Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

The overall goal of this set of experiments is to measure phaco somal pH in living cells using ratio metric fluorescence microscopy. This method can help answer key questions in the field of phagocyte biology, such as identifying key molecules in pathogen interactions or the effect of genetic disruption or drugs on phagocytosis. The main advantage of this technique is that unlike population-based methods such as facts or spectroscopy effects, such as changes in particle binding migration, or in the heterogeneity of the individual phagosomes can be detected.

The implication of this technique extend to our therapies against pathogenic chemical organisms because many of these microorganisms can alter fragoso pH in order to avoid distraction within phagosomes. To begin this procedure, add 20 milligrams of dried Xan to 10 milliliters of sterile PBS vortex and heat in a boiling water bath for 10 minutes. Then cool and centrifuge at 2000 Gs for five minutes.

After that, remove the supernatant and resuspend the Xan in one milliliter of PBS. Sonicate it for 10 minutes in a water bath. Sonicate then transfer 500 microliters of the resuspended Xan to two 1.5 milliliter tubes and centrifuge them at 11, 000 Gs for five minutes.

Repeat the wash with 500 microliters of PBS. Then wash the Xan twice in 500 microliters of freshly prepared 0.1 molar sodium carbonate solution at pH 9.3 and RESUSPENDED in 490 microliters of sodium carbonate solution after the final wash. Subsequently, add 10 microliters of FZ dissolved in DMSO at 10 milligrams per milliliter to the Xan vortex.

Cover with foil and incubate with agitation for four hours at room temperature To remove the unbound eye first wash with 0.5 milliliters of DMSO plus 150 microliters of PBS, including sonication between the wash steps. Next wash with 0.5 milliliters of DMSO plus 300 microliters of PBS, then washed with 0.5 milliliters of DMSO plus 450 microliters of PBS followed by PBS alone. After that, add one microliter of Xan to 500 microliters of PBS and vortex.

Then add 10 microliters of diluted xma sand to the edge of the hemo cytometer chamber where it meets the cover slip mount the hemo cytometer on a brightfield microscope equipped with a 20 x objective. Focus on the upper left hand corner containing 16 squares, and count all the xmas sand particles in this area. Using a hand tally counter, repeat the procedure on the lower right hand corner.

Then calculate the Xan concentration using this equation. Next, opsonize the labeled Xan with the rabid anti Xan antibody at a one to 100 dilution vortex and incubate the sample for one hour in a 37 degree Celsius water bath. Subsequently count the xmas sand using a hemo cytometer then diluted to 100 times 10 to the sixth particles per milliliter.

After isolating the primary mouse neutrophils, add two times 10 to the fifth of neutrophils to the center of a 35 millimeter glass bottom dish. Then spread the drop by adding 50 microliters of medium and incubated at 37 degrees Celsius for five minutes to allow the cells to adhere. Afterward, add one milliliter of HBSS warm to 37 degrees Celsius containing myelo peroxidase inhibitor.

Mount the dish on a wide field fluorescence microscope, equipped with a 37 degree Celsius heating system and a 40 x oil objective. Incubate the cells without imaging for 10 minutes to allow the cells and equipment to equilibrate. Next, adjust the microscope settings to acquire a brightfield transmission image with 440 nanometers or 490 nanometers excitation and 535 nanometers emission every 30 seconds.

After two minutes, add 10 times 10 to the six ized fitzy zy sand to the center of the dish, and continue imaging for 30 minutes. After 30 minutes, stop the time-lapse acquisition and start a timer. Move the stage and capture 10 or more snapshots in different fields of view to image other vaga zones within five minutes.

In this procedure, thaw the calibration solutions and warm to 37 degrees Celsius in a water bath. Check the pH with a pH meter and record it. Next mount a peristaltic pump on the glass bottom dish prior to image acquisition.

Then perform live video microscopy at the end of the 30 minute acquisition period. Turn the pump on to remove the solution in the dish. Subsequently, add one milliliter of the first calibration solution and stop the pump.

Wait for five minutes until the signal stabilizes. Repeat the procedure with each calibration solution. To analyze the time-lapse movies and phagosome snapshots, open the images with image J.Subtract the background in the 490 channel by drawing a small square ROI in an area without cells.

