Preparation of Supplemented Leishmania Translational Extract from Cell Concentrate

To begin, harvest the overnight cultures of Leishmania tarentolae. Measure the harvest optical density of the cultures at 600 nanometers, using a 1:10 dilution in culture medium. Calculate the target concentrate volume to achieve a final optical density of 300 at 600 nanometers.

Transfer the harvested cell culture into suitable centrifuge bottles. Centrifuge the bottles at 2, 500 G for 10 minutes at four degrees Celsius. Then carefully decant the supernatant into a receptacle for culture waste.

Wash the cell pellet in 500, 200, and 20 milliliters of SEB buffer. Then pour the SEB resuspended concentrate into a suitable washed volumetric cylinder. Make up the volume to the target volume using cold SEB and then gently mix using a pipette.

To lyse the cell concentrate, transfer it into a pre-cooled nitrogen cavitation device. Pressurize to 70 bars and incubate for 45 minutes on ice. Next, open the vent on the cavitation device and expel the lysate into a cleaned vacuum receiver flask placed on ice.

Then transfer the lysate into centrifuge tubes and centrifuge at 10, 000 G for 15 minutes at four degrees Celsius. After transferring the supernatant to fresh tubes, centrifuge the lysate at 30, 000 G for 15 minutes at four degrees Celsius. Then transfer the supernatant to a fresh centrifuge tube placed on ice.

Set up four PD-10 gravity-fed gel filtration columns per 10 milliliters of lysate in a rack format that allows them to drip into a suitable collection tray. Add 2.5 milliliters of lysate into each column. Once it passes into the column, add 0.5 milliliters of elution buffer to settle the lysate.

Remove the waste tray and place a fresh collection tray beneath the columns. Then add 2.5 milliliters of elution buffer to elute the gel-filtered lysate. Collect the gel-filtered lysate in a fresh centrifuge tube.

Next, measure the absorbance of the filtered lysate at 280 nanometers with a NanoDrop spectrophotometer. Add five times feeding solution to the lysate in a 2:5 ratio. Then vortex the mixture thoroughly.

Aliquot the mixture into 1.5 milliliter microcentrifuge tubes and then snap-freeze the tubes in liquid nitrogen for future use.