High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment. Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria. This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.


Introduction
Large-scale non-specific fluid uptake by macropinocytosis is important in several biological contexts 1 , including antigen sampling by immune cells 2 , pathogen entry into host cells 3 , cancer cell proliferation 4 and the spreading of prion diseases 5 . In mammalian and Dictyostelium cells, actin 6,7 , PI(3,4,5)P3 8,9,10 (although the exact nature of the lipid differs between the two 11 ), activated Ras 12,13 , and activated Rac 14,15 are important for efficient fluid uptake by macropinocytosis, although there remain many unanswered questions about how the macropinocytic patch is formed, organized and eventually internalized. Discovering more proteins important for macropinocytosis, and subsequent determination of how they are important in the various biological contexts, will give a more comprehensive understanding of macropinocytosis and potentially allow development of targeted treatments for a range of conditions.
Dictyostelium is an ideal model system for studying macropinocytosis. The high level of constitutive macropinocytosis in standard laboratory strains means that fluid uptake is over 90% due to macropinocytosis 6 . This allows macropinocytosis to be measured solely by determining fluid uptake, unlike mammalian cells where the proportion of fluid uptake due to macropinocytosis is much lower. That macropinocytosis is so well defined and easily visualized 12 in this system similarly offers distinct advantages for investigating core conserved components of the macropinosome over other systems where there may be multiple regulatory signals 16,17 .
The standard technique used to measure macropinocytosis by mammalian cells involves fixing cells after pulsing with dextran for a short period of time followed by microscopy to determine the area of a cell that is occupied by dextran-positive vesicles 18 . This technique does not however account for the possibility of macropinosomes shrinking upon entering the cell, which has been reported in Dictyostelium 19 , and only takes into account single planes of the cell, meaning the volume internalized is unclear. An alternative technique, of counting the number of macropinosomes internalized in a given time, has the same downsides 20 . Using Dictyostelium avoids these issues; however, existing techniques for measuring fluid uptake by Dictyostelium are relatively labor intensive, using a large amount of both cells and dextran 21 . Cells are shaken at high density in fluorescent dextran and samples removed at various time-points for determination of the internalized fluorescence using a fluorimeter. Cells prepared this way can be analyzed by flow cytometry to gain single cell, rather than population-level, resolution 22 1. Cultivate cells either on SM agar plates in conjunction with bacteria such as Klebsiella aerogenes, or in nutrient medium such as HL5 as described 23 . NOTE: Growing knockout mutants that are defective in macropinocytosis on bacteria helps prevent accumulation of secondary mutations that may increase the rate of macropinocytosis. Keep the passage number below 4 after plating cells from stocks for optimum results. Mutants should be stored as frozen stocks as soon as possible after isolation. 2. To cultivate cells on SM agar plates, grow K. aerogenes to confluence in SM medium. Add 200-300 μL of bacteria onto an SM agar plate and spread. Take a sterile loop of cells (when transferring from another bacterial plate) and spread at one edge of the plate. Incubate at 22 °C for up to a week. 3. Dissolve Tetra-methyl-rhodamine isothiocyanate (TRITC-) dextran to 50 mg/mL in water, filter using 0.22 μm filters into 1 mL aliquots and store at -20 °C. Aliquots can be kept indefinitely. NOTE: 155 kDa is the typical size used with this method, as it can be bought cheaply in bulk, but smaller dextrans measure macropinocytosis just as effectively in Dictyostelium 24 . Different non-quenchable dextrans, such as cascade blue or Alexa-647, are alternatives if different fluorophores are required.

