This work was kindly funded by the Wellcome Trust, the Biotechnology and Biological Sciences Research Council, and the Irwin Joffe Memorial Fellowship.

Ethics statement: All animal work was conducted with the license and approval of the United Kingdom Home Office. Human participation in this research was approved by the Joint UCL/UCLH Committees on the Ethics of Human Research. All participants provided informed consent.
1. Preparation of C. albicans
<ol>
	<li>Grow a Candida disc (see Materials List) on a YPD agar plate as per the manufacturer&#39;s instructions. Pick a colony and add it to 15 mL of YPD broth. Incubate it in a shaking incubator at 30 &#176;C and 200 rpm until the broth is cloudy (usually about 2 days, or to a concentration of roughly over 1 x 109/mL).</li>
	<li>Spin down the Candida medium and resuspend it in 50 mL of phosphate-buffered saline (PBS). Centrifuge at 3,000 x g for 10 min. Repeat this step twice.</li>
	<li>Place the tube containing 50 mL of PBS/Candida medium in a water bath preheated to 60 &#176;C so that the entire tube is submerged for 1 h.
	<ol>
		<li>To confirm that the Candida are heat-killed, streak a sample onto a YPD agar plate and incubate overnight at 30 &#176;C. Adjust the heat-killed (HK) Candida concentration to 5-9 x 108/mL, depending on Candida growth, and store it in 1 mL aliquots in a -20 &#176;C freezer. 
		NOTE: All cell counts in this protocol were performed using an automated cell counter.</li>
	</ol>
	</li>
</ol>
2. S-1 Coupling to Heat-killed (HK) Candida
<ol>
	<li>Prepare an aliquot of carboxy-S-1 succinimidyl ester (50 &#181;g) by diluting it in 100 &#181;L of high-grade dimethyl sulfoxide (DMSO). Vortex well to mix.</li>
	<li>Prepare 1 mL of 1 x 108 HK Candida in 0.1 M sodium bicarbonate (pH 8.3) in a 15 mL tube.</li>
	<li>Add 100 &#181;L of carboxy-S-1 one drop at a time to the HK Candida while mixing on a vortex at roughly 2,000 rpm (medium to high speed). Wrap aluminum foil around the tube and place it on a roller at room temperature for 1 h.</li>
	<li>Wash the HK Candida-S-1(HKC-S-1) three times by centrifugation at 2,250 x g for 10 min each. For the first two washes, resuspend the pellet in 15 mL of 0.1 M sodium bicarbonate (pH 8.3). After the third wash, resuspend it in 1 mL of balanced salt solution (BSS) buffer (see Table 1).</li>
	<li>Transfer the HKC-S-1 suspension into tubes in 100 &#181;L aliquots and store them at -20 &#176;C. 
	NOTE: Use tubes with low surface binding material, if possible, as HKC-S-1 particles can stick to the wall of normal centrifuge tubes.</li>
	<li>Opsonize HKC-S-1
	<ol>
		<li>For human neutrophils, add 100 &#181;L of human IgG serum to 100 &#181;L of thawed HKC-S-1.</li>
		<li>For mouse neutrophils, add 50 &#181;L of normal mouse serum and 50 &#181;L of mouse Candida immune serum (generated from C57B6 mice injected with HK Candida)5 to 100 &#181;L of HKC-S-1.</li>
		<li>Mix it on a heat-shaker at 37 &#176;C and 1,100 rpm for 60-90 min, and then on a 4 &#176;C roller for 2 h.</li>
		<li>Wash three times in BSS buffer by centrifugation at 17,200 x g for 1 min each. Resuspend in 100 &#181;L of BSS buffer.</li>
	</ol>
	</li>
</ol>
3. Isolation of Neutrophils
<ol>
	<li>For human peripheral blood neutrophils, take 15 mL of blood by venipuncture from a healthy donor and place it into a 20 mL syringe containing 90 &#181;L of 1,000 IU/mL heparin sodium solution (60 &#181;L of heparin/10 mL of blood).</li>
	<li>Using a pipette tip, inject 1.5 mL of 10% dextran solution into the syringe. Invert it gently 3 times and leave it standing upright on the bench.</li>
	<li>Wait 30-60 min, until the blood is split roughly in half, with a top buffy coat layer that is straw-colored and a bottom layer containing erythrocytes.</li>
	<li>Carefully push out the top layer through a needle or remove it with a pipette tip. Put it in a 15 mL tube. Avoid taking out the red layer. Using a 5 mL pipette, add 3-4 mL of density gradient medium (see Materials List) to the bottom of the tube under the buffy coat layer to obtain two distinct layers. Centrifuge at 913 x g for 10 min. 
	NOTE: If using mouse neutrophils (see reference17 for mouse neutrophil isolation), use the cell suspension that has been flushed from the bone marrow instead.</li>
	<li>Pour off the supernatant, leaving behind a red pellet. Gently vortex to disturb the pellet. Add 7 mL of distilled, autoclaved H2O to the pellet and invert for 20 s to resuspend the pellet. Add 7 mL of 2x saline and invert a few times to mix, lysing the remaining red blood cells.</li>
	<li>Centrifuge at 300 x g for 5 min. Pour off the supernatant and resuspend the pellet in BSS buffer to approximately 4 x 106/mL. 
	NOTE: For optimal microscopy of the cells, keep the cell suspension concentration between 1.5-6 x 106/mL.</li>
</ol>
4. Preparation of Slides
<ol>
	<li>Pre-treat an 8-well microscopy plate (see Materials List) with 200 &#181;L of poly-L-lysine (0.01% solution) in each well for 40-60 min at room temperature.</li>
	<li>Remove the poly-L-lysine (can be reused) and wash the wells twice with 200 &#181;L of distilled H2O.</li>
	<li>Add 200 &#181;L of cell suspension prepared in step 3.6 to each well. Incubate at room temperature for 30-60 min.</li>
	<li>Prepare an aliquot of 5-(and-6)-carboxy S-1 acetoxymethyl (S-1-AM) ester (50 &#181;g) by adding 100 &#181;L of high-grade DMSO to one tube. Vortex to mix. When not in use, store at -20 &#176;C.</li>
	<li>In a small tube, add 1.7 mL of BSS buffer and 20 &#181;L of S-1-AM. Vortex to mix.</li>
