To begin, place frozen Naegleria gruberi trophozoites at 37 degrees Celsius for three minutes. Culture the trophozoites in PYNFH media at 25 degrees Celsius until approximately 80%confluent. Place the confluent flask on ice for five to 10 minutes to release the adhered trophozoites from the plastic.
Then gently swirl the flask to dislodge any remaining adherent cells. Replace the media with sterile encystment media to encyst the Naegleria gruberi cells. After 48 hours, remove the cysts from the flask.
Centrifuge the cysts at 600 x g for 10 minutes at four degrees Celsius. After centrifugation, resuspend the cysts in two milliliters of PYNFH media with 10%fetal calf serum to excyst the cells. Incubate the cysts at 25 degrees Celsius for 72 hours until trophozoites emerge and reach 80%confluency.
To transfect the trophozoites, first plate one milliliter of the trophozoites into a six-well, flat bottom tissue culture plate. Next, incubate the plasmid transfection reagent mixture at room temperature for 10 minutes. Pipette out approximately 800 microliters of medium from the trophozoites monolayers just prior to transfection.
Then add 200 microliters of the plasmid transfection reagent mixture to the trophozoites. Gently swirl the plate every 15 minutes to prevent the media from aggregating at the edges of the plate and drying out. After an hour, add 10 units of DNase in one milliliter of 10X DNase buffer and add it to the wells.
Incubate the plate at 37 degrees Celsius for 15 minutes to remove residual plasmid DNA. Now wash the plate once with one milliliter of growth medium, then add two milliliters of fresh media. Incubate the plate at 25 degrees Celsius for 24 to 36 hours.
When incubation is complete, split the contents of a well into a new plate at a 1:2 ratio. Then collect the trophozoites from the remaining wells for further analysis. The Naegleria gruberi trophozoites were amoeboid and adherent to the culture flasks under standard culture conditions.
Their incubation on ice resulted in round and non-adherent trophozoites. Incubation of the trophozoites in encystment media caused them to become encysted.
