We thank the Medical Research Council UK for core funding (U105115237) to RRK.

1. Preparation of Cells and Materials
<ol>
	<li>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.</li>
	<li>To cultivate cells on SM agar plates, grow K. aerogenes to confluence in SM medium. Add 200&#8211;300 &#956;L 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 &#176;C for up to a week.</li>
	<li>Dissolve Tetra-methyl-rhodamine isothiocyanate (TRITC-) dextran to 50 mg/mL in water, filter using 0.22 &#956;m filters into 1 mL aliquots and store at -20 &#176;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.</li>
</ol>
2. Converting Qualitative Measurements into Quantitative (Optional)
<ol>
	<li>Perform a fluid uptake assay using cells in suspension.
	<ol>
		<li>Pellet axenically growing cells at 300 x g for 3 min, discard the supernatant and resuspend in nutrient medium to 1x107 cells/mL. Add TRITC-dextran to 0.5 mg/mL and shake at 180 rpm, 22 &#176;C.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Determine the volume of fluid internalized by the cells.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Dilute the remainder of the cells from each time point separately into 0.5 mL ice cold KK2. Filter through a 70 &#181;m 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).</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Measuring Fluid Uptake
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58434/58434fig1.jpg" /> 
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 &#181;L into three wells per sample of a flat bottom 96-well plate. Incubate at 22 &#176;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 &#181;L to each sample (excluding the 0 min uptake control wells), and incubate at 22 &#176;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 &#181;L of 5 mM sodium azide dissolved in KK2MC to detach the cells. Take to flow cytometer for measurement of internalized fluorescence. <a href="https://www.jove.com/files/ftp_upload/58434/58434fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>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 &#181;g/mL of dihydrostreptomycin, 100 &#181;g/mL ampicillin and 50 &#181;g/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.
	<ol>
		<li>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.</li>
		<li>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 &#956;L into each well, using three wells for each condition. Incubate at 22 &#176;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.</li>
	</ol>
	</li>
	<li>Load cells with TRITC-dextran (Figure 1B).
	<ol>
		<li>Dilute the dextran to 1 mg/mL in the medium used (this can be increased to 2.5&#8211;5 mg/mL when assessing cells with very low uptake). Add 50 &#956;L to each well (giving a final concentration of 0.5 mg/mL TRITC-dextran) and return the plate to 22 &#176;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.</li>
	</ol>
	</li>
	<li>Prepare cells for flow cytometry (Figure 1C).
	<ol>
		<li>Immediately prior to washing, add 50 &#956;L dextran-containing media to the 0 min uptake controls.</li>
		<li>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.</li>
		<li>Add 100 &#956;L of ice-cold 5 mM sodium azide dissolved in KK2MC (KK2 + 2 mM MgSO4 and 100 &#956;M 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.</li>
	</ol>
	</li>
	<li>Measure fluid uptake by flow cytometry
	<ol>
		<li>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).</li>
		<li>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 &#956;L of the cell suspension and run.</li>
	</ol>
	</li>
	<li>Analyze flow cytometry results
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
4. Performing Fluid Uptake Time-courses
<ol>
	<li>Set up cells as in section 3, with three wells for each time point and strain/condition.</li>
	<li>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).</li>
	<li>At 0 min, add 50 &#956;L of dextran-containing medium and immediately decant and wash the plate as in section 3.3. Analyze as described in section 3.5.</li>
</ol>
5. Dose Response Curves
<ol>
	<li>Set up cells as described in section 3, with three wells for each strain and condition.</li>
	<li>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.</li>
	<li>Create a dilution series of the compound of interest in 200 &#956;L dextran containing medium per sample (Figure 4A). Vortex to mix. Add 50 &#956;L of the medium to each sample well for 1 h.</li>
	<li>Wash and analyze as described in section 3.3 onwards.</li>
</ol>
6. Phagocytosis and Membrane Uptake
<ol>
	<li>To measure membrane uptake, prepare the cells as described in section 3. Add 50 &#956;L of medium containing 20 &#956;M FM 1-43 for a final concentration of 10 &#956;M. 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.</li>
	<li>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.
	<ol>
		<li>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 &#956;m) or 1 x 108 beads/mL (1 and 1.5 &#956;m) 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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dictyostelium+myosin+I+double+mutants+exhibit+conditional+defects+in+pinocytosis.">Dictyostelium myosin I double mutants exhibit conditional defects in pinocytosis.</a> Journal of Cell Biology. 131, 1205-1221 (1995).</li><li>Mercer, J., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccinia+virus+uses+macropinocytosis+and+apoptotic+mimicry+to+enter+host+cells.">Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells.</a> Science. 320 (5875), 531-535 (2008).</li><li>Paschke, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+genetic+engineering+of+both+wild+type+and+axenic+strains+of+Dictyostelium+discoideum.">Rapid and efficient genetic engineering of both wild type and axenic strains of Dictyostelium discoideum.</a> PLoS One. 13 (5), e0196809 (2018).</li><li>Bloomfield, G., Tanaka, Y., Skelton, J., Ivens, A., Kay, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+duplications+in+the+genomes+of+laboratory+stocks+of+Dictyostelium+discoideum.">Widespread duplications in the genomes of laboratory stocks of Dictyostelium discoideum.</a> Genome Biology. 9 (4), R75 (2008).</li><li>Fey, P., Dodson, R. J., Basu, S., Chisholm, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+stop+shop+for+everything+Dictyostelium:+dictyBase+and+the+Dicty+Stock+Center+in+2012.">One stop shop for everything Dictyostelium: dictyBase and the Dicty Stock Center in 2012.</a> Methods in Molecular Biology. 983, 59-92 (2013).</li></ol>