Converting Qualitative Measurements into Quantitative (Optional)
1. Perform a fluid uptake assay using cells in suspension.
1. Pellet axenically growing cells at 300 x g for 3 min, discard the supernatant and resuspend in nutrient medium to 1x10 7 cells/mL. Add TRITC-dextran to 0.5 mg/mL and shake at 180 rpm, 22 °C. 2. Dilute 0.8 mL samples into 0.7 mL of ice-cold KK 2 at 0, 30, 60, 90, 120 min. Wash once in 1.5 mL of ice-cold KK 2 buffer 23 in a benchtop centrifuge, resuspend to 1 mL in the same buffer and store on ice. NOTE: washing immediately or once all samples have been collected yields equivalent results. NOTE: This is the reference for all future fluorescence measurements on the flow cytometer: before using again run the beads and adjust the settings so they have the same fluorescence. This will give a very narrow defined peak. Putting a gate around the fluorescence peak can make this easier. 5. Repeat three times and plot the cell fluorescence data obtained in sections 2.2 and 2.3 against each other. Plot a line of best fit and use the equation to convert the qualitative data from the flow cytometry into quantitative units. 1. Set up a flat-bottomed 96-well tissue-culture plate ( Figure 1A). Use cells grown on bacteria and to transfer them into HL5 medium (containing 100 µg/mL of dihydrostreptomycin, 100 µg/mL ampicillin and 50 µg/mL kanamycin) 24 h before the assay; this allows macropinocytosis to be upregulated from the low levels seen in cells grown on bacteria 24 . Alternatively, dilute cells directly from axenic culture, incubating for 24 h before the assay, to reduce errors due to the cells being diluted from different densities.

Measuring Fluid Uptake
1. Harvest cells from the feeding front into 25 mL of KK 2 in a 50 mL centrifuge tube. Dissociate cells by vortexing and pellet at 300 x g for 3 min. Discard the supernatant, resuspend the pellet and wash 3 times at 300 x g for 3 min in 50 mL of KK 2 , discarding the supernatant each time to remove the bacteria. 2. Determine the cell density (using a hemacytometer or other cell counting system) and dilute into HL5 containing antibiotics to 1 x 10 5 cells/mL. Pipette 50 μL into each well, using three wells for each condition. Incubate at 22 °C for 24 h. Remember to set up a 0 min uptake control. NOTE: An alternative medium, e.g. SIH, VL6 can be used instead. Figure 1B).

Load cells with TRITC-dextran (
1. Dilute the dextran to 1 mg/mL in the medium used (this can be increased to 2.5-5 mg/mL when assessing cells with very low uptake). Add 50 μL to each well (giving a final concentration of 0.5 mg/mL TRITC-dextran) and return the plate to 22 °C for 1 h, as this allows significant dextran accumulation but exocytosis of dextran has not yet begun. NOTE: A repeater pipette allows this step to be done more rapidly, reducing error. Figure 1C). 1. Immediately prior to washing, add 50 μL dextran-containing media to the 0 min uptake controls. 2. Decant the medium and pat dry on a tissue. Wash by submerging the plate in ice-cold KK 2 , then decant. NOTE: some strains with adherence defects may become detached during this step, e.g. a knockout of both homologs of Talin (talA-/ talB-) 25 . Use caution when working with cell lines that attach poorly and aspirate media if required. 3. Add 100 μL of ice-cold 5 mM sodium azide dissolved in KK 2 MC (KK 2 + 2 mM MgSO 4 and 100 μM CaCl 2 ).

Prepare cells for flow cytometry (
CAUTION: Sodium azide is extremely toxic. Use a dust mask and safety glasses when working with the powder. Always wear gloves and lab coat. Avoid heat and do not mix with acid. NOTE: Cells rapidly detach, and exocytosis is prevented 24 . A microscope can be used to check this. 4. Measure fluid uptake by flow cytometry 1. If planning to convert relative values to absolute ones, use beads to standardize flow cytometer settings (see section 2). Alternatively, use cells loaded with dextran as in section 2 to ensure that the forward scatter and side scatter are set appropriately to isolate cells (Figure 2A), ensuring the machine is not blocked, (Figure 2B) and adjust the parameters to measure the internalized fluorescence ( Figure 2C).  (Figure 2A). Calculate the median fluorescence of the cells using the statistics option. Set these parameters using one sample and apply to all samples. NOTE: The forward scatter/side scatter profiles can vary between samples when using different mutants or inhibitors. 2. Determine the mean of the three wells for each condition and subtract the 0 min uptake control. Either convert to quantitative values using the equation determined in section 2 or normalize to the control.

