High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

1. Preparation of Cells and Materials

1. Cultivate cells either on SM agar plates in conjunction with bacteria such as Klebsiella aerogenes, or in nutrient medium such as HL5 as described²³.
   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 Dictyostelium²⁴. Different non-quenchable dextrans, such as cascade blue or Alexa-647, are alternatives if different fluorophores are required.

2. 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 1×10⁷ 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 KK₂ at 0, 30, 60, 90, 120 min. Wash once in 1.5 mL of ice-cold KK₂ buffer²³ 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.
2. Determine the volume of fluid internalized by the cells.
   1. Set the fluorimeter to measure fluorescence using an excitation wavelength of 544 nm and emission wavelength of 574 nm with a 10 nm slit. Set up a dilution series of TRITC-dextran and use it to measure the fluorescence for given volumes of dextran. Create a calibration curve.
   2. Measure the fluorescence internalized by the cells in section 2.1 using 0.9 mL of the cell suspension. Calculate the volume of fluid internalized per cell using the calibration curve²⁴.
3. Dilute the remainder of the cells from each time point separately into 0.5 mL ice cold KK₂. Filter through a 70 µm cell strainer into 5 mL polystyrene flow cytometry tubes. Set the flow cytometer so that cells from each time point are distinct from each other and record their fluorescence (see sections 3.4 and 3.5).
4. Measure the fluorescence of calibration beads in the flow cytometer using the settings above and record it.
   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.

3. Measuring Fluid Uptake

Figure 1: Schematic of high-throughput measurement of macropinocytosis. (A) Grow Dictyostelium on an SM plate seeded with K. aerogenes bacteria (yellow). Harvest cells from the feeding front (orange), avoiding cells that are already developed (green), into 25 mL of KK₂ buffer. Vortex to dissociate, pellet by 3 min centrifugation at 300 x g, then wash 3 times in 50 mL of KK₂ buffer, discarding the supernatant each time. Resuspend to 1 x 10⁵ cells/mL in HL5 growth medium and add 50 µL into three wells per sample of a flat bottom 96-well plate. Incubate at 22 °C for 24 h. (B) Dilute TRITC-dextran to 1 mg/mL in HL5 growth medium from a stock solution of 50 mg/mL. Add 50 µL to each sample (excluding the 0 min uptake control wells), and incubate at 22 °C for 1 h, after which add the dextran to the 0 min uptake control. (C) Immediately decant the media, pat the plate on a tissue to remove excess medium and submerge in a bath of ice-cold KK₂ buffer, filling the wells to wash. Decant the buffer and pat dry again. Add 100 µL of 5 mM sodium azide dissolved in KK₂MC to detach the cells. Take to flow cytometer for measurement of internalized fluorescence. Please click here to view a larger version of this figure.

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 bacteria²⁴. 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.
   1. Harvest cells from the feeding front into 25 mL of KK₂ 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 KK₂, 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 10⁵ 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.
2. Load cells with TRITC-dextran (Figure 1B).
   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.
3. Prepare cells for flow cytometry (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 KK₂, 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 KK₂MC (KK₂ + 2 mM MgSO₄ and 100 μM CaCl₂).
      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 prevented²⁴. 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).
   2. Attach high-throughput sampling system and add plate. Set up a protocol to measure the fluorescence (for TRITC-dextran use a yellow-green laser to excite and measure in the 582 nm channel or similar) of up to 65 μL of the cell suspension and run.
5. Analyze flow cytometry results
   1. Gate on the cells using forward scatter and side scatter (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.

4. Performing Fluid Uptake Time-courses

1. Set up cells as in section 3, with three wells for each time point and strain/condition.
2. Add dextran labelled medium to the different wells sequentially. A typical time-course measures fluid uptake at 0, 30, 60, 90, 120 and 180 min. Add dextran-containing medium for the 180 min time point wells, followed 60 min later by adding it to the wells for the 120 min time point, etc. (Figure 3A).
3. At 0 min, add 50 μL of dextran-containing medium and immediately decant and wash the plate as in section 3.3. Analyze as described in section 3.5.

5. Dose Response Curves

1. Set up cells as described in section 3, with three wells for each strain and condition.
2. Dilute the compound of interest into medium containing 1 mg/mL dextran at double the desired maximum final concentration of the compound. Prepare a second tube containing medium with dextran with the same proportion of vehicle as the first. Vortex to mix.
3. Create a dilution series of the compound of interest in 200 μL dextran containing medium per sample (Figure 4A). Vortex to mix. Add 50 μL of the medium to each sample well for 1 h.
4. Wash and analyze as described in section 3.3 onwards.

6. Phagocytosis and Membrane Uptake

1. To measure membrane uptake, prepare the cells as described in section 3. Add 50 μL of medium containing 20 μM FM 1-43 for a final concentration of 10 μM. After loading, wash and prepare cells for flow cytometry as in section 3.3. Measure in the 585 nm channel or similar, using a blue laser to excite.
   NOTE: As membrane trafficking is more rapid than fluid-phase, 10 min is a more appropriate time point to use than 1 h²⁶. Alternative dyes that fluoresce only when incorporated into the membrane can be used instead.
2. Use either fluorescently labelled beads or bacteria to measure phagocytosis. Fluorescent yeast cannot be used, as they are inseparable from Dictyostelium by forward and side scatter²⁴. Set up cells as described in section 3 and analyze as described²⁷.
   1. To measure phagocytosis of beads add yellow-green labelled beads as described in section 3.2 to a final concentration of 5 x 10⁷ beads/mL (1.75 and 2 μm) or 1 x 10⁸ beads/mL (1 and 1.5 μm) for 1 h before washing and preparing for flow cytometry as in section 3.3. Measure in the 525 nm channel or similar, using a blue laser to excite.
      Note: Higher concentrations will lead to cell detachment.
   2. To measure phagocytosis of bacteria, add Texas-Red labelled Escherichia coli bioparticles as described in section 3.2 to 1 x 10⁸ bacteria/mL for 1 h before washing and preparing for flow cytometry as in section 3.3. Measure in the 610 nm channel or similar, using a yellow-green laser to excite.