The methodology used here is based on the reaction catalysed by hydrogenases of the functional hyphae. They will cleave the yellow tetrazolium salt MTT ([3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) to its purple derivative MTT-formazan, which can be dissolved in isopropanol and quantified by spectrophotometry.
- Effector cells (monocytes, macrophages, neutrophils)
- Yeast Nitrogen Base media with 2% glucose media (YNB) (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, kept in HBSS for up to 3 weeks.
- 50-ml polypropylene tubes
- 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
- 0.5% Sodium deoxycholate (Sigma Chemical CoO,. St. Louis, U.S.A.)
- RPMI-1640 w/ phenol red (Biochrom KG., Berlin, Germany)
- 5 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide; Sigma Chemical CO. St. Louis, U.S.A.) in RPMI-1640 w/v phenol red (10 x stock). Alternatively, RPMI-1640 with phenol red can be used.
- 10x MTT stock may be kept at refrigerator temperature in the dark for a week.
- 1x MTT (0.5 mg/ml in RPMI). 1 x MTT working solution can only be kept for one day.
- Acidic isopropanol (95 ml isopropanol + 5 ml HCl 1N)
- Sterile disposable 5- and 10-ml plastic pipettes
- 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)
- Sterile disposable 23G needles (Becton Dickinson)
1. Suspend conidia in 1x Yeast Nitrogen Base medium (YNB) with 2% glucose at a final concentration of 7.5x104 conidia/ml.
2. Aliquot 200 µl of conidia suspension into each well of a 96-well plate, final concentration of 15,000 conidia/well. The plate layout will vary depending upon experimental conditions e.g. number of isolates tested, the effector to target ratio (E/T ratio), choice of effector cell. Effector cell numbers are based on the numbers of conidia originally seeded into each well of the 96-well plate, e.g. 5/1 E/T ratio = 75,000 effector cells / 15,000 target hyphae. These values should be determined experimentally. Control wells into which no effector cells will be added should also be included.
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. e.g. for E/T ratios 1/1, 5/1, 10/1 and 20/1 a stock solution of 7.5x106/ml should be prepared, giving 15,000 cells in 2 µl; 75,000 cells in 10 µl; 150,000 cells in 20 µl and 300,000 cells in 40 µl.
5. With a 23G needle, aspirate the medium from each well and add effector cells at the appropriate E/T ratio. To avoid drying of the hyphae this step must be performed one row at a time and in stages. Using a 12-channel pipette add 110 µl of HBSS+ to each row of aspirated wells. Effector cell volumes and remaining volumes of HBSS+ may be added according to the table below, the final volume being 150 µl.
|E/T ratio||Volume of effector cell stock
|Volume of HBSS+
6. Incubate for two hours in 5% CO2 and at 37°C.
7. NOTE: ALL THE ASPIRATIONS SHOULD BE CARRIED OUT WITH A 23G NEEDLE.
8. 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.
9. Add 60 µl of 0.5% Sodium deoxycholate. This step must be performed quickly and carefully to avoid killing any viable hyphae.
10. Aspirate and add carefully 200 µl of sterile distilled water.
11. Aspirate and repeat step 10 twice.
12. Aspirate and add 200 µl of 1x MTT solution.
13. Incubate in 5% CO2 and at 37°C for about 3 hours.
14. Aspirate the wells dry and store at -20°C overnight.
15. Add 120 µl of acidic isopropanol to each well. Swirl until the blue colour is dissolved.
16. Transfer 100 µl from each well to a 96-well plate.
17. Read plate on the Elisa reader at a wavelength of 550 nm, blanked against 100 µl of acidic isopropanol.
18. % Killing = 100 - [(mean O.D. of E/T ratio x 100)/mean O.D. control].