Granulocyte oxidative burst

Materials	and Preparations
	2 days before experiment, streak out gonococci and select the colony phenotype one day before experiment.
	Chemiluminescense buffer (CL-Buffer): 8 g NaCl; 0.2 g KCl; 0.62 g KH2PO4; 1.14 g Na2HPO4; 2.5 ml 40% glucose; 50 mg BSA; solve in 800 ml bidest. H2O; adjust pH to 7.2 and fill it up to 1 liter; steril filter and store at 4°C.
	0.2 mg/ml PMA (Phorbol-12-myristat-13-acetat; positive control) in DMSO
	Luminol (2 mg/ml in 5% DMSO; stored at -20°C)
	Spectrofluorometer (Thermo Varioskan Flash) Pre-heat up to 37°C (Adjust under settings/Options/Temperature).

Procedure

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• After granulocyte isolation (check protocol). Resuspend in CL-Buffer and count the granulocytes manually; adjust the cell density to 1 x 106 cells/ml.
• Transfer 700 µl (7x105) granulocytes in sterile 1.5 ml Eppis.
• Add inhibitor or solvent and incubate for 15 min on rotating wheel (speed 4) at 37°C. PRE-HEAT THE SPECTROFLUOROMETER AT 37°C
• Place 3 x 200 µl granulocyte suspension in three triplicate wells (2x105 cells) in a 96-well plate (white, flat bottom).
• Add 2 µl (equivalent: 4 µg) luminol per well (luminescense background measurement).
• Infect with gonococci (or other bacteria) at MOI 50 (1 x 10⁷ bacteria/ well). Bacteria are suspended before in CL-buffer at 4 x 10⁸ bacteria/ml, so that 25 µl of bacterial suspension in CL-buffer are added to each well. As positive and negative control, add 1 µl PMA (1µg/ml final concentration; positive control) or plain 25 µl CL-buffer (negative control).
• Shake the plate with granulocytes and bacteria carefully to uniformly distribute the cells.
• Put the 96-well plate in the spectrofluorometer.
• Measure the luminescence every 2 min for a total of 100 min (Kinetic Loop - Normal luminescence (i.e. without filter) Dynamic Range: medium, each measuring time 1000 ms).