Representative Results
Once the technique has been performed and cells are loaded with dextran and ready for analysis (Figure 1), ensure the flow cytometer is not blocked and adjust the forward scatter/side scatter profile to look like the cells shown in Figure 2A. If the machine is blocked, it will look more like that shown in Figure 2B and must be unblocked before continuing. Ensure the parameters show control axenic cells have high internalized dextran fluorescence at longer time points and low internalized fluorescence at shorter ones ( Figure 2C).
When looking for differences between mutants, it is likely that there will be one of three phenotypes. The mutants could have normal fluid uptake, they could have a partial defect, or fluid uptake could be completely abolished. Figure 2D shows a strain with normal fluid uptake, in this case the standard laboratory strain Ax2, a mutant with a ~50% decrease in fluid uptake (Ax2 rasG-

Discussion
Whereas other methods to assess fluid uptake are low throughput, washing the cells in situ and the use of sodium azide to detach cells are the critical steps in this method, which allow high-throughput measurement of macropinocytosis, membrane uptake, or phagocytosis by Dictyostelium. As the cells are attached to a surface and the medium is not, they can be left attached while the medium around them is first thrown off and then changed by immersion in buffer and thrown off again. Sodium azide, which depletes cellular ATP and depolarizes the membrane 30 , is then used to detach the cells, and also prevents exocytosis without affecting cell viability 24 . While using flow cytometry to measure macropinocytosis by Dictyostelium gives a very accurate measurement of fluid uptake very quickly, to establish the reason why a particular strain or condition has altered fluid uptake, further investigation using microscopy is required 24 . It should also be noted that previously published results have, in some cases, shown a difference in fluid uptake by mutant strains grown either on a surface (as in this case), or in shaking suspension (as in the standard protocol) 31 . Using this method may mean that, in rare cases, apparent fluid uptake defects are missed. Additionally, when measuring phagocytosis, only low concentrations of particles can be used. The maximum rate of phagocytosis that can be determined with this technique is far below the real maximum, although it is still possible to measure relevant differences in phagocytosis between strains and conditions 24 . To determine the maximum rate of phagocytosis, uptake must be measured in shaking suspension by an alternative protocol 27 . Cells that have phagocytosed beads have increased side scatter, so this should be corrected for accordingly when setting up the flow cytometer.
Flow cytometry can be used to measure fluid uptake in mammalian cells 32 , however the higher proportion of fluid phase uptake by other endocytic pathways than seen in Dictyostelium is a concern. In addition, cells are typically detached using trypsin at 37 °C, allowing further endocytic progression of internalized dextran. Ice-cold sodium azide does not cause macrophages to detach from a surface (Williams, unpublished observation), making this technique not applicable to mammalian cells without further optimization.
High throughput measurement of macropinocytosis has the potential to be used to screen quickly and cheaply for the effects of inhibitors, genetic mutation or gene knockdown on Dictyostelium cells. Mutants should always be compared to their direct parent only. If the reader has no prior preference for Dictyostelium strain, non-axenic strains such as DdB or NC4 are more "wild-type" than axenic ones and can be manipulated as effectively as axenic strains 33 . Otherwise, Ax2 strains are the axenic strains with the fewest genome duplications 34 , while many strains of Ax4 are Talin A knockouts and should be avoided if possible 23 . Most previously published strains can be ordered from the Dicty Stock Center 35 . This technique allows greater investigative possibilities than was previously possible into the effects of different conditions, inhibitors and mutations on macropinocytosis by Dictyostelium.