Salmonella infection assay for autophagy visualisation and quantification - version 2.0, Nov 2011

Purpose: To infect cells with Salmonella Typhimurium for quantification of autophagy or immunofluorescent staining.

Day before assay:

Set up O/N culture of Salmonella Typhimurium SL1344 DsRed2 in 3 ml LB + amp and shake at 37 C, 250 rpm.
Plate HeLa cells (GFP-LC3 expressing if measuring autophagy) in 12-well plates on coverslips at 1x105 cells per well.  If transfecting and waiting a further 24/48 hours before infection, plate at a lower density. Highly-confluent cells are more difficult to efficiently infect.

Day of assay:

1. Dilute O/N bacterial culture 1:33 in 3 ml of LB + amp and shake for 3h at 37C.

2. Change media on cells to antibiotic-free DMEM (10% serum).

3. Place sufficient antibiotic-free DMEM (10% serum) for all wells to be infected in 37 C water bath to warm up (if you use cold media, the bugs will not invade).

4. Also warm up twice as much DMEM (10% serum) containing 100 ug/ml gentamycin.

5. Dilute your 3h bacterial subsulture 1:100 into warmed antibiotic-free DMEM and mix well.

6. Remove medium from wells and add 1 ml of bacteria-containing medium.  Incubate for 20 mins. (You can also spin at room temperature for 5 minutes at 700xg to increase invasion rates and better synchronise invasion.)

7. Wash once with high-gentamycin DMEM (100 ug/ml) and incubate in high-gentamycin DMEM for 40 minutes.

8. Wash once with PBS and fix in 4% formalin in PBS for 30 mins.

9. Wash once with PBS, permeabilise with 0.1% Triton X100, 1% BSA in PBS for 2 minutes.

(If staining for other markers or epitopes, perform staining as normal here)

10. Wash with PBS and move coverslips to Parafilm.  Add 100 ul of 633 phalloidin (1:40 dilution of stock) and leave in the dark for 20 mins.

11. Wash 3 times with PBS and mount (if using a liquid mountant, e.g. Polymount, seal with nail varnish.