Name: imaging the neutrophil phagosome and cytoplasm using a ratiometric ph indicator
Uploaded: 2017-04-05T14:00:00.000+00:00
Duration: 12 min 42 s
Description: Watch this Scientific Journal Video about Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator at JoVE.com

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><tbody><tr><td>&lt;em&gt;Candida albicans&lt;/em&gt; ATCC 10231 Vitroids&amp;nbsp; 80 CFU</td><td>Sigma-Aldrich</td><td>RQC14003-10EA</td><td>Place directly on a spread agar plate according to the manufacturer&#039;s instructions</td></tr><tr><td>YPD broth</td><td>Sigma-Aldrich</td><td>Y1375</td><td>Follow manufacturer&#039;s instructions, various providers exist</td></tr><tr><td>Dextran Clinical grade MW = 200,000-300,000</td><td>MP Biomedicals</td><td>2101514</td><td>Use gloves&amp;nbsp;</td></tr><tr><td>Heparin Sodium solution for infusion (preservative free), 1,000 IU/mL&amp;nbsp;</td><td>Fannin, Wockhardt UK Ltd</td><td>FP1077</td><td>We purchased from UCH inpatient pharmacy</td></tr><tr><td>SNARF-1 Carboxylic Acid, Acetate, Succinimidyl Ester - Special Packaging</td><td>Thermo Scientific/Invitrogen</td><td>SS22801</td><td>Use gloves</td></tr><tr><td>5-(and-6)-Carboxy SNARF-1, Acetoxymethyl Ester, Acetate</td><td>Thermo Scientific/Invitrogen</td><td>C1272</td><td>Use gloves</td></tr><tr><td>Vivaglobin human IgG&amp;nbsp; 160 mg/mL solution</td><td>CSL Behring</td><td>received as a gift</td><td>Various providers exist</td></tr><tr><td>Normal mouse serum</td><td>Abcam</td><td>ab7486</td><td>Various providers exist</td></tr><tr><td>Lymphoprep (density gradient medium)</td><td>Alere Ltd</td><td>1114547</td><td>Keep sterile and away from direct sunlight, can be substituted by Ficoll-Paque</td></tr><tr><td>Nigericin</td><td>Sigma-Aldrich</td><td>N7143</td><td>Toxic, use gloves, various providers exist</td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>47036</td><td>Toxic, use gloves, various providers exist</td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td>Toxic, use gloves, 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>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>Magnesium sulphate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>M7506</td><td>Various providers exist</td></tr><tr><td>Potassium dihydrogen phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>P9791</td><td>Various providers exist</td></tr><tr><td>Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>C1016</td><td>Various providers exist</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>D9434</td><td>Various providers exist</td></tr><tr><td>Absolute Ethanol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>2860</td><td>Various providers exist</td></tr><tr><td>Diphenylene iodonium (DPI)</td><td>Sigma-Aldrich</td><td>D2926</td><td>Various providers exist</td></tr><tr><td>Zinc chloride</td><td>Sigma-Aldrich</td><td>208086</td><td>Various providers exist, do not dilute in phosphate based buffers as it will precipitate</td></tr><tr><td>Poly L lysine</td><td>Sigma</td><td>P4707</td><td>Various providers exist</td></tr><tr><td>Laser scanning confocal microscope (Zeiss LSM 700)</td><td>Carl Zeiss</td><td></td><td>The microscope must be able to excite at 555 nm and measure emission at two channels simultaneously (560-600 nm, &gt;610 nm)</td></tr><tr><td>Ibidi &amp;micro; slide 8 well ibiTreat</td><td>Thistle Scientific</td><td>IB-80826</td><td>Other appropriate imaging dishes can be substituted</td></tr><tr><td>Soniprep 150</td><td>MSE</td><td></td><td>Any appropriate sonicator will suffice</td></tr><tr><td>TC20 Automated Cell Counter</td><td>BioRad</td><td></td><td>Various providers exist</td></tr><tr><td>1.5 mL Protein LoBind Tubes</td><td>Sigma-Aldrich</td><td>Z666505-100EA</td><td>Specially used as &lt;em&gt;Candida&lt;/em&gt; 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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 ...

imaging-neutrophil-phagosome-cytoplasm-using-ratiometric-ph

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.0K Views.  University College London.  Dit manuscript beschrijft een eenvoudige methode om de pH en phagosomal gebied en het cytoplasmatische pH van mens en muis neutrofielen via de ratiometrische indicator seminaphthorhodafluor (SNARF) -1 of S-1 te meten. Dit wordt bereikt met behulp van levende cellen confocale fluorescentie microscopie en beeldanalyse. 

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

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 ...

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 ...

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major immune cells and only phagocytes in the brain, recent studies have shown that astrocytes also participate in various phagocytic processes, such as developmental synapse elimination and clearance of amyloid beta plaques in Alzheimer&#39;s disease (AD). Despite these findings, the efficiency of astrocyte engulfment and degradation of their targets is unclear compared with that of microglia. This lack of information is mostly due to the lack of an assay system in which the kinetics of astrocyte- and microglia-mediated phagocytosis are easily comparable. To achieve this goal, we have developed a long-term live-imaging in vitro phagocytosis assay to evaluate the phagocytic capacity of purified astrocytes and microglia. In this assay, real-time detection of engulfment and degradation is possible using pH indicator-conjugated synaptosomes, which emit bright red fluorescence in acidic organelles, such as lysosomes. Our novel assay provides simple and effective detection of phagocytosis through live-imaging. In addition, this in vitro phagocytosis assay can be used as a screening platform to identify chemicals and compounds that can enhance or inhibit the phagocytic capacity of astrocytes. As synaptic pruning malfunction and pathogenic protein accumulation have been shown to cause mental disorders or neurodegenerative diseases, chemicals and compounds that modulate the phagocytic capacity of glial cells should be helpful in treating various neurological disorders.

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

This protocol presents an in vitro live-imaging phagocytosis assay to measure the phagocytic capacity of astrocytes. Purified rat astrocytes and microglia are used along with pH indicator-conjugated synaptosomes. This method can detect real-time engulfment and degradation kinetics and provides a suitable screening platform to identify factors modulating astrocyte phagocytosis.

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

Neuroscience

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. ...

Quantifying NET Formation Using Dual-Dye Live Cell Analysis

Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that neutrophils play in the pathogenesis of cancer, autoimmune diseases, and various acute and chronic inflammatory conditions by contributing to the initiation and perpetuation of immune dysregulation through multiple mechanisms, including the formation of neutrophil extracellular traps (NETs), which are structures crucial in antimicrobial defense. Limitations in techniques to quantify NET formation in an unbiased, reproducible, and efficient way have restricted our ability to further understand the role of neutrophils in health and diseases. We describe an automated, real-time, high-throughput method to quantify neutrophils undergoing NET formation using a live cell imaging platform coupled with a membrane permeability-dependent dual-dye approach using two different DNA dyes to image intracellular and extracellular DNA. This methodology is able to help assess neutrophil physiology and test molecules that can target NET formation.

Author Spotlight: Quantifying Neutrophil Extracellular Traps in Disease and Drug Screening Using Dual-Color Live-Cell Imaging

We present an automated high-throughput method to quantify neutrophil extracellular traps (NETs) utilizing the live cell analysis system, coupled with a membrane permeability-dependent dual-dye approach.

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that ...

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 ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

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