The authors have no conflicts of interest to disclose.

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 membrane30, is then used to detach the cells, and also prevents exocytosis without affecting cell viability24.
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 required24. 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 conditions24. To determine the maximum rate of phagocytosis, uptake must be measured in shaking suspension by an alternative protocol27. 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 cells32, 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 &#176;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 &#34;wild-type&#34; than axenic ones and can be manipulated as effectively as axenic strains33. Otherwise, Ax2 strains are the axenic strains with the fewest genome duplications34, while many strains of Ax4 are Talin A knockouts and should be avoided if possible23. Most previously published strains can be ordered from the Dicty Stock Center35.
This technique allows greater investigative possibilities than was previously possible into the effects of different conditions, inhibitors and mutations on macropinocytosis by Dictyostelium.

Large-scale non-specific fluid uptake by macropinocytosis is important in several biological contexts1, including antigen sampling by immune cells2, pathogen entry into host cells3, cancer cell proliferation4 and the spreading of prion diseases5. In mammalian and Dictyostelium cells, actin6,7, PI(3,4,5)P38,9,10 (although the exact nature of the lipid differs between the two11), activated Ras12,13, and activated Rac14,15 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 macropinocytosis6. 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 visualized12 in this system similarly offers distinct advantages for investigating core conserved components of the macropinosome over other systems where there may be multiple regulatory signals16,17.
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 vesicles18. This technique does not however account for the possibility of macropinosomes shrinking upon entering the cell, which has been reported in Dictyostelium19, 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 downsides20. 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 dextran21. 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, resolution22, although this remains low-throughput.
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. This method was designed to overcome the limitations of the above methods and allow simultaneous comparison of the fluid uptake of large numbers of strains/conditions while using fewer resources and reducing the labor involved.

