Name: measuring phagosome ph by ratiometric fluorescence microscopy
Uploaded: 2015-12-07T14:00:00.000+00:00
Duration: 14 min 39 s
Description: Watch this Scientific Journal Video about Measuring Phagosome pH by Ratiometric Fluorescence Microscopy at JoVE.com

The authors are financially supported by the Swiss National Science Foundation through an operating grant N&#176; 31003A-149566 (to N.D.), and The Sir Jules Thorn Charitable Overseas Trust through a Young Investigator Subsidy (to P.N.).

Ethics Statement:&#160;All animal manipulations were performed in strict accordance with the guidelines of the Animal Research Committee of the University of Geneva.
1. Preparation of Phagocytic Targets
<ol>
	<li>Add 20 mg of dried zymosan to 10 ml sterile phosphate buffered saline (PBS). Vortex and heat in a boiling water bath for 10 min. Cool and centrifuge at 2,000 x g for 5 min.</li>
	<li>Remove the supernatant, resuspend in 1 ml PBS and sonicate for 10 min in a water bath sonicator. Transfer 500 &#181;l to two 1.5 ml tubes. Centrifuge at 11,000 x g for 5 min.</li>
	<li>Repeat the wash with 500 &#181;l PBS. The boiled zymosan may be aliquoted, frozen and stored at -20 &#176;C.</li>
	<li>Wash zymosan twice in 500 &#181;l freshly prepared 0.1 M Na2CO3, pH 9.3 as above (step 1.2).</li>
	<li>Resuspend in 490 &#181;l after the final wash. Add 10 &#181;l of 10 mg/ml FITC dissolved in dimethyl sulfoxide (DMSO), vortex, cover with foil, and incubate with agitation for 4 hr at RT.</li>
	<li>To remove unbound dye, wash as above (step 1.2), first with 0.5 ml DMSO + 150 &#181;l PBS, then 0.5 ml DMSO + 300 &#181;l PBS, then 0.5 ml DMSO + 450 &#181;l PBS, then PBS alone.</li>
	<li>Count zymosan using a haemocytometer. Add 1 &#181;l of zymosan to 500 &#181;l PBS, vortex, then add 10 &#181;l to the edge of the haemocytometer chamber, where it meets the coverslip placed over the central grid. Mount the haemocytometer on a bright-field microscope equipped with a 20X objective.</li>
	<li>Focus on the upper left-hand corner containing 16 squares and count all zymosan particles in this area using a hand tally counter. Repeat with the lower right-hand corner. Calculate the zymosan concentration using the following equation: Zymosan concentration = Count/2 x 500 x 104 zymosan/ml. Labeled zymosan may be aliquoted, frozen and stored at -20 &#176;C.</li>
	<li>Opsonize the labeled zymosan with a rabbit anti-zymosan antibody at 1:100 dilution. Vortex and incubate for 1 hr in a 37 &#176;C water bath. Wash with 500 &#181;l PBS as above (step 1.2). 
	Note: Sonication is highly recommended, particularly after opsonization, as zymosan tends to form clumps that can make analysis more difficult later on. Opsonization with 65% v/v rat or human serum may be used as an alternative.</li>
	<li>Count zymosan using a haemocytometer as described in steps 1.7-1.8, and dilute to 100 x 106 particles/ml. Opsonization with other phagocytic stimulators, such as complement or no opsonization, may be alternatively performed.</li>
</ol>
2. Live Video Microscopy
<ol>
	<li>Isolate primary mouse neutrophils as described in detail elsewhere38.
	<ol>
		<li>Alternatively, use any phagocytic cell type. Keep neutrophils on ice in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 5% fetal calf serum (v/v), 200 U penicillin/streptomycin, at a concentration of 10 x 106 cells/ml until ready for use. Other cell types may be seeded 1-4 days before.</li>
	</ol>
	</li>
	<li>Add 2 x 105 (20 &#181;l) of neutrophils to the center of a 35 mm glass-bottom dish, spread the drop by adding 50 &#181;l of medium and incubate at 37 &#176;C for 5 min to allow cells to adhere.</li>
	<li>Add 1 ml of Hank&#8217;s balanced salt solution (HBSS) warmed to 37 &#176;C containing the specific MPO inhibitor 4-aminobenzoic hydrazide (4-ABH, 10 &#181;M). Non-specific MPO inhibitors, such as sodium azide (5 mM), may also be used but have been recently suggested to exert additional non-specific effects on pH 39.</li>
	<li>Mount the dish on a widefield fluorescence microscope equipped with a 37 &#176;C heating system and a 40X oil objective. Incubate cells without imaging for 10 min to allow cells and equipment to equilibrate.</li>
	<li>Adjust the microscope settings to acquire a bright-field transmission image [phase or differential interference contrast (DIC) if available] with 440 nm or 490 nm excitation and 535 nm emission every 30 sec.</li>
	<li>Adjust exposure time and acquisition frequency according to the microscope system to avoid too much photobleaching and phototoxicity and depending on the experimental questions being asked. Begin image acquisition.</li>
