ESO wikiadmin at 08:07, 2 June 2025

2025-06-02T08:07:34Z

← Older revision		Revision as of 10:07, 2 June 2025
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			{{Protokoll-Mix\|Inhalt=Medium: DMEM; 10% CS<br>
			PBS<br>Trypsin/EDTA<br>0.5% Saponine in 1xPBS<br>Gentamicin (50 ug/mL) in DMEM 0.5% CS}}

	== Procedure ==		== Procedure ==

ESO wikiadmin: Created page with "== 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: Deter..."

2025-06-02T08:04:05Z

Created page with "== 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: Deter..."

New page

== 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

Gentamicin Protection Assay - Revision history

ESO wikiadmin at 08:07, 2 June 2025