<table><tbody><tr><td>LSR_II flow cytometer</td><td>BD Biosciences</td><td>-</td><td>Other Flow cytometers can also do this role, e.g. the LSRFortessa by BD</td></tr><tr><td>TRITC-dextran (155 kDa)</td><td>Sigma-Aldrich</td><td>T1287</td><td>Other non-quenchable dextrans, and other sizes are also fine</td></tr><tr><td>HL5 medium</td><td>Formedium</td><td>HLGCFG</td><td></td></tr><tr><td>96-well tissue culture plate</td><td>Corning</td><td>3596</td><td>Any flat-bottom tissue culture treated 96-well plate will work</td></tr><tr><td>Dihydrostreptomycin sulfate</td><td>Sigma-Aldrich</td><td>PHR1517</td><td></td></tr><tr><td>Ampicillin sodium</td><td>Formdium</td><td>AMP50</td><td></td></tr><tr><td>Kanamycin monosulfate</td><td>Sigma-Aldrich</td><td>60615</td><td></td></tr><tr><td>Sodium azide</td><td>VWR</td><td>103694M</td><td></td></tr><tr><td>Magnesium sulfate hydrate</td><td>VWR</td><td>25169.295</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>VWR</td><td>1.02382.0250</td><td></td></tr><tr><td>Potassium dihydrogen phopshate</td><td>VWR</td><td>1.04877.1000</td><td></td></tr><tr><td>Di-potassium hydrogen phosphate</td><td>VWR</td><td>1.05104.1000</td><td></td></tr><tr><td>Fluorimeter</td><td>Perkin-Elmer</td><td>LS 50 B</td><td></td></tr><tr><td>FM1-43</td><td>Thermofisher</td><td>T35356</td><td></td></tr><tr><td>Fluoresbrite YG-carboxy microspheres 1.00 &amp;micro;m</td><td>Polysciences</td><td>15702-10</td><td></td></tr><tr><td>Fluoresbrite YG-carboxy microspheres 1.50 &amp;micro;m</td><td>Polysciences</td><td>09719-10</td><td></td></tr><tr><td>Fluoresbrite YG-carboxy microspheres 1.75 &amp;micro;m</td><td>Polysciences</td><td>17687-5</td><td></td></tr><tr><td>Fluoresbrite YG-carboxy microspheres 2.00 &amp;micro;m</td><td>Polysciences</td><td>09847-5</td><td></td></tr><tr><td>Texas Red &lt;em&gt;E. coli&lt;/em&gt; bioparticles</td><td>Thermofisher</td><td>E2863</td><td></td></tr><tr><td>Flow-set fluorospheres</td><td>Beckman Coulter</td><td>6607007</td><td>Calibration Beads</td></tr><tr><td>SM agar</td><td>Formedium</td><td>SMACFG</td><td></td></tr><tr><td>0.22 &amp;micro;m syringe filter</td><td>Elkay Laboratory Products</td><td>E25-PS22-50S</td><td></td></tr><tr><td>10 mL Syringe</td><td>Becton Dickinson</td><td>302188</td><td></td></tr><tr><td>Round-bottom polystyrene tubes</td><td>Corning</td><td>352058</td><td>Use a tube that will fit onto your flow cytometer.</td></tr><tr><td>70 &amp;micro;m cell strainer</td><td>Falcon</td><td>352350</td><td></td></tr><tr><td>50 mL centrifuge tube</td><td>Sarstedt</td><td>62.547.004</td><td></td></tr><tr><td>Repeating pipette</td><td>Eppendorf</td><td>M4-SK</td><td></td></tr><tr><td>5 mL repeating pipette tips</td><td>Eppendorf</td><td>30089650</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D2650-100ML</td><td></td></tr><tr><td>LY294002</td><td>Cayman Chemical Company</td><td>70920</td><td></td></tr><tr><td>Nocodazole</td><td>Sigma-Aldrich</td><td>M1404-2MG</td><td></td></tr></tbody></table>

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.