Using the square polygon selection tool and clicking plugins. Then BG subtraction afterward. Upload the same ROI on the 440 channel by clicking edit.

Then selection followed by restore selection and repeat the background subtraction procedure. Make a 32 bit ratio image of the 490 channel divided by the 440 channel by first clicking process. Then image calculator.

After that, select the appropriate images in the image one and image two dropdown menus. Select divide in the operation dropdown menu and check the 32 bit result checkbox. After that, change the color coding lookup table to a ratio compatible color coding by clicking image, then up tables.

Then rainbow RGB. Set a threshold for the ratio image to eliminate zero and infinity pixels by clicking image. Then adjust followed by threshold.

Adjust the scroll bars so that the Xan appears in red and click apply, and make sure that the set background pixels to nan checkbox is selected. If background and Xan signal values produce very similar ratio values resulting in a noisy ratio image, add back a small amount of background to the 440 channel by clicking process. Then math.

Then add, then remake the ratio image to obtain a cleaner result. Alternatively, omit the 440 channel background subtraction. Next, combine this ratio stack with a brightfield channel into a multi-channel composite by clicking image color.

Then merge channels. Select the brightfield image in the gray channel dropdown menu and the ratio image in the green channel dropdown menu. Then select the keep source images checkbox.

Afterward, scan the time-lapse movies or snapshots comparing the brightfield and ratio channels to determine which phagosomes are to be analyzed, draw an ROI around the phagosomes using the oval polygon selection tool and add them to the ROI manager by clicking analyze tools. Then ROI manager, followed by add subsequently measure their Xan intensity ratio by clicking more multi measure and deselect the one row per slice checkbox. For time-lapse movies.

Track the FGA zones will one at a time by drawing an ROI type control M to measure each time point and move the ROI when necessary to track the intensity values over time. Then copy the measurements from the results window to a spreadsheet to convert fluorescence to pH values. After measuring the 490 to 440 ratio for phagosomes in the calibration time-lapse movies in a spreadsheet, plot the ratio values over time.

Then calculate the average ratio values for all phagosomes within the field of view at five time points within the middle of the time period corresponding to each pH calibration solution. Plot these average 490 to 440 ratio values against the actual measured pH by combining at least three independent calibrations into a single graph. Then perform a nonlinear regression analysis in order to obtain an equation to transform the ratio values to pH.

This figure shows how the phagosomes and ingested Xan are distinguished. The left panel shows a merged brightfield image and a 490 to 440 Fitz zy and ratio. Whereas the right panel shows the brightfield image alone.

Phagosomes can be distinguished from the external particles that either do not co localize with cells or whose fluorescence does not match up with a dark center surrounded by a brighter ring characteristic of phagosomes in the brightfield image shown. Here are the typical time courses of wild type and HV CN one knockout neutrophil FGA omal pH in the wild type neutrophils, the pH's neu neutral or slightly alkaline during early time points Following ingestion, the phagosomes of HV CN one knockout neutrophils can either be alkaline or acidic compiling. Snapshots taken 30 to 35 minutes after the addition of targets allows the average population smal pH to be calculated from a large number of phagosomes in the histograms of the Fitz Xan ratios shown here.

We could see the differences in smal pH between wild type and HVCN one knockout neutrophils Once mastered, this technique can be done in six hours. Starting from the optimization step while attempting this procedure, it's important to remember to optimize the imaging conditions. First, optimizing the antibody concentration for the optimization step can also be helpful to ensure robust phagocytosis Following this procedure.

Other methods like measuring Ross production or measuring bacterial killing can be performed in order to answer additional questions like whether smal Ross or the ability of the phagosome to kill bacteria is altered along with the pH after its development. This technique paved the way for researchers in the field of phagocyte biology to explore what factors influence the ability of phagocytic cells to kill or process the ingested materials to elicit further immune responses. After watching this video, you should have a good understanding of how to perform geometric fluorescence microscopy in order to measure physiological parameters of phagosomes in real time in living cells.

Don't forget that working with UNIFOR inhibitors and living cells can be extremely hazardous, and precautions such as wearing gloves should always be taken while performing this procedure.