	<li>After 2 min, add 10 x 106 (50 &#181;l) opsonized FITC-zymosan to the center of the dish. Adjust target:cell ratios depending on the type of phagocyte and target used.</li>
	<li>Continue imaging for 30 min. After 30 min stop the time-lapse acquisition and start a timer. Move the stage and capture 10 or more snapshots in different fields of view to image other phagosomes, all within 5 min.</li>
</ol>
3. Calibration and Control Experiments
<ol>
	<li>Prepare the pH calibration solutions (recipes are described in Table 1) prior to the day of experiment and freeze in 10 ml aliquots. Thaw the calibration solutions and warm to 37 &#176;C in a water bath. Check the pH with a pH meter and record the actual pH of solutions.</li>
	<li>Mount a peristaltic pump on the glass bottom dish prior to image acquisition and perform live video microscopy as described above (section 2).</li>
	<li>At the end of the 30 min acquisition period turn the pump on to remove the solution within the dish, add 1 ml of the first calibration solution and stop the pump.</li>
	<li>Wait until the signal stabilizes, usually 5 min. Repeat with each calibration solution.
	<ol>
		<li>Perform at least one calibration per experimental day, and calibrate starting with the highest pH and working sequentially to the lowest, as well as starting from the lowest pH and working sequentially towards the highest.</li>
	</ol>
	</li>
	<li>As a control, add 100 &#181;l of 100 &#181;M NADPH oxidase inhibitor diphenyl propionium iodide (DPI) diluted in HBSS to cells in 900 &#181;l HBSS in the glass bottom dish, and incubate for 10 min.</li>
	<li>Start acquisition and add zymosan as above (step 2.7). After 15 min of phagocytosis, add 10 &#181;l of 10 &#181;M V-ATPase inhibitor concanamycin A (ConcA) diluted in HBSS and continue imaging for an additional 15 min.</li>
</ol>
4. Analysis
<ol>
	<li>Analysis of time-lapse movies and phagosome snapshots. 
	Note: The following instructions are specific for ImageJ. Step-by-step instructions may vary widely depending of the software used, but the functions described in this section are available in most professional image analysis software.
	<ol>
		<li>Open the images with professional image analysis software. Subtract the background in the 490 channel by drawing a small square ROI with the square polygon selection tool in an area where there are no cells, and clicking Plugins&#62; BG Subtraction from ROI. Then upload the same ROI on the 440 channel by clicking Edit&#62; Selection&#62; Restore Selection and repeat.</li>
		<li>Make a 32-bit ratio image of the 490 channel divided by the 440 channel by clicking Process&#62;Image Calculator&#8230; , selecting the appropriate images in the Image1 and Image2 drop-down menus, then selecting &#8216;Divide&#8217; in the Operation drop-down menu, and checking the 32-bit (float) result check-box.</li>
		<li>Change the color-coding look-up table to a ratio-compatible color-coding by clicking Image&#62; Lookup Tables&#62; Rainbow RGB. Threshold the ratio image to eliminate 0 and infinity pixels by clicking Image&#62; Adjust&#62; Threshold&#8230;. Adjust the scroll bars so that zymosan appears in red and click Apply, making sure the Set background pixels to NaN check box is selected.</li>
		<li>Combine this ratio stack with the bright-field channel into a multi-channel composite by clicking Image&#62; Color&#62; Merge Channels, and selecting the bright-field image in the gray channel dropdown menu and the ratio image in the green channel dropdown menu, and selecting the Keep source images check-box. Scan the time-lapse movies or snapshots to determine which phagosomes are to be analyzed. The bright-field channel is useful for this.</li>
		<li>Draw regions of interest (ROI) using the oval polygon selection tool around phagosomes, and add them to the ROI manager by clicking Analyze&#62; Tools &#62; ROI Manager&#62; Add. Measure their zymosan intensity ratio by clicking More&#62; Multi-Measure and de-selecting the One row per slice check-box.</li>
		<li>For time-lapse movies, track phagosomes one at a time by drawing an ROI, typing Ctrl+M to measure each time point, and moving the ROI when necessary to track intensity values over time. Copy the measurements from the Results window into a spreadsheet software. 
		Note: Analysis of 15 (5 time courses per experiment x 3) independent experiments is ideal, but more or less may be required depending on the variability observed. Saving the ROIs is always recommended. Some software packages allow image registration and dynamic update of ROIs, facilitating this part of the analysis.</li>
	</ol>
	</li>
	<li>Conversion from fluorescence to pH values
	<ol>
