Encyclopedia of Experiments: Immunology
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Transcript
Prepare serial dilutions of heat-inactivated serum samples in sterile RPMI1640 MOPS medium. Add 100 microliters of the selected serum dilution to each well of the 96-well microtiter plate.
In column 10, add only RPMI1640-MOPS medium for the fungus-only positive control. Add a 1-to-50 dilution of serum to all wells in column 11 to serve as the no-fungus plus serum negative control. After covering the plate with a lid and aluminum foil, incubate the plate for 24 hours at 37 degrees Celsius.
The next day, after aspirating the serum carefully using a multichannel pipette, tap the inverted plate gently to remove any residual serum. Repeat the PBS wash three times, and air-dry the plate for 30 minutes at room temperature inside a biological safety cabinet to dry any excess PBS.
Then, dissolve 25 milligrams of XTT in 50 milliliters of filter-sterilized Ringer's lactate. Aliquot 10 milliliters in separate tubes. Cover it with aluminum foil, and store it at minus 80 degrees Celsius. Next, dissolve 8.6 milligrams of menadione in 5 milliliters of acetone, and after distributing 50 microliters in 100 separate microtubes, store the aliquots at minus 80 degrees Celsius.
Take 10 milliliters of XTT, and add 1 microliter of menadione to obtain a 1-micromolar working solution. Add 100 microliters of the XTT menadione solution per well of the 96-well microtiter plate. Next, after covering the plate with a lid and aluminum foil, incubate the plate for two hours at 37 degrees Celsius in the dark. Finally, transfer 80 microliters of the colored supernatant from each well into a fresh 96-well plate, and read the plate at 490 nanometers.
