This procedure is used to quantify the O2- released by phagocytes in response to a given stimulus.
- Effector cells (monocytes, macrophages, neutrophils)
- Yeast Nitrogen Base media with 2% glucose media (Difco Laboratories, Detroit, U.S.A.). YNB is made and stored as a 10x concentrated stock solution and should be diluted in sterile distilled water before the experiment
- Conidial stock of test fungal isolates
- Complete Medium (CM; human serum (25% v/v) in RPMI-1640 w/v phenol red)
- 96-well flat-bottom cell culture cluster (Corning Inc., NY., U.S.A.)
- Adjustable 20-µl, 200-µl and 1000-µl pipettes and sterile disposable tips
- Adjustable 200-µl 12-channel pipette and sterile disposable tips
- Sterile disposable 1.5- and 5-ml tubes
- Sterile disposable 5- and 10-ml plastic pipettes
- 2 µg/ml stock PMA (Phorbol Myristate Acetate, Sigma Chemical Co., St. Louis, U.S.A.)
- 1M Cytochrome C (Sigma Chemical CO. St. Louis, U.S.A.)
- Hank's Balanced Salt Solution, without phenol red, calcium or magnesium (HBSS- Gibco, BRL, Life Technologies Ltd, Paisley, Scotland)
- Hank's Balanced Salt Solution, without phenol red, with calcium and magnesium (HBSS+ Gibco, BRL, Life Technologies Ltd, Paisley, Scotland)
1. Suspend conidia in 1x Yeast Nitrogen Base medium (YNB) with 2% glucose at a final concentration of 1.5x106 conidia/ml.
2. Aliquot 200 µl of conidia suspension into each well of a 96-well plate, final concentration of 300,000 conidia/well. The plate layout will vary depending upon experimental conditions e.g. number of isolates tested. Control wells should also be included (see below).
|Experimental parameters||Hyphae||Serum||Effector cells||PMA|
3. Incubate conidia at 32°C for approximately 16 hours. The conidia should be fully germinated, hyphae should be 100-120 µm long and have just reached confluence. Overgrowth should be avoided as this increases the risk of aspirating the lawn when washing. The time to confluence must be determined experimentally for each individual isolate tested. When confluence has been achieved, plates may be stored at refrigeration temperature for a maximum of 2 hours.
4. Estimate the total number of effector cells required and adjust the stock solution of effector cells in HBSS- as appropriate.
5. Aspirate wells in rows to avoid drying of hyphae and effector cells. A 12-channel pipette should be used during all of the remaining steps to facilitate processing of entire rows of the 96-well plate.
6. Opsonize the hyphae by incubating in 100 µl of 50% human serum in HBSS+.
7. Incubate the hyphae at 37°C for 30 minutes.
8. Aspirate and wash twice with 200 µl HBSS+.
9. Aspirate and add HBSS+, with 15 µl Cytochrome C, soluble stimuli if needed (e.g., 100 ng/ml PMA) and 300,000 effector cells, in that order. HBSS+ volume should be adjusted, according to the other variables, to a final total volume of 300 µl.
10. Incubate for 1 hour at 37°C in 5% CO2.
11. Transfer 2x140 µl to 96-well tissue culture plate.
12. Centrifuge at 2,000 rpm for 10 minutes if hyphae have been picked up and transferred with the supernatant.
13. Transfer 200 µl to a 96-well plate.
14. Read plate on the Elisa reader at a wavelength of 550 nm blanked against 200 µl of blanking wells (which contain HBSS and cytochrome C, but no stimuli or cells). nM O2- = OD · 660 / 29.5