Labelled E coli

This method will produce highly-fluorescent, heat-killed E. coli for phagocytosis assays. This assay has been optimised for ML35, but should work for other strains of E. coli. Method can be scaled linearly to produce larger numbers of bacteria. Similar methods can be used to label other types of bacteria, including both Gram-positive and Gram-negative, but alternative killing methods may be required . Can also be performed on live bacteria.

Labelling Procedure

1. Grow ML35 overnight to stationary phase in LB media, 37°C with 200 RPM shaking. 5 mL culture size is recommended, but smaller cultures can be made.
2. Place 100 µl of the overnight culture into a 1.5 mL snap-cap tube and heat kill by incubating at 70°C for 10 minutes, without agitation.
3. Rinse sells 2x with 1 mL PBS, using a 30 sec/6000 RPM spin in the minifuge.
4. Suspend in 100 µL of PBS and add 0.5 µL of Cell Proliferation Dye eFluor 670. (see notes 1,2)
5. Incubate at room temperature for 20 minutes, protected from light.
6. Quench excess dye by addition of 900 µL of LB broth, followed by a 3 min incubation at room temperature.
7. Pellet cells using a 30 sec/6000 RPM spin in the minifuge.
8. Suspend bacterial pellet in 100 µL of PBS.

Use

For experiments where bacteria are centrifuged onto cells:

1. Add 10-25 µL of the bacterial suspension per well of a 12-well plate
2. Spin plate at 1000 RPM for 1 min to force bacteria into contact with the phagocytes
3. Return cells to 37°C incubator and continue experiment as planned.

For live cell imaging experiments

1. Add 25-50 µL of the bacterial suspension to the Leiden chamber and mix well
2. Incubate and image at 37°C as per normal procedures

Notes

• Note 1: The amount of dye does not need to be scaled linearly with the rest of the protocol. 0.5 µL of dye is enough to label up to ~500 µL of the stationary-phase culture.
• Note 2: Other protein-reactive dyes can be used to provide bacteria that fluoresce at different wavelengths, but the dye volumes for these dyes and incubation timing will need to be determined on a dye-by-dye basis