ESO wikiadmin: Created page with "{| class="wikitable" |+ !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 in..."

2025-05-20T14:03:29Z

Created page with "{| class="wikitable" |+ !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 in..."

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{| class="wikitable"
|+
!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

Bacterial invasion assay by flow cytometry - Revision history