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

2025-02-27T12:35:01Z

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

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{| class="wikitable"
|+
!Materials
!and Preparations
|-
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|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.
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|0.2 mg/ml PMA (Phorbol-12-myristat-13-acetat; positive control) in DMSO
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|Luminol (2 mg/ml in 5% DMSO; stored at -20°C)
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|Spectrofluorometer (Thermo Varioskan Flash) Pre-heat up to 37°C (Adjust under settings/Options/Temperature).
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=== 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).

Granulocyte oxidative burst - Revision history