		<li>Measure the 490/440 ratio for phagosomes in the calibration time-lapse movies as described in section 4.1.</li>
		<li>In a spreadsheet, plot the ratio values over time, and calculate the average ratio values from all phagosomes within the field of view (usually, between 5-20) from 5 time-points within the middle of the time period corresponding to each pH calibration solution. (See also the red boxes in Figure 5A.)</li>
		<li>Plot these average 490/440 ratio values against the actual measured pH. Combine at least 3 independent calibrations into a single graph. Use a statistical or numerical software package to perform a nonlinear regression (best fit curve), usually to a sigmoidal equation. 
		Note: For example, in Figure 5B the equation used was a Boltzmann sigmoidal [Y = Bottom + ((Top-Bottom)/(1+EXP((V50-X)/Slope))], where Y values are the 490/440 ratios, X are the pH values and the Top, Bottom, V50 and slope parameters are calculated by the software.</li>
		<li>Use the obtained equation to transform ratio values to pH. 
		Note: For example, in Figure 5B the equation obtained for the calibration curve in the presence of sodium azide is 490/440 Ratio = 1.103 + [(2.89-1.103)/(1+EXP((6.993-pH)/0.9291)]. Solving for pH gives the following equation: pH = 6.993 - 0.9291*[LN((1.178/(Ratio-1.103))-1))].</li>
	</ol>
	</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma-Interferon-inducible+lysosomal+thiol+reductase+(GILT)+maintains+phagosomal+proteolysis+in+alternatively+activated+macrophages.">gamma-Interferon-inducible lysosomal thiol reductase (GILT) maintains phagosomal proteolysis in alternatively activated macrophages.</a> J Biol Chem. 289 (46), 31891-31904 (2014).</li><li>Sanjurjo, L., 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=The+scavenger+protein+apoptosis+inhibitor+of+macrophages+(AIM)+potentiates+the+antimicrobial+response+against+Mycobacterium+tuberculosis+by+enhancing+autophagy.">The scavenger protein apoptosis inhibitor of macrophages (AIM) potentiates the antimicrobial response against Mycobacterium tuberculosis by enhancing autophagy.</a> PLoS One. 8 (11), e79670 (2013).</li><li>Yuan, Z., Zhi, L., Li-Juan, Z., Hong-Tao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Utility+of+Chloroquine+in+Cancer+Therapy.">The Utility of Chloroquine in Cancer Therapy.</a> Curr Med Res Opin. , 1-12 (2015).</li><li>Ohkuma, S., Poole, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+probe+measurement+of+the+intralysosomal+pH+in+living+cells+and+the+perturbation+of+pH+by+various+agents.">Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents.</a> Proc Natl Acad Sci U S A. 75 (7), 3327-3331 (1978).</li><li>El Chemaly, A., 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=VSOP/Hv1+proton+channels+sustain+calcium+entry,+neutrophil+migration,+and+superoxide+production+by+limiting+cell+depolarization+and+acidification.">VSOP/Hv1 proton channels sustain calcium entry, neutrophil migration, and superoxide production by limiting cell depolarization and acidification.</a> J Exp Med. 207 (1), 129-139 (2010).</li><li>Levine, A. P., Duchen, M. R., Segal, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HVCN1+channel+conducts+protons+into+the+phagocytic+vacuole+of+neutrophils+to+produce+a+physiologically+alkaline+pH.">The HVCN1 channel conducts protons into the phagocytic vacuole of neutrophils to produce a physiologically alkaline pH.</a> bioRxiv. , (2014).</li><li>Al-Fageeh, M. B., Smales, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+and+regulation+of+the+cellular+responses+to+cold+shock:+the+responses+in+yeast+and+mammalian+systems.">Control and regulation of the cellular responses to cold shock: the responses in yeast and mammalian systems.</a> Biochem J. 397 (2), 247-259 (2006).</li><li>Yui, N., 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=Basolateral+targeting+and+microtubule-dependent+transcytosis+of+the+aquaporin-2+water+channel.">Basolateral targeting and microtubule-dependent transcytosis of the aquaporin-2 water channel.</a> Am J Physiol Cell Physiol. 304 (1), C38-C48 (2013).</li><li>Griffiths, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+phagosome+individuality+and+membrane+signalling+networks.">On phagosome individuality and membrane signalling networks.</a> Trends Cell Biol. 14 (7), 343-351 (2004).</li><li>Poburko, D., Santo-Domingo, J., Demaurex, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+regulation+of+the+mitochondrial+proton+gradient+during+cytosolic+calcium+elevations.">Dynamic regulation of the mitochondrial proton gradient during cytosolic calcium elevations.</a> J Biol Chem. 286 (13), 11672-11684 (2011).</li></ol>

