The overall goal of this confocal microscopy technique is to image the pH and area of the neutrophil phagocytic vacuole and cytoplasm. This method enables the monitoring and quantification of dynamic changes in the pH in the phagocytic vacuole and in the cytoplasm, and also the volume of the phagocytic vacuole. These measurements change with the activity of the NADPH oxydase, and can be used as surrogate markers of its function and of ion fluxes across the membrane of the phagocytic vacuole.
The main advantage of this technique is that both the phagosome and cytoplasm can be simultaneously imaged with a dye that has a wide pH range. To begin this procedure, prepare an aliquot of carboxy-S-1 succinimidyl ester by diluting 50 micrograms of it in 100 microliters of high grade DMSO. Vortex it well to mix.
Next, prepare one milliliter of one times 10 to the eighth heat killed or HK Candida in 0.1 molar sodium bicarbonate in a 15 milliliter tube. Add 100 microliters of carboxy-S-1 one drop at a time to the HK Candida while mixing on a vortex at 2, 000 RPM. After that, wrap the tube with aluminum foil and place it on a roller at room temperature for one hour.
After an hour, wash the HKC-S-1 three times by centrifugation at 2, 250 times g for 10 minutes each time. For the first two washes, re-suspend the pellet in 15 milliliters of 0.1 molar sodium bicarbonate. After the third wash, re-suspend it in one milliliter of BSS buffer.
Transfer HKC-S-1 suspension into the tubes of 100 microliter aliquots and store them at minus 20 degrees Celsius. To opsonize HKC-S-1 for human neutrophils, add 100 microliters of human IGG serum to 100 microliters of thawed HKC-S-1. Mix it on a heat shaker at 37 degrees Celsius and 1, 1000 RPM for 60 to 90 minutes, and then on a roller, at four degrees Celsius for two hours.
After that, wash the sample three times in BSS buffer by centrifugation at 17, 000 times g for one minute each. Subsequently, re-suspend the sample in 100 microliters of BSS buffer. For human peripheral blood neutrophils, take 15 milliliters of blood by venipuncture from a healthy donor and transfer it into a 20 milliliter syringe containing 90 microliters of heparin sodium solution at 1, 000 IU per milliliter.
Using a pipette tip, inject 1.5 milliliters of 10%dextran solution into the syringe. Invert it gently three times and then leave it standing upright on the bench for 30 to 60 minutes. After 30 to 60 minutes, the blood should split roughly in half, with a straw color top buffy coat layer and a bottom layer containing erythrocytes.
Carefully push out the top layer through a needle or remove it with a pipette tip, then transfer it to a 15 milliliter tube and avoid taking out the red layer. Using a five milliliter pipette, add three to four milliliters of density gradient medium to the bottom of the tube, below the buffy coat layer, to obtain two distinct layers. Subsequently, centrifuge the sample at 900 times g for 10 minutes.
Pour off the supernatant and leave the red pellet behind. Following that, gently vortex to disturb the pellet. Next, add seven milliliters of distilled autoclaved water to the pellet and invert it for 20 seconds to re-suspend the pellet.
Following that, add seven milliliters of 2x saline and invert it a few times to mix, lysing the remaining red blood cells, then centrifuge the sample at 300 times g for five minutes. Pour off the supernatant and re-suspend the pellet in BSS buffer to approximately four times 10 to the sixth per milliliter. In this procedure, pre-treat an eight well microscopy plate with 200 microliters of poly-L-lysine in each well for 40 to 60 minutes at room temperature, then remove the poly-L-lysine and wash the wells twice with 200 microliters of distilled water.
Next, add 200 microliters of the prepared cell suspension to each well and incubate it at room temperature for 30 to 60 minutes. After that, prepare an aliquot of S-1-AM ester by adding 100 microliters of high grade DMSO to a tube and vortex to mix. In a small tube, add 1.7 milliliters of BSS buffer and 20 microliters of S-1-AM and vortex the mixture.
Afterward, wash the wells twice with 200 microliters of BSS buffer and replace the buffer with S-1-AM solution. Take care not to disturb the cell monolayer attached to the well's bottom by gently pipetting the liquid down the wall of the wells. Subsequently, incubate the sample at room temperature for at least 25 minutes.
Following that, wash the wells twice with 200 microliters of BSS buffer. To test an inhibitor, make up a master mix of the appropriate drug in BSS buffer and wash the wells twice with it. Next, sonicate the opsonized HKC-S-1 for approximately three seconds at five microns and add 10 microliters of it to each well, then incubate the plate at 37 degrees Celsius for 15 to 20 minutes, making the cells ready to be imaged for snapshots of phagocytosis.
Using a confocal microscope, adjust the laser wavelength so that the cells are excited at 555 nanometers and the emission is detected by two detectors at 560 to 600 nanometers, and over 610 nanometers. Next, view the cells using a 63X oil immersion lens. Use a tile scan image on continuous setting to view the center tile.
Using the fluorescence intensity and the gain of detector channels, adjust the focus and intensity of the laser and the gain of the two channels to optimize the image. After that, split the image using the settings to view both channels. Before the start of the experiment, check that there is no saturation of fluorescence intensity in the cytoplasm or vacuoles, which would appear as red dots.
If so, reduce the laser intensity so that there are a minimal number of red dots, but the cells and phagosomes are bright and clear enough to see. In this procedure, open ImageJ and load the image file chosen for analysis onto the toolbar. To combine the two channels, use Image, Color, then Make Composite.
Right click to choose a file to store the results. Next, click on the Line Tool on the toolbar and double click to increase the width to two. Draw a line across the width of a phagosome then right click to measure pH.
Cytoplasmic pH can be measured in the same way by drawing a line across the cytoplasm. After finishing the measurements, right click to choose Save File. To measure the phagosome area, use the fourth icon along the toolbar to draw freehand around the area.
Subsequently, right click to select Measure Area. Here 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.
This image shows mouse-bound marrow neutrophils lacking the Hvcn1 channel 20 minutes after phagocytosis. The phagosomes appear very red, alkaline and swollen. The red arrow here points to an intracellular Candida, while this arrow points to an extracellular Candida.
This image shows wild type mouse bone marrow neutrophils that have ingested Candida. They are much less alkaline than Hvcn1 knockout neutrophils. This image shows human peripheral blood neutrophils at the same time point after phagocytosis.
They appear slightly more alkaline than the mouse wild type cells, but the phagosomes are still not as large and red as the Hvcn1 knockout cells, and this image shows human neutrophils that have phagocytosed Candida in the presence of five micromolar diphenylene iodonium. Once mastered, this technique can be done in approximately four to five hours if it is performed properly. While attempting this procedure, it's important to remember to be careful when isolating the neutrophils so that they are not activated.
In order to determine whether the effects one's seeing on changes in vacuolar or cytosolic pH or vacuolar volume are due to changes in respiratory burst, then this respiratory burst can be measured in other ways that directly measure free radical production or oxygen consumption. After watching this video, you should have a good understanding of how to isolate human neutrophils then stain and image the phagosome and cytoplasm. Don't forget that working with human blood samples can be extremely hazardous, and precautions such as wearing gloves and protective clothing should always be taken while performing this procedure.
