JNAR Protocol

Bacterial killing assay protocol

Núria Crua· Postdoc· University of Lausanne (UNIL)

Published Jul 28, 2026

~75h 40m total· 0 contributions

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JNAR-ID: JNAR-97451750

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PublishedLast updated Jul 28, 2026

Objective

To quantify the intracellular killing capacity of host cells against a specific bacteria by measuring colony-forming units (CFU) after infection.

Classification

Sample typeCells

TechniqueCell Culture

Experimental contextIn vitro

Biological modelBacteria

Reproducibility status

Reproducible

No reproducibility blockers or risks reported. The protocol runs end-to-end.

17 healthy

⏱ Total 75 h 40 min2 sections17 steps

Day 0: Bacterial Inoculum and Cell Plating

48 h

Seed a 5 mL culture from the bacterial glycerol stock (-80°C) and grow overnight (16–24 h) at 37°C with shaking at 240 RPM.24 h

Plate 100,000 cells in 100 μL of RPMI + FCS (without antibiotics but with required cytokines) in a flat-bottom plate.

Incubate the plate at 37°C overnight in a cell culture incubator.24 h

Day 1: Infection and Killing Assay

27 h 40 min

From the overnight inoculum, transfer 400 μL and 600 μL into 5 mL LB and grow at 37°C with shaking (240 RPM) to reach OD600 = 0.4–0.5 (approx. 2.5 h).2 h 30 min

Measure OD600 of 1 mL culture to determine bacterial concentration

Critical step

WarningDetermine the optical density (OD) corresponding to the desired bacterial concentration (CFU/mL) for each bacterial strain.

Prepare the desired MOI (e.g., 1:50 for BMDM) in RPMI + FCS without antibiotics.

Critical stepAdjust the multiplicity of infection (MOI) according to your experimental conditions and bacterial strain.

Centrifuge the bacterial inoculum at 3000g for 5 min at RT. Discard supernatant and resuspend the pellet in RPMI + FCS without antibiotics.5 min

Remove old medium and add 100 μL of medium containing bacteria.

Centrifuge the plate for 5 min at 300 g at RT to enhance bacterial contact with cells.5 min

Critical stepCentrifugation promotes efficient bacterial uptake by host cells.

Incubate the plate at 37°C with 5% CO2 for 1 h to allow bacterial internalization.1 h

After 1 h, remove supernatant and wash wells 3 times with warm PBS to remove unattached bacteria.

Add 50 mg/mL Gentamicin in RPMI + FCS to each well to kill extracellular bacteria.

At the desired post-infection time point, carefully remove the culture supernatant and wash each well three times with room temperature (RT) PBS.

Critical stepWashing removes residual gentamicin and any remaining extracellular debris before lysis.

Add 100 µL of 0.1% NP-40 in PBS to each well to lyse the host cells and release intracellular bacteria. Incubate for 5 minutes at room temperature to ensure complete cell lysis.

TipMix gently until bubbles form, then wait 5 minutes for complete lysis.

Prepare serial dilution plate: Label tubes -1 to -6. Add 180 μL PBS to each tube. Add 20 μL from previous dilution to next tube (except -1, which receives 20 μL lysate). Discard 20 μL from the last tube (-6).

Serial Dilution Setup

Tube	Addition	Final Volume (μL)
-1	20 μL lysate + 180 μL PBS	200
-2	20 μL from -1 + 180 μL PBS	200
-3	20 μL from -2 + 180 μL PBS	200
-4	20 μL from -3 + 180 μL PBS	200
-5	20 μL from -4 + 180 μL PBS	200

15.1

Transfer 20 µL of the lysed bacterial suspension into tube -1. Mix thoroughly, then transfer 20 µL from tube -1 to tube -2. Repeat this serial dilution until the desired dilution is reached.

15.2

Discard 20 μL from the last dilution to maintain consistent volume across dilutions.

Plate 100 μL from the desired d onto agar plates.

Critical stepPlating multiple dilutions ensures at least one plate will have countable colonies (30–300 CFU).

Incubate the plates at 37°C overnight. Count CFU the next day.24 h