	<li>Wash the wells twice with 200 &#181;L of BSS buffer, and then replace the buffer with the S-1-AM solution. Take care to not disturb the monolayer of cells attached to the bottom of the well by gently pipetting down the walls of the wells. Incubate at room temperature for at least 25 min.</li>
	<li>Wash the wells twice with 200 &#181;L of BSS buffer. To test an inhibitor, make up a &#34;master mix&#34; of the appropriate drug in BSS buffer. Wash the wells twice in the inhibitor solution. 
	NOTE: Make sure to use the same amount of drug vehicle (e.g., DMSO or ethanol) in the control well as was used for the inhibitor. If testing the effect of zinc chloride, use BSS buffer lacking KH2PO4 (the HEPES can buffer the solution alone), as zinc precipitates with phosphate.</li>
	<li>Sonicate the opsonized HKC-S-1 (section 6.2) for approximately 3 s at 5 amplitude &#181;m. Add 10 &#181;L to each well. Incubate the plate at 37 &#176;C for 15-20 min to allow phagocytosis, making the cells ready to be imaged for snapshots of phagocytosis.</li>
</ol>
5. Confocal Microscopy
<ol>
	<li>Using a confocal microscope, adjust the laser wavelength so that the cells are excited at 555 nm and the emission is detected by two channels, or detectors: 560-600 nm and over 600 nm. 
	NOTE: Specific microscope parameters will differ for each microscope and need to be optimized by the researcher.</li>
	<li>View the cells using a 63X lens with oil. Use a tile-scan image on continuous setting to view the center tile. Using the fluorescence intensity and gain of detector channels, adjust the focus and intensity of the laser and the gain of the two channels to optimize the image.</li>
	<li>Split the image using the settings to view both channels; check that there is no saturation of fluorescence intensity in the cytoplasm or vacuoles. Saturation appears as red dots. Reduce the laser intensity so that there is a minimal number of red dots, but the cells and phagosomes are bright enough to see clearly.</li>
	<li>When satisfied, click on &#34;Start experiment.&#34; A 9-tile scan image is generated. Save the image as a complex file recommended by the software that contains a composite image of the two channels (not jpeg or tiff).</li>
</ol>
6. Calibration Experiments
<ol>
	<li>Construct the pH standard curve with Candida alone.
	<ol>
		<li>Prepare the buffers from pH 3.0 to 13.0, as per Table 1.</li>
		<li>Take one aliquot (100 &#181;L) of HKC-S-1. Spin it down for 1 min at 17,200 x g. Resuspend it in 500 &#181;L of the assigned buffer.</li>
		<li>Starting from the lowest pH range, add the suspension to one pre-treated well (from step 4.2). Place the slide on the confocal microscope on a heated stage set to 37 &#176;C.</li>
		<li>Record the fluorescence every min for 10 min. Repeat for each buffer.</li>
	</ol>
	</li>
	<li>Saponin standard curve.
	<ol>
		<li>Isolate 1 x 107 human neutrophils5 and resuspend them in 1 mL of the lowest pH buffer.</li>
		<li>Follow the method described in section 4 for measuring phagosomal pH in neutrophils.</li>
		<li>After incubation with the Candida suspension for 20 min, add 0.3% saponin (15 &#181;L of 10% stock to one pre-treated well (from step 4.2)) to the dish, and then incubate for 20 min at 37 &#176;C. Take an image every minute for 10 min. Repeat for each buffer.</li>
	</ol>
	</li>
	<li>Cytosolic pH standard curves using the high K+/nigericin technique5,18,19.
	<ol>
		<li>Make up the buffers described in Table 1, from pH 4.0 to 13.0.</li>
		<li>Follow steps 4.1-4.7, leaving only neutrophils in the pre-treated wells &#8212; each well to contain a different buffer. Add the slide to the 37 &#176;C heated stage and record the fluorescence every min for 10 min.</li>
		<li>Note: It may take some time for the cytosolic pH to stabilize with the external solution, but after this point, the S-1 ratio value becomes constant. Measuring the extracellular HKC-S-1 in each experiment can serve as a control to check that any inhibitors added do not interfere with the S-1 emitted fluorescence or affect the buffer&#39;s pH.</li>
	</ol>
	</li>
</ol>
7. Image Analysis
NOTE: Here, instructions for image analysis (quantitation of fluorescence) using the free software ImageJ is provided. Use of ImageJ is recommended.
<ol>
	<li>Download and install ImageJ version &#62;1.46r as per the developer&#39;s instructions (https://imagej.nih.gov/ij/). Install the supplementary code file attached with this protocol called &#8220;Phagosomal measurement.&#8221;</li>
	<li>Open Image J and load the image file chosen for analysis onto the toolbar. To combine the two channels, use Image&#62;Color&#62;Make Composite.</li>
	<li>Right-click to choose a file in which to store results.</li>
	<li>Click on the line tool on the tool bar and double-click to increase the width to &#34;2.&#34;</li>
	<li>Draw a line across the width of a phagosome, and then right-click to &#34;measure pH&#34;. The average intensity of the two channels is acquired, and their ratio is calculated automatically by the code file in ImageJ.</li>
	<li>After finishing the measurements, right-click to choose &#34;save file.&#34;</li>
	<li>To measure the phagosome area, use the fourth icon along the tool bar to draw free-hand around the area. Right-click to select &#34;measure area.&#34; 
	NOTE: The results are saved and a log file is generated in the directory selected. Results may be processed using spreadsheet, R, or an equivalent software. The relationship between fluorescence ratio to pH is approximately sigmoidal, and the ratio values generated by the ImageJ analysis can be converted to pH using a generalized logistic or sigmoid function or by linear or cubic spline interpolation.</li>
	<li>To measure cytoplasm, draw a line across the cytoplasm and right-click to &#8220;measure pH&#8221;.</li>
</ol>

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The authors declare that they have no competing financial interests.

Once the appropriate reagents, microscope settings, and calibration experiments are set up, this method is relatively simple to perform. The critical steps include: labeling the Candida with S-1 to ensure that there is no overloading of the dye, calibrating, and analyzing the image.
S-1 is a reagent suited to more alkaline pH environments, which is particularly important for neutrophils21 but limits its use in certain cell types. For more acidic environments, such as macrophage phagosomes, SNARF-4, or S-4, is more suitable because of its lower pKa22. Moreover, for more accurate cytoplasmic readings, it is better to use S-4, as the standard curve for S-1 shows that fluorescence ratios begin to plateau below pH 6 (Figure 3). Other dyes, such as 2&#39;,7&#39;-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein (BCECF) or pHrodo Red may also be more suitable in a context that is expected to be acidic. Yet the cytoplasm staining is still necessary for correct identification of the phagosomes containing Candida.
An important feature of a phagosomal pH indicator is that it is not irreversibly altered by the reactive phagosomal environment. S-1 seems to be resistant to the neutrophil milieu. This is shown by Levine et al.5 (see Supplementary Video 4 of reference5) which demonstrate the phagocytosis and subsequent release of an S-1-labeled Candida particle by an Hvcn1-/- neutrophil. When phagocytosed, the particle turns from yellow/orange to red (neutral to alkaline pH), but when the particle is released by the neutrophil, it returns to its original color.
It is important to mention some of the limitations associated with using S-1. The fact that this dye has two emission spectra allowing ratiometric measurement is an advantage, but specialist equipment is needed to acquire images; the microscope used for the experiments must be able to record two images simultaneously or with an insignificant time delay. The authors assume that the researcher attempting this protocol has experience using confocal microscopy, or has access to a trained professional. We cannot list all the specific microscope parameters as they will differ for each microscope and need to be optimized by the researcher. The acetoxymethyl ester conjugated to S-1 that allows the dye to diffuse into the cell cytoplasm is degraded by non-specific esterases in the cell cytoplasm to form the fluorescent molecule. Esterases, such as alkaline phosphatase, are present in human serum and fetal bovine serum, which are used to supplement cell culture media. Accordingly, the medium in which the cells are loaded with S-1-AM (section 4.5) must not contain serum. This may prove challenging if using cells that require a more nutrient-rich medium to sustain them than the balanced salt solution used throughout this protocol. Similarly, other fluorescent medium components, such as phenol red, may interfere with S-1 measurements.
The error bars in Figure 3 indicate that there is some variation in the ratio measurements at each pH. A suitable number of repeats of each experiment (at least n = 3) and as many individual measurements in each single experiment are needed to overcome the inter-vacuolar variation. It is thus advisable to measure the pH of at least 100 phagosomes for each condition and as many phagosomal areas that appear to contain only one Candida. The phagosomes to be measured for quantitation should be those that have completely engulfed a Candida particle (i.e., those completely surrounded by cytoplasm). To mitigate against unintentional biases in the selection of cells/phagosomes for quantitation, all analyses should be performed while blind to the experimental conditions.
Here, we describe the isolation of neutrophils by dextran sedimentation of whole blood followed by centrifugation of the plasma layer through a density gradient. We use this technique as it quickly and efficiently produces a pure (&#62;95%) population of neutrophils, although there are other methods available, such as whole blood centrifugation through other density gradient formulas or negative selection of neutrophils using specialist kits with antibodies or magnetic beads. However, the latter can be prohibitively expensive for most groups who isolate neutrophils routinely. In addition, we use the anticoagulant heparin in the blood-collecting tube, whereas other researchers may be more accustomed to using ethylenediaminetetraacetic acid (EDTA) or acid citrate sodium (ACD). As there are many different methods to choose from, it is up to the personal preference of the researcher.
Furthermore, when isolating and manipulating neutrophils they should be handled with some care to avoid excessive activation. Precautionary steps include: only using plastic ware, no glass; filter-sterilize all buffers to remove any contaminating endotoxin; when spinning neutrophils make sure the centrifuge is well balanced to avoid excessive vibrations; limit as much as possible the time neutrophils remain in a pellet after centrifugation; do not maintain neutrophils in solution of more than 5 x 106/mL; and perform the experiment as soon as possible after isolation.
This method can be adapted to measure changes in the pH and phagosomal area over time by using a heated stage set to 37 &#176;C on the microscope and taking snapshots once every 30-60 s from the same position, as described for the calibration steps. It could also theoretically be adapted for higher-throughput experiments, such as in 96-well plates, and for flow cytometry experiments, where S-1 can be used as a pH indicator23. However, in these settings, the emphasis on individual cell activity is replaced by a more global effect on the cell population.
This method aims to provide a relatively simple experimental setup upon which individual researchers can adapt to suit their area of interest. Researchers may want to explore neutrophil phagosomal pH and area whilst also measuring movement of other ions, for example, intracellular calcium concentration. There are several fluorescent Ca2+ indicators readily available for confocal microscopy, such as Indo-1, which also has dual emission spectra at 400 and 475 nm24. These emission wavelengths do not overlap with S-1 emission spectra, but the excitation wavelength is at the ultraviolet (UV) end of the spectrum, which can be damaging to cells, and a UV laser is not commonplace on all microscopes. A comprehensive review of the different indicators to measure intracellular calcium flux is covered by Takahashi et al.25 and Hillson et al.26.
In conclusion, there are other methods available to measure phagosomal pH using different fluorescent dyes as Nunes et al. have demonstrated13 as well as other groups27,28. Other researchers have also used S-1 to measure cytosolic pH29 or phagosomal pH14. However, this protocol is unique in measuring simultaneously the pH of both cytosol and phagosome, which provides the opportunity to observe changes in phagosomal cross-sectional area and to distinguish between wildtype and Hvcn1-/- mouse neutrophils, and human neutrophils with and without a working NADPH oxidase.

The neutrophil is the most abundant innate immune cell in the body. Its main function consists of patrolling the bloodstream and engulfing and digesting the foreign particles that it may encounter in a process known as phagocytosis1,2. The particles are degraded in an intracellular compartment called the phagosome. The activation of neutrophil NADPH oxidase, the isoform NOX2, initiates a cascade of biochemical reactions that culminates in the death of the pathogen. NOX2 protein subunits proceed to form an electron transport chain complex in the phagosomal membrane3. Once activated, it transports electrons from NADPH across the membrane to molecular oxygen inside the phagosome, producing superoxide anions and further reactive oxygen species. This is known as the respiratory burst, and it is thought to be essential for efficient microbial killing and digestion2. However, this exclusive movement of negative charge across the membrane would soon inactivate NOX2 if it were not compensated for by positive charge moving in and/or negative charge moving out of the phagosome. It has been well established that the majority of charge compensation in the neutrophil is carried out by the proton channel HVCN14,5. This channel allows for the passive movement of protons down their electrochemical gradient from the cytosol into the phagosome. Proton concentration is reflected by pH, so for a given level of oxidase activity, measuring the pH in the phagosome can provide information on the relative participation of protonic and non-protonic pathways in charge compensation.
The human neutrophil phagosome has an alkaline pH of approximately 8.5 for 20-30 min after phagocytosis5. This implies the existence of additional non-proton ion channels in NOX2-induced charge compensation, as the fusion and release of the contents of the acidic granules and sole compensation by HVCN1 would maintain an acidic environment, in contrast to that observed. The movement of ions to compensate this negative charge may also exert changes in phagosome size via osmosis. These may be ions present in the neutrophil at high physiological concentrations: potassium ions have been shown to move into the phagosome6, and chloride ionic movement is another candidate important for neutrophil function7.
The regulation of pH in the phagosome is vital for antimicrobial protease activity5. Myeloperoxidase (MPO) appears to have optimal activity at pH 6, while for cathepsin G and elastase, the optimal levels are pH 7-9 and pH 8-10, respectively5. Therefore, transient change in phagosomal pH may provide activity niches for different enzymes to function. Understanding how pH is involved in neutrophil microbial killing may provide useful information for the design of novel neutrophil-augmenting microbial agents.
The neutrophil phagosome is a highly reactive environment. This makes it difficult to accurately assess pH, because dyes may be easily oxidized, leading to technical artefacts. Historically, fluorescein isothiocyanate (FITC) has been the dye of choice to measure intracellular pH8,9. However, there are some disadvantages for its use in measuring neutrophil phagosomal pH. It has a pKa of 6.410, meaning that it can only accurately be used to assess pH levels from 5 to 7.58, as it saturates at pH &#60; 811. As the neutrophil phagosomal pH can become much more alkaline5, FITC cannot capture the full range of potential pH changes. A further significant problem with FITC in the context of neutrophils is that it is thought to be photobleached by MPO12. The MPO inhibitor, sodium azide, can be used to limit photobleaching13, but it has been shown that sodium azide directly lowers the phagosomal pH in an MPO-independent manner and is thus inappropriate for use in such assays5.
Compared to other intracellular dyes, S-1 has a relatively high pKa of 7.510. In acidic conditions, the molecule is protonated and produces an emission signal between 560 and 600 nm when excited at 488 nm or above. When the molecule is deprotonated in more alkaline conditions, the emission wavelength is over 600 nm. A ratio of the fluorescence intensities at these two wavelengths indicates the emission shift, which is more is reliable than single fluorescence measurements, as it is unaffected by fluorophore concentration and cell structure. S-1 can be conjugated to antigenic material, such as zymosan14, although heat-killed (HK) Candida albicans is preferred, as the larger surface area gives a more consistent fluorescence reading.
We have also used a modification of this method to study temporal changes in pH (Figure 3)5. This method for measurement of cytosolic pH can be easily applied to other cell types, as described elsewhere15,16, and cells with more alkaline phagosomes14.

<table border="0" cellpadding="0" cellspacing="0" width="851">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Candida albicans ATCC 10231 Vitroids&#160; 80 CFU</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">RQC14003-10EA</td>
			<td style="width:360px;">Place directly on a spread agar plate according to the manufacturer&#39;s instructions</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">YPD broth</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">Y1375</td>
			<td style="width:360px;">Follow manufacturer&#39;s instructions, various providers exist</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Dextran Clinical grade MW = 200,000-300,000</td>
			<td style="width:99px;">MP Biomedicals</td>
			<td style="width:119px;">2101514</td>
			<td style="width:360px;">Use gloves&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:275px;">Heparin Sodium solution for infusion (preservative free), 1,000IU/ml&#160;</td>
			<td style="width:99px;">Fannin, Wockhardt UK Ltd</td>
			<td style="width:119px;">FP1077</td>
			<td style="width:360px;">We purchased from UCH inpatient pharmacy</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:275px;">SNARF-1 Carboxylic Acid, Acetate, Succinimidyl Ester - Special Packaging</td>
			<td style="width:99px;">Thermo Scientific/Invitrogen</td>
			<td style="width:119px;">SS22801</td>
			<td style="width:360px;">Use gloves</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:275px;">5-(and-6)-Carboxy SNARF-1, Acetoxymethyl Ester, Acetate</td>
			<td style="width:99px;">Thermo Scientific/Invitrogen</td>
			<td style="width:119px;">C1272</td>
			<td style="width:360px;">Use gloves</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Vivaglobin human IgG&#160; 160mg/ml solution</td>
			<td style="width:99px;">CSL Behring</td>
			<td style="width:119px;">received as a gift</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Normal mouse serum</td>
			<td style="width:99px;">Abcam</td>
			<td style="width:119px;">ab7486</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Lymphoprep (density gradient medium)</td>
			<td style="width:99px;">Alere Ltd</td>
			<td style="width:119px;">1114547</td>
			<td style="width:360px;">Keep sterile and away from direct sunlight, can be substituted by Ficoll-Paque</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Nigericin</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">N7143</td>
			<td style="width:360px;">Toxic, use gloves, various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Saponin</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">47036</td>
			<td style="width:360px;">Toxic, use gloves, various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Dimethyl sulphoxide (DMSO)</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8418</td>
			<td style="width:360px;">Toxic, use gloves, various providers exist</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Tris(hydroxymethyl)aminomethane (Tris)</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">T1503&#160;</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Potassium chloride (KCl)</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9333</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Sodium chloride (NaCl)</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7653</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Magnesium chloride (MgCl2)</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Magnesium sulphate (MgSO4)</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7506</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Potassium dihydrogen phosphate (KH2PO4)</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9791</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Calcium chloride (CaCl2)</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1016</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Glucose</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9434</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Absolute Ethanol&#160;</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">2860</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Diphenylene iodonium (DPI)</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">D2926</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Zinc chloride</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">208086</td>
			<td style="width:360px;">Various providers exist, do not dilute in phosphate based buffers as it will precipitate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Poly L lysine</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">P4707</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:275px;">Laser scanning confocal microscope (Zeiss LSM 700)</td>
			<td style="width:99px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:360px;">The microscope must be able to excite at 555nm and measure emission at two channels simultaneously (560-600nm, &#62;610nm)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Ibidi &#181; slide 8 well ibiTreat</td>
			<td style="width:99px;">Thistle Scientific</td>
			<td style="width:119px;">IB-80826</td>
			<td style="width:360px;">Other appropriate imaging dishes can be substituted</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:275px;">Fluorodish Cell Culture Dish-35mm, 23mm well</td>
			<td style="width:99px;">World Precision Instruments&#160;</td>
			<td style="width:119px;">FD35-100</td>
			<td style="width:360px;">Other appropriate imaging dishes can be substituted, different sizes are available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Soniprep 150</td>
			<td style="width:99px;">MSE</td>
			<td style="width:119px;"></td>
			<td style="width:360px;">Any appropriate sonicator will suffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">TC20 Automated Cell Counter</td>
			<td style="width:99px;">BioRad</td>
			<td style="width:119px;"></td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">1.5ml Protein LoBind Tubes</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z666505-100EA</td>
			<td style="width:360px;">Specially used as Candida can stick to normal eppendorfs</td>
		</tr>
	</tbody>
</table>

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.

