View source for Granulocyte FACS phagocytosis assay

{| class="wikitable"
|+
!Material
!
|-
|
|Freshly isolated granulocytes
|-
|
|Phagocytosis buffer (PBS, 0.9 mM CaCl<sub>2</sub>; 0.5 mM MgCl<sub>2</sub>; 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 ===

# Isolate granulocytes according to protocol. During granulocyte isolation, label bacteria with fluorescein so that isolated granulocytes can be infected without delay.
# 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).
# Isolated granulocytes are resuspended in phagocytosis buffer and adjusted to a concentration of 1X106/ml in eppi-tubes.
# 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. 
# 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.
# 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 % NaN<sub>3</sub>) and analysed by FACS.
# 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.
# 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.