Gentamicin Protection Assay

Materials

Medium: DMEM; 10% CS
PBS
Trypsin/EDTA
0.5% Saponine in 1xPBS
Gentamicin (50 ug/mL) in DMEM 0.5% CS

Procedure

• Aspirate cell culture medium with a pasteur pipette
• Rince attaching cells carefully with cold PBS, aspirate PBS
• Add 1.5 mL Trypsin/EDTA and study the detachment of the cells in the microscope
• Inactivate Trypsin/EDTA with 3.5 mL medium, transfer the cell suspension into a 15 mL centrifuge tube
• Pipette 20 mL of the cell suspension into a Neubauer counting chamber
• Centrifuge the remaining cells at 600 rpm for 3 min
• During centrifugation: Determine the cell number using a Neubauer counting chamber
• After centrifugation, carefully aspirate the supernatant and resuspend the cells in fresh medium at a density of 5x10 5 cells/mL
• Seed the cells in a 24-well plate, 1 mL/well (each group makes 4 wells)
• Infection:
• Collect the bacteria from plate and resuspend in medium (DMEM, 0.5% CS)
• Measure the OD 550 in a photometer
• Estimate the bacterial density (cfu/mL) with the help of the existing calibration curve and calculate the required volume of bacterial suspension per sample:
• multiplicity of infection (MOI) of 30 means that we will add 30 bacteria per 1 cell
• Infect the cells and incubate them for the desired time (1h or 2h) at 37°C
• Aspirate the medium carefully and replace with 1 mL/well gentamicin (50 μg/mL in medium) and further incubate for 45 min at 37°C
• Apirate gentamicin carefully and lyse the cells with 1 mL/well 0.5 % Saponin (in 1x PBS) for 15 min to recover the intracellular bacteria
• For all samples: make serial dilutions and plate the dilutions 10 -2 and 10 -3 onto agar plates