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-14) and one with abolished fluid uptake (Ax3 gefB-28). Obtain the average median fluid uptake (section 3.5) and use it to either calculate the volume of fluid internalized (as in Figure 3B) or compare the data to a control (as in Figure 4B and 4C).
When performing a fluid uptake time course, as in Figure 3A, the internalized fluorescence should increase for 60&#8211;90 min, after which the dextran begins to be exocytosed and a plateau is reached (Figure 3B). Using 60 min as the standard time point when comparing macropinocytosis in different mutants/conditions therefore allows a good signal to be achieved, and no signal is lost due to exocytosis. Mutants where exocytosis is severely obstructed may take longer to reach a plateau29.
When treating cells with inhibitors that are effective against macropinocytosis in Dictyostelium (set up as in Figure 4A), the dextran internalized in 1 h will go down to almost nothing at higher inhibitor concentrations in the majority of cases (Figure 4B). Some inhibitors may not be 100% effective, however, e.g. nocodazole only inhibits up to 50% of fluid uptake by macropinocytosis when added acutely (Figure 4C). If the inhibitors are not effective, the cells will internalize a similar amount of dextran as the control, and a decrease in fluorescence will not be seen. This technique allows a large range of different inhibitors and inhibitor concentrations to be screened for effects on fluid uptake by macropinocytosis very quickly, reducing the time spent optimizing the inhibitor treatment.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58434/58434fig2.jpg" /> 
Figure 2: Set up of flow cytometer and representative data. (A) The forward scatter (FSC) and side scatter (SSC) profiles of cells should be set so the cells can be easily distinguished. An example of how Ax2 cells should look is shown. (B) If the flow cytometer is blocked, as in this example, the cells have very low side scatter. The laser will not excite the fluorophores properly and the machine should be unblocked before continuing. Any data obtained while the machine was blocked should be discarded. (C) The fluorescence should be set so that a 0 min uptake sample has low fluorescence, which increases when cells have been incubated for longer in the fluorescent medium, as shown in this example taken from Williams &#38; Kay 201824. (D) Examples of cells that have been incubated with TRITC dextran for 1 h with normal macropinocytosis (Ax2, green), reduced macropinocytosis (Ax2 rasG-, HM172614, orange) and abolished macropinocytosis (Ax3 gefB-, HM177628, blue). <a href="https://www.jove.com/files/ftp_upload/58434/58434fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58434/58434fig3.jpg" /> 
Figure 3: Performing fluid uptake time-courses in 96-well plates. (A) Dextran should be added to each set of samples sequentially, with the same finish time. Then wash the wells, detach and measure the internalized fluorescence by flow cytometry. Example times to add the dextran are shown here. (B) Fluid uptake time-course of Ax2 cells performed in 96-well plates. Taken from Williams &#38; Kay 201824, error bars show the standard error of three independent experiments. <a href="https://www.jove.com/files/ftp_upload/58434/58434fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58434/58434fig4.jpg" /> 
Figure 4: Fluid uptake dose response curves. (A) Add the compound of interest, in this case the PI3K inhibitor LY294002, to HL5 containing 1 mg/mL TRITC-dextran at double the desired final maximum concentration. Mix with HL5 growth medium + 1 mg/mL dextran containing vehicle alone in various proportions to generate a dilution series of 200 &#181;L medium per condition. Add to wells as normal for 1 h before washing and measuring internalized fluorescence. (B) Fluid uptake dose response curve for Ax2 cells incubated with the LY294002-containing medium from A. Adapted from Williams &#38; Kay 201824. Fluid uptake is normalized to an untreated control. Error bars show the standard error of three independent experiments. (C) Fluid uptake dose response curve for Ax2 cells incubated with the nocodazole. Adapted from Williams &#38; Kay 201824. Fluid uptake is normalized to an untreated control. Error bars show the standard error of three independent experiments. <a href="https://www.jove.com/files/ftp_upload/58434/58434fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry at JoVE.com

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

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 ...

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JoVE Journal

Biology

7.7K Views.  MRC-Laboratory of Molecular Biology, Cambridge, UK. Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

Video: High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Immunology and Infection

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells maintain organismal homeostasis and immunity. Simultaneously, and in contrast to its homeostatic role, it can also drive various pathologies, including cancer and viral infections. Unlike other modes of endocytosis, the tools developed for studying the maturation of macropinosomes remain underdeveloped. Here the protocol describes newly developed tools for studying the redox environment within the lumen of early and maturing macropinosomes. Methodologies for using ratiometric fluorescence microscopy in assessing the pH, production of reactive oxygen species, and the degradative capacity within the lumen of individual macropinosomes in live cells are described. Single organelle measurements offer the advantage of revealing spatiotemporal heterogeneity, which is often lost with population-based approaches. Emphasis is placed on the basic principles of dual fluorophore ratiometric microscopy, including probe selection, instrumentation, calibration, and single-cell versus population-based methods.

We describe protocols for measuring pH, oxidative events, and protein digestion in individual macropinosomes in live cells. An emphasis is placed on dual-fluorophore ratiometric microscopy and the advantages it offers over population-based techniques.

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells ...

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in neurological diseases including neurodegeneration and stroke. CNS-MPs are involved in phagocytic clearance of pathological proteins, debris and neuronal synapses, each with distinct underlying molecular pathways. Characterizing these phagocytic properties can provide a functional readout that compliments molecular profiling of microglia using traditional flow cytometry, transcriptomics and proteomics approaches. Phagocytic profiling of microglia has relied on microscopic visualization and in vitro cultures of mouse neonatal microglia. The former approach suffers from limited sampling while the latter approach is inherently poorly reflective of the true in vivo state of adult CNS-MPs. This paper describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. CNS-MPs are acutely isolated from adult mouse brain using mechanical dissociation followed by density gradient centrifugation, incubated with fluorescent microspheres or fluorescent A&#946; fibrils, washed, and then labeled with panels of antibodies against surface markers (CD11b, CD45). Using this approach, it is possible to compare phagocytic properties of brain-resident microglia with CNS-infiltrating macrophages and then assess the effect of aging and disease pathology on these phagocytic phenotypes. This rapid method also holds potential to functionally phenotype acutely-isolated human CNS-MPs from post-mortem or surgical brain specimens. Additionally, specific mechanisms of phagocytosis by CNS-MP subsets can be investigated by inhibiting select phagocytic pathways.

