 μL of the suspension to the first row in triplicate (2% and 4% Triton X-100 as positive controls).
Mix each row and dilute serially, incubating at 37°C for 1 hour.
Optical Densitometry
Centrifuge at 2,000 rpm for 5 minutes, aspirate the supernatant, and measure OD at 600 nm.
KM mice (Mus musculus)
Fungal solution (1.5×10⁹ CFU/mL)
Chlorpromazine solution (for anesthesia)
Velcro and sterile tools
Model Preparation
Create a wound on the back skin of anesthetized mice using velcro (1.5 cm x 2 cm).
Apply fungal suspension on the wound and incubate for 12 hours.
Treatment
Apply antifungal peptide or control solution to the wound every 12 hours for 3 treatments.
Dissection and Homogenization
12 hours after the final treatment, dissect and homogenize the mouse skin tissue for CFU quantification.