The authors have nothing to disclose.

Although more time consuming than alternative methods, such as spectroscopy and FACS, which employ a similar strategy of using a pH sensitive dye coupled to targets but measure the average pH of a population of phagosomes, microscopy offers several advantages. First is that internal and external bound, but not internalized, particles can easily be distinguished without having to add other chemicals, such as trypan blue or antibodies, to quench or label external particles, respectively. Second is that following the cells ...

Phagocytosis, the process through which innate immune cells engulf large particles, evolved from the eating mechanism of single-celled organisms, and involves binding to a target, enveloping it with a membrane and pinching the membrane off to form a vacuole within the cytosol called a phagosome. While the phagosomal membrane is derived from the plasma membrane, active protein and lipid sorting, as well as fusion with endomembranes during phagosome formation, transform the phagosome into a distinct organelle within the cell with degradative properties that allow the killing, neutralization and breakdown of the ingested material1-3. This process, called phagosomal maturation, relies on the delivery of a host of proteolytic and microbicidal enzymes, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which transfers electrons into phagosomes producing the strong oxidant O2- and its derivative reactive oxygen species (ROS) 2,4. 
The luminal pH of phagosomes has a profound influence on several events required for phagosome function. First, pH influences trafficking of endomembranes in general, as pH-dependent conformational changes of transmembrane trafficking regulators alters the recruitment of trafficking determinants such as Arfs, Rabs and vesicular coat-proteins, which in turn define which vesicles may fuse with phagosomes 5-8. Second, the ionic composition of the phagosomal lumen is also transformed as phagosomes mature, and some ion transporters, such as the Na+/H+ exchanger or ClC family Cl-/H+ antiporters, which promote phagocytic function, rely on H+ translocation 9,10. Similarly, ROS production is intimately linked with phagosomal pH. ROS and its toxic oxidant byproducts have long been recognized as crucial for phagosomal killing in neutrophils 4,11,12, and have been shown to play critical roles in other phagocytes including macrophages, dendritic cells (DCs) and amoeba 13-16. The NADPH oxidase is an electrogenic enzyme that releases H+ in the cytosol as NADPH is consumed, and that requires the simultaneous transfer of H+ through companion HVCN1 channels alongside the transported electrons into the phagosomal lumen, in order to alleviate the massive depolarization that would otherwise lead to self-inhibition of the enzyme 17-21. Finally, several proteolytic enzymes have optimal activity at different pH, so time-dependent phagosomal pH changes can influence which enzymes are active and when. The importance of phagosomal pH is highlighted by organisms such as Mycobacterium tuberculosis, Franciscella tularensis and Salmonella typherium that subvert phagocytic killing at least in part by altering phagosomal pH 22-24.
In mammals the main phagocytes are neutrophils, macrophages and dendritic cells, and depending on cell type, time-dependent phagosomal pH changes can vary widely, and appear to play different roles. In macrophages a strong and rapid acidification mediated by the ATP-dependent proton pump vacuolar ATPase (V-ATPase) is one of the key factors mediating killing 25-27, resembling the mechanisms present in amoeba that use phagocytosis as an eating mechanism 28. In these cells activation of acidic proteases is thought to play a key role. In contrast, neutrophil killing relies more on ROS as well as HOCl produced by myeloperoxidase (MPO)11, and the pH remains neutral or alkaline during the first 30 min acidifying only later 29,30. Neutral pH has been suggested to favor the activity of oxidative proteases such as certain cathepsins. In DCs phagosomal pH is controversial, with some reporting acidification and others neutral or alkaline pH 31,32, but ROS and pH may profoundly influence the ability of these cells to present antigens to T cells, one of their main functions 33. 
Importantly, hormones, chemokines and cytokines may produce signaling events that induce maturation and changes in phagocyte behavior, and in turn influence phagosomal pH 34,35. Similarly, drugs, for example the antimalarial chloroquine, which is also considered for anti-cancer therapies 36, may affect phagosomal pH and therefore immune outcomes. Thus, a variety of cell biologists, immunologists, microbiologists and drug developers may be interested in measuring phagosomal pH when seeking to understand the mechanisms underlying the effects of a particular genetic disruption, bioactive compound or microorganism on innate and adaptive immune responses. 
Here we describe a method for measuring phagosomal pH in neutrophils that allowed us to show the importance of the HVCN1 channel in regulating neutrophil phagosomal pH 19. The method is based on the ratiometric property of fluorescein isothiocyanate (FITC) whose fluorescence emission at 535 nm is pH sensitive when excited at 490 nm but not 440 nm 37. When this dye is chemically coupled to a target, in this case zymosan, it can be followed using wide-field fluorescence microscopy, where cells are imaged as they phagocytose, and phagosomal pH changes are measured in real time as the phagosome matures. The actual pH is then gleaned by performing a calibration experiment where cells that have phagocytosed are exposed to solutions of different pH that contain the ionophores nigericin and monensin, that allow the rapid equilibration of the pH within phagosomes with the external solution. Ratio values are then compared to the known pH of solutions, a calibration curve is constructed by nonlinear regression and the resulting equation used to calculate pH from the ratio value.

<table><tbody><tr><td>Zymosan A powder</td><td>Sigma-Aldrich</td><td>Z4250</td><td>Various providers exist</td></tr><tr><td>Fluorescein isothiocyanate</td><td>Sigma-Aldrich</td><td>F7250</td><td>Various providers exist</td></tr><tr><td>Anti-zymosan antibody (Zymosan A Bioparticles opsonizing reagent)</td><td>Life Technologies</td><td>Z2850</td><td>Sigma-Aldrich O6637 is an equivalent product. Alternatively 25% serum can be used as an opsonizing reagent.</td></tr><tr><td>4-Aminobenzoic hydrazide (4-ABH)</td><td>Santa Cruz</td><td>sc-204107</td><td>Toxic, use gloves, various providers exist</td></tr><tr><td>Diphenyleneiodonium chloride (DPI)</td><td>Sigma-Aldrich</td><td>D2926</td><td>Toxic, use gloves, various providers exist</td></tr><tr><td>Concanamycin A (ConcA)</td><td>Sigma-Aldrich</td><td>27689</td><td>Toxic, use gloves, various providers exist</td></tr><tr><td>Nigericin</td><td>Sigma-Aldrich</td><td>N7143</td><td>Toxic, use gloves, various providers exist</td></tr><tr><td>Monensin</td><td>Enzo</td><td>ALX-380-026-G001</td><td>Toxic, use gloves, various providers exist</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Life Technologies</td><td>14200-075</td><td>Toxic, use gloves, various providers exist</td></tr><tr><td>Hank&#039;s balance salt solution</td><td>Life Technologies</td><td>14025092</td><td>Ringer&#039;s balanced salt solution or other clear physiological buffers may be substituted.</td></tr><tr><td>Sodium carbonate (Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S7795</td><td>Various providers exist</td></tr><tr><td>2-(N-Morpholino)ethanesulfonic acid (MES)</td><td>Sigma-Aldrich</td><td>M3671&amp;nbsp;</td><td>Various providers exist</td></tr><tr><td>4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)</td><td>Sigma-Aldrich</td><td>H3375</td><td>Various providers exist</td></tr><tr><td>N-Methyl-D-glucamine (NMDG)</td><td>Sigma-Aldrich</td><td>M2004&amp;nbsp;</td><td>Various providers exist</td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&amp;prime;,N&amp;prime;-tetraacetic acid (EGTA)</td><td>Sigma-Aldrich</td><td>3777</td><td>Various providers exist</td></tr><tr><td>Tris(hydroxymethyl)aminomethane (Tris)</td><td>Sigma-Aldrich</td><td>T1503&amp;nbsp;</td><td>Various providers exist</td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma-Aldrich</td><td>P9333</td><td>Various providers exist</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S7653</td><td>Various providers exist</td></tr><tr><td>Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>M8266</td><td>Various providers exist</td></tr><tr><td>Absolute Ethanol (EtOH)</td><td>Sigma-Aldrich</td><td>2860</td><td>Various providers exist</td></tr><tr><td>Glass-bottom 35 mm petri dishes (Fluorodish)</td><td>World Precision Instruments</td><td>FD35-100</td><td>Ibidi &amp;micro;-clear dishes or coverslips with appropriate imaging chambers may be sustituted</td></tr><tr><td>Sonicating water bath</td><td>O. Kleiner AG</td><td></td><td>A sonicator may be used instead, various instrument providers exist</td></tr><tr><td>Heamocytometer</td><td>Marienfeld GmbH</td><td></td><td>Various instrument providers exist</td></tr><tr><td>Widefield live imaging microscope</td><td>Carl Zeiss AG</td><td></td><td>Various instrument providers exist, but the microscope must be able to image 440/535 and 490/535 excitation/emission respective. Spinning disk confocal set-ups with brightfield capabilities may substituted, but zymosan tend to go out of focus more often.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Peristaltic pump (Dynamax RP-1)</td><td>Rainin</td><td></td><td>Various instrument providers exist</td></tr><tr><td>pH meter</td><td>Schott Gerate GmbH</td><td></td><td>Various instrument providers exist</td></tr><tr><td>Manual Counter</td><td>Milian SA</td><td></td><td>Various instrument providers exist</td></tr></tbody></table>