Assessment of phagocytic properties of microglia and brain macrophages can provide a valuable functional dimension to molecular profiling studies. We describe a validated protocol that uses flow cytometry to rapidly and reliably quantify phagocytosis of fluorescent microspheres and amyloid beta fibrils by acutely-isolated microglia and brain macrophages from mouse models.

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in ...

Neuroscience

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Flow Cytometry-Based Assays for Microglial Engulfment of Synaptic Materials

Quantification of Microglial Engulfment of Synaptic Material Using Flow Cytometry

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry.
In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1+ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodo&#160;Red-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.

Author Spotlight: Advancing Understanding Through Technological Innovations in Psychoneuroimmunology

Here we present two protocols to quantify microglial engulfment of vGLUT1-positive synapses and pHRodo&#160;Red-labeled crude synaptosomes using flow cytometry.

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this ...

Quantitative Assessment of Macropinocytosis in mTORC1-Hyperactive Cells using Flow Cytometry

Macropinocytosis is a highly conserved, actin-dependent endocytic process that allows the uptake of extracellular material, including proteins and lipids. In proliferating cells, macropinocytosis can deliver extracellular nutrients to the lysosome, processed into critical macromolecule building blocks. Recent studies have highlighted the dependence of multiple cancers on macropinocytosis, including breast, colorectal and pancreatic cancer. Ras mutations are thought to be the driver events behind macropinocytosis initiation, leading to the activation of cellular anabolic processes via the mTORC1 signaling pathway. Interestingly, mTORC1 can also be activated by macropinocytosis independently of Ras. Therefore, macropinocytosis represents a metabolic vulnerability that can be leveraged to target macropinocytic tumors by limiting their access to nutrients therapeutically.
In Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM), mTORC1-hyperactivation leads to enhanced macropinocytosis and metabolic reprogramming. Here, we describe a flow cytometry-based protocol to assess macropinocytosis in mammalian cells quantitatively. TSC2-deficient MEFs are employed, which exhibit aberrant activation of mTORC1 and have been shown to have increased macropinocytosis compared to TSC2-expressing cells. Cells treated with pharmacologic inhibitors of macropinocytosis are incubated with fluorescently labeled, lysine-fixable, 70 kDa dextran, or fluorescently labeled bovine serum albumin (BSA) assayed by flow cytometry. To date, robust image-based techniques have been developed to quantitatively assess macropinocytosis in tumor cells in vitro and in vivo. This analysis provides a quantitative assessment of macropinocytosis in multiple experimental conditions and complements existing image-based techniques.

This protocol provides experimental tools to evaluate macropinocytic uptake of nutrients (carbohydrate and protein) by mTORC1-hyperactive cells. Detailed steps to quantify the uptake of fluorescently labeled dextran and bovine serum albumin (BSA) are described.

Macropinocytosis is a highly conserved, actin-dependent endocytic process that allows the uptake of extracellular material, including proteins and lipids. ...

Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and cell debris, through bulk endocytosis. This pathway plays an essential role in a variety of cellular processes, including the regulation of immune responses and cancer cell metabolism. Given this importance in biological function, examining cell culture conditions can provide valuable information by identifying regulators of this pathway and optimizing conditions to be employed in the discovery of novel therapeutic approaches. The study describes an automated imaging and analysis technique using standard laboratory equipment and a cell imaging multi-mode plate reader for the rapid quantification of the macropinocytic index in adherent cells. The automated method is based on the uptake of high molecular weight fluorescent dextran and can be applied to 96-well microplates to facilitate assessments of multiple conditions in one experiment or fixed samples mounted onto glass coverslips. This approach is aimed at maximizing reproducibility and reducing experimental variation while being both time-saving and cost-effective.

Automated assays using multi-well microplates are advantageous approaches for identifying pathway regulators by allowing the assessment of a multitude of conditions in a single experiment. Here, we have adapted the well-established macropinosome imaging and quantification protocol to a 96-well microplate format and provide a comprehensive outline for automation using a multi-mode plate reader.

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and ...

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

This protocol demonstrates dextran imaging in live cells using continuous uptake and inverse images to optimize visualization of ruffling, macropinosome maturation, and analysis of dextran and other cell labelings.

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

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Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Quantification of Microglial Engulfment of Synaptic Material Using Flow Cytometry

Quantitative Assessment of Macropinocytosis in mTORC1-Hyperactive Cells using Flow Cytometry

Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis