Granulocyte FACS phagocytosis assay

Material
	Freshly isolated granulocytes
	Phagocytosis buffer (PBS, 0.9 mM CaCl2; 0.5 mM MgCl2; 5 mM glucose; 1% heat inactivated FCS)
	5-(6)-carboxyfluorescein succinimidyl ester (FAM SE; CFSE, Fluorescein, FITC)
	Trypan Blue solution

Preparations

Two days before experiment: streak out gonococci on selective agar plates and select colony morphology one day before infection, when you streak on regular GC plates.

Procedure

1. Isolate granulocytes according to protocol. During granulocyte isolation, label bacteria with fluorescein so that isolated granulocytes can be infected without delay.
2. Bacteria are taken from plates (cultures not older than 18 h), suspended in PBS and bacterial suspension is set to 4 x 108 bacteria/ml. Bacteria are labeled with 5- (6)-carboxyfluorescein succinimidyl ester (FAM SE; CFSE) (2 mg/ml stock in DMSO) using a 1:2000 dilution (0.5 µl in 1ml of bacterial suspension) for 20 min in the dark at RT on a shaker. Centrifuge at 4000 rpm for 4 min and wash the pellet 3 times with phosphate- buffered saline (PBS).
3. Isolated granulocytes are resuspended in phagocytosis buffer and adjusted to a concentration of 1X106/ml in eppi-tubes.
4. If required, add pharmacological inhibitors at the desired concentration to the granulocytes and rotate (8 rpm) on a wheel rotating platform for 10 min at 37°C prior to infection.
5. Take 50 µl of labelled bacteria (2 x 107 bacteria) to infect 1 x 106 (1 ml) of granulocytes (resulting in a multiplicity of infection/MOI of 20). Leave one granulocyte sample uninfected. Incubate for 15 min at 37°C with 8 rpm.
6. Phagocytosis is stopped by addition of ice-cold PBS. Samples are washed and taken up in ice-cold FACS-buffer (PBS, 2 % h.i. FCS, 0.05 % NaN₃) and analysed by FACS.
7. Set up FACS parameters (FSC, SSC) with uninfected control, gate on granulocyte population. Set channel voltage on the flow cytometer while measuring CFSE fluorescence (488 nm excitation; 525 nm emission) using negative/ positive control samples. Measure 10.000 cells for each sample. Immediately after running of each tube, add 500 µl neutrophil-supension to 500 µl of trypan blue solution (0.4 mg/ml) for a final trypan blue concentration of 0.2 mg/ml. Mix and measure the fluorescence signals again.
8. To determine the number of phagocytosed bacteria use the percentage of CFSE- positive cells (percentage of cells with intracellular bacteria) and multiply this number by the mean fluorescence intensity of the CFSE-positive cell population. This number is refered to as the uptake index.