measuring phagosome ph by ratiometric fluorescence microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

The following are representative results for an experiment where the phagosomal pH of primary mouse neutrophils isolated from the bone-marrow of wild-type or Hvcn1-/- mice were compared. For a successful experiment, it is important to obtain enough phagosomes within the field of view during the whole duration of the time-lapse movie, while avoiding too many phagosomes, which will later be more difficult to segment during the image analysis. Figure 1 shows examples of...

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Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

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Immunology and Infection

13.9K Views.  University of Geneva. Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy. 

Video: Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

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 ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

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 ...

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

Biology

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

Neuroscience

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

TIRF Microscopy-Based Visualization of Phagosome Formation and Closure

For non-covalent fixation of the SRBCs, pour 2 milliliters of the IgG-opsonized cells into each of the poly-lysine-coated dishes, and centrifuge the samples in a swinging rotor centrifuge. Discard the supernatants, and wash the particles one time with 2 milliliters of PBS plus 10% BSA.
Then, incubate the particles with 2 milliliters of fresh PBS plus 10% BSA for 30 minutes at room temperature, followed by three washes with 2 milliliters of PBS. After the last wash, replace the PBS with 2 milliliters of 37 degrees Celsius serum-free microscopy medium.
Place an SRBC-coded dish onto the microscope stage. Then, scrape the transfected cells of interest from the bottom of the culture dish and pipette the cells a few times to achieve a single-cell suspension. Add the transfected cells to the SRBC-coated dish.
Start the live acquisition software. Find a cell that expresses the fluorescently tagged proteins and adjust the position of the dish so that a cell of interest is in the middle of the field. Acquire 500 images at different angles from 0 to 5 degrees at 0.01-degree increments at one excitation wavelength.
To determine the critical angle for the incident light to be totally reflected at the glass-medium interface and generate an evanescent wave, open the image sequence in the appropriate imaging software. Select a region of interest in the cell with uniform fluorescence.
Then, under the Image tab, select Stacks and plot Z-axis profile. To plot the Z-axis profile mean fluorescence intensity measured in the region of interest with the function of the angles on the X-axis. Any angle value on the x-axis superior to the critical angle can then be used during the microscopy session to obtain a TIRF signal.