Figure 1 presents snapshots of neutrophils from different origins to demonstrate varying phagosomal environments. To ease quantitative analysis, it is important to seed the wells with an appropriate number of cells: too many will cause the cells to layer over each other, making it difficult to view enclosed phagosomes accurately; too few will, of course, provide fewer results, particularly as not every neutrophil will phagocytose. Figure 2 is an image that is over-saturated; this can be assessed by splitting the image between its two channels (using the microscope software recommended in the Materials List or an equivalent) &#8212; red dots show where maximum fluorescence has been detected. This can be countered by reducing the intensity of the laser. Calibration curves using the various buffer systems are shown in Figure 3, adapted from Levine et al.5. The error bars show that there is some variation in fluorescence between readings. Figure 4 gives an example of how the data for phagosomal pH and area could be presented. This approach allows each individual measurement to be displayed with an over-laying boxplot. However, the data could also be displayed in a histogram bar chart.
<img alt="Figure 1" src="/files/ftp_upload/55107/55107fig1.jpg" /> 
Figure 1: Appropriate snapshot images of neutrophils from humans and mice. To the far right is a qualitative visual key of the approximate color of the S-1-stained phagosomes corresponding to the pH. The yellow color indicates more acidity, while red indicates more alkalinity. (A) shows Hvcn1-/- mouse bone marrow neutrophils 20 min after phagocytosis. The phagosomes appear very red, alkaline, and swollen. The red arrow in the bottom right part of the image points to an intracellular Candida, while the arrow in the top left points to an extracellular particle. (B) shows wildtype mouse bone marrow neutrophils that have ingested Candida; they are much less alkaline than Hvcn1-/- cells. (C) shows human peripheral blood neutrophils at the same point after phagocytosis. They appear slightly more alkaline than the mouse wildtype cells, but the phagosomes are still not as large and red as the Hcvn1-/- cells. (D) shows human neutrophils that have phagocytosed Candida in the presence of 5 &#181;M diphenylene iodonium (DPI). All the phagosomes are very acidic, with a pH of 6 or less; the drug inhibits the NADPH oxidase, so there is no compensatory ion movement. The protons released from the acidic granules that fuse to the phagosome cause the acidic pH20, and increased recruitment of the V-ATPase to the phagosomal membrane upon treatment with DPI9. The cytoplasm also appears more alkaline compared to the cells from the other conditions. <a href="http://cloudflare.jove.com/files/ftp_upload/55107/55107fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55107/55107fig2.jpg" /> 
Figure 2: Over-saturation of Hvcn1-/- mouse bone marrow neutrophils. It is important to exclude from the analysis images in which the fluorescence data are over-saturated. As described in section 5.3, the composite image is split into two images (top left, red arrow) with both channels presented individually. In this software, range indicator is checked on (bottom left, red arrow), then any pixels which are over-saturated are bright red. There is over-saturation present in both channels (1 and 2). A magnification of some cells and extracellular Candida is shown at the bottom right, with arrows pointing to affected areas. Analyzing these points would lead to a false ratiometric measurement. <a href="http://cloudflare.jove.com/files/ftp_upload/55107/55107fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55107/55107fig3.jpg" /> 
Figure 3: Standard calibration curves for the conversion of S-1 fluorescence ratios to pH measurements. The standard curves for Candida alone in the different buffers (labeled as &#34;Tris&#34;) and when the cells are permeabilized with saponin (&#34;Saponin&#34;) are very similar, showing that the S-1 reading inside and outside the cell (phagosome and extracellular medium) are comparable. The error bars represent the mean &#177; SD. The S-1 ratio/pH curve is shortened when tested in the cytoplasm (&#34;Cytoplasm&#34;), which should be taken into consideration in the image analysis. This figure has been modified from Levine et al.5. <a href="http://cloudflare.jove.com/files/ftp_upload/55107/55107fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55107/55107fig4.jpg" /> 
Figure 4: Quantification of phagosomal pH and area. This figure presents an example of data presentation. The graphs were generated using the programming software R. A: shows phagosomal ratio and corresponding pH for A (Hvcn1-/- mouse bone marrow neutrophils), B: wildtype mouse bone marrow neutrophils, C: human peripheral blood neutrophils, and D: human neutrophils with 5 &#181;M DPI n = 3/300. Individual measurements are shown as small squares, with an overlaying boxplot with median and interquartile range. A red bar represents the mean. As seen in the images in Figure 1, Hvcn1-/- cells have very alkaline phagosomes in comparison to wildtype mouse and human neutrophils. They also have a bigger phagosomal area (Figure 4B, n = 3/300). Human neutrophil phagosomes are slightly more alkaline and larger than wildtype mouse neutrophils, while human neutrophils incubated with DPI have very acidic and small phagosomes. <a href="http://cloudflare.jove.com/files/ftp_upload/55107/55107fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table>
