This method can help answer key questions in the Dictyostelium and macropinocytosis fields such as whether a particular gene, protein or molecular pathway is involved in macropinocytosis. The main advantage of this technique is that many variables such as time, genetic mutations, inhibitor concentrations and media compositions can be tested in parallel, giving results a single-cell resolution. To begin this procedure, obtain cultivated cells on SM agar with bacteria as outlined in the text protocol.
Harvest cells from the feeding front into 25 milliliters of KK2 buffer in a 50 milliliter centrifuge tube. Vortex the tube to dissociate the cells and centrifuge at 300 G for three minutes. Next, discard the supernatant and resuspend the pellet in 50 milliliters of fresh KK2 buffer.
Repeat this wash three times by centrifuging, removing the supernatant and resuspending in 50 milliliters of KK2 buffer for each wash. After this, use a hemocytometer or other cell counting device to determine the cell density. Dilute the cells into HL5 medium containing antibiotics to a concentration of 100, 000 cells per milliliter.
Pipette 50 microliters of the diluted cell solution into each well of a flat-bottomed 96-well tissue culture plate, using three wells for each condition. Incubate at 22 degrees Celsius for 24 hours. Then, dilute TRITC-dextran to one milligram per milliliter in the medium used.
Add 50 microliters to each well for a final TRITC-dextran concentration of 0.5 milligrams per milliliter in each well. Incubate at 22 degrees Celsius for one hour. To begin preparing the cells for flow cytometry, add 50 microliters of dextran-containing media to the zero minute update controls.
Next, decant the medium from the plate and pat dry on a tissue. Wash the plate by submerging it in ice-cold KK2 buffer and then decant the buffer from the plate. Add 100 microliters of a solution containing ice-cold five millimolar sodium azide dissolved in KK2MC to each well.
Measure the fluid update by flow cytometry as outlined in the text protocol. Prepare a fresh flat-bottomed 96-well tissue culture plate as previously described, with three wells for each strain and condition. For a typical time course, plan on measuring fluid uptake at zero, 30, 60, 90, 120 and 180 minutes.
Add 50 microliters of dextran-labeled medium to the 180 minute time point wells. 60 minutes later add dextran-labeled medium to the 120 minute time point wells. Continue adding the medium like this sequentially for the entire plate.
At zero minutes, add 50 microliters of dextran-containing medium to the zero minute control wells. Then immediately decant the medium. Wash the plate by submerging it in ice-cold KK2 buffer then decant the buffer from the plate.
Analyze the plate using flow cytometry as outlined in the text protocol. Prepare a fresh flat-bottomed 96-well tissue culture plate as previously described, with three wells for each strain and condition. Prepare medium containing dextran at a concentration of one milligram per milliliter.
Use this medium to dilute the compound of interest to a concentration that is double the desired maximum final concentration. Then prepare a second tube with medium containing dextran at the same proportion of vehicle. Mix both solutions by vortexing.
Use these solutions to create a dilution series of the compound of interest in 200 microliters of dextran-containing medium per sample. Mix each sample by vortexing. Add 50 microliters of this medium to each sample well.
After one hour, decant the medium from the plate. Wash the plate by submerging it in ice-cold KK2 buffer and then decant the buffer from the plate. Analyze the plate using flow cytometry as outlined in the text protocol.
Representative flow cytometry data shows that when the cytometer is not blocked and the forward scatter and side scatter profiles of the cells are set properly, the cells can be easily distinguished. When looking for differences between mutants, it is likely that there will be one of three phenotypes. The mutants could have a normal fluid uptake, they could have a partial defect or fluid uptake could be completely abolished.
The average median fluid uptake is used to calculate the volume of fluid internalized. In a fluid uptake time course, the internalized fluorescence increases for 60 to 90 minutes. After which the dextran begins to be exocytosed and a plateau is reached.
The average median fluid uptake is also used when comparing data against a control, such as when treating cells with inhibitors that are effective against macropinocytosis. In treated cells, the dextran internalized in one hour is seen to decrease and almost drops to zero at higher inhibitor concentrations. Not all inhibitors are completely effective, such as nocodazole, which only inhibits up to 50%of fluid uptake by macropinocytosis.
Following this procedure other methods, like microscopy can be performed in order to answer additional questions like whether fluid uptake may be different in a particular condition, such as whether cells with reduced macropinocytosis make fewer or smaller macropinosomes.
