Bacterial invasion assay by flow cytometry
| Materials (for 6 cm dishes) |
|---|
| CFSE |
| FACS buffer (1% (F)CS in PBS, 50-100 mL (ice-cold) |
| Trypsin/EDTA (1mL/dish) |
| Medium with serum according to cell type (3mL/dish) |
| 15 mL PP falcon tubes |
| Trypan blue solution, 0.4 mg/mL |
| Eppendorf tubes 1.5mL (1 per dish) |
Procedure
Bacteria:
- 2 days before the experiment, streak bacteria from glycerol stock on selective GC or TSB agar plates (with antibiotics when needed) and incubate at 37°C o/n
- Approx. 16h before experiment, select one clone and streak onto a fresh agar plate (without antibiotics) covering the whole area
Invasion assay:
- Collect bacteria from plate (with a wet swab) and resuspend in PBS
- FITC-labelling of bacteria according separate protocol: FBA-staining of bacteria
- Infect the cells (in 6 cm dishes) with desired MOI and incubate for desired time at 37°C
- Aspirate medium, wash once with PBS, and detach the cells by adding 1 mL Trypsin/EDTA
- Inactivate Trypsin/EDTA by adding 3 mL medium
- Separate cells by pipetting up and down several times, transfer the cell suspension into 15 mL centrifuge tube, and pellet the cells by centrifugation (3 min, 600 rpm)
- Resuspend pellet in 1 mL ice-cold FACS buffer and transfer the cells to 1.5mL Eppendorf tubes, then wash cells twice again (table-top centrifuge: 2 min, 2000 rpm, 4°C)
- Resuspend the cells in 1 mL FACS buffer and distribute to 2 FACS tubes (500 µL/tube), put on ice and keep dark until acquisition
- Further dilute the samples if necessary before acquisition
- to quench fluorescence of adhering/extracellular bacteria, add trypan blue (final concentration: 0.2 mg/mL) directly before acquiring the sample