	<tbody>
		<tr>
			<td colspan="4">Balanced salt solution (BSS) buffer</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>156 mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>3 mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>2 mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>1.25 mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>2 mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>10 mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>10 mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">pH 7.4 with NaOH or HCl</td>
		</tr>
		<tr>
			<td colspan="4">YPD broth</td>
		</tr>
		<tr>
			<td>YPD broth</td>
			<td>50 g</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>1 L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Autoclave at 121 &#176;C for 15 min</td>
		</tr>
		<tr>
			<td colspan="4">For YPD agar: before autoclaving add 15 g/L agar</td>
		</tr>
		<tr>
			<td colspan="4">10% Dextran solution</td>
		</tr>
		<tr>
			<td>Dextran clinical grade</td>
			<td></td>
			<td>50 g</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td>4.5 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td>500 mL</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Add to glass bottle and autoclave at 121 &#176;C for 15 min</td>
		</tr>
		<tr>
			<td colspan="4">2x Saline solution</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td>18 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water&#160;</td>
			<td></td>
			<td>1 L</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Add to glass bottle and autoclave at 121 &#176;C for 15 min</td>
		</tr>
		<tr>
			<td colspan="4">10% Saponin stock</td>
		</tr>
		<tr>
			<td>BSS buffer</td>
			<td></td>
			<td>50 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td></td>
			<td>5 g</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Heat BSS buffer to 37 &#176;C, add saponin and mix.</td>
		</tr>
		<tr>
			<td colspan="4">Add 0.1% sodium azide as preservative, store mixture at 4 &#176;C.</td>
		</tr>
		<tr>
			<td colspan="4">Calibration buffers</td>
		</tr>
		<tr>
			<td colspan="4">Candida standard curve</td>
		</tr>
		<tr>
			<td colspan="4">First make up 0.15 M stock of each buffer</td>
		</tr>
		<tr>
			<td colspan="4">Make up 0.15 M NaCl solution</td>
		</tr>
		<tr>
			<td colspan="4">15 mL final volume: 5 mL 0.15 M of desired buffer solution + 10 mL 0.15 M NaCl solution</td>
		</tr>
		<tr>
			<td>pH 3</td>
			<td>100 mM NaCl</td>
			<td>50 mM glycine</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 4</td>
			<td>100 mM NaCl</td>
			<td>50 mM acetate</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 5</td>
			<td>100 mM NaCl</td>
			<td>50 mM acetate</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 6</td>
			<td>100 mM NaCl</td>
			<td>50 mM acetate</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 7</td>
			<td>100 mM NaCl</td>
			<td>50 mM Tris</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 8</td>
			<td>100 mM NaCl</td>
			<td>50 mM Tris</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 9</td>
			<td>100 mM NaCl</td>
			<td>50 mM Tris</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 10</td>
			<td>100 mM NaCl</td>
			<td>50 mM glycine</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 11</td>
			<td>100 mM NaCl</td>
			<td>50 mM phosphate</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 12</td>
			<td>100 mM NaCl</td>
			<td>50 mM phosphate</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 13</td>
			<td>100 mM NaCl</td>
			<td>50 mM phosphate</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Cytosolic standard curve</td>
		</tr>
		<tr>
			<td colspan="4">Use previously made 0.15 M stock buffer solutions</td>
		</tr>
		<tr>
			<td colspan="4">Make up 0.15 M KCl solution</td>
		</tr>
		<tr>
			<td colspan="4">15 mL final volume: 5 mL 0.15 M of desired buffer + 10 mL 0.15 M KCl solution</td>
		</tr>
		<tr>
			<td colspan="4">10 mM stock of nigericin in ethanol, add 15 &#181;L to each final volume solution</td>
		</tr>
		<tr>
			<td>pH 3</td>
			<td>100 mM KCl</td>
			<td>50 mM glycine</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td>pH 4</td>
			<td>100 mM KCl</td>
			<td>50 mM acetate</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td>pH 5</td>
			<td>100 mM KCl</td>
			<td>50 mM acetate</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td>pH 6</td>
			<td>100 mM KCl</td>
			<td>50 mM acetate</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td>pH 7</td>
			<td>100 mM KCl</td>
			<td>50 mM Tris</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td>pH 8</td>
			<td>100 mM KCl</td>
			<td>50 mM Tris</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td>pH 9</td>
			<td>100 mM KCl</td>
			<td>50 mM Tris</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td>pH 10</td>
			<td>100 mM KCl</td>
			<td>50 mM glycine</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td>pH 11</td>
			<td>100 mM KCl</td>
			<td>50 mM phosphate</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td>pH 12</td>
			<td>100 mM KCl</td>
			<td>50 mM phosphate</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td>pH 13</td>
			<td>100 mM KCl</td>
			<td>50 mM phosphate</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td colspan="4">pH all calibration solutions with either HCl or NaOH</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of buffers. This table describes the appropriate compositions of the different buffers used in the protocol.

Supplementary code file. This file contains a number of macros, written by A. P. Levine, which are necessary for image analysis. The authors would be happy to try to address any queries associated with using this code. <a href="https://www.jove.com/files/ftp_upload/55107/Phagosomal_measurement.txt" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator at JoVE.com

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

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 ...

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JoVE Journal

Immunology and Infection

10.0K Views.  University College London. 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.

Video: Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

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 ...

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.

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 ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

"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 ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, periodontitis, mucosal infections and, most notably, early childhood caries. In all of these conditions, the pH in the biofilm matrix impacts microbe-host interactions and thus the disease progression. The present protocol describes a confocal microscopy-based method to monitor pH dynamics inside cross-kingdom biofilms comprising Candida albicans and Streptococcus mutans. The pH-dependent dual-emission spectrum and the staining properties of the ratiometric probe C-SNARF-4 are exploited to determine drops in pH in extracellular areas of the biofilms. Use of pH ratiometry with the probe requires a meticulous choice of imaging parameters, a thorough calibration of the dye, and careful, threshold-based post-processing of the image data. When used correctly, the technique allows for the rapid assessment of extracellular pH in different areas of a biofilm and thus the monitoring of both horizontal and vertical pH gradients over time. While the use of confocal microscopy limits Z-profiling to thin biofilms of 75 &#181;m or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.

The protocol describes the cultivation of cross-kingdom biofilms consisting of Candida albicans and Streptococcus mutans and presents a confocal microscopy-based method for the monitoring of extracellular pH inside these biofilms.