The overall goal of this assay is to measure granulocyte and monocyte phagocytosis and oxidative burst activity in whole blood. This method can help answer key questions in the field of immunology, such as how does a particular dietary intervention affect the immune response to exercise. The main advantage of this technique is that it is a simple, straightforward assay that can be easily modified to account for the available laboratory resources and research interests.
We have been using this measure of innate immune function for the past two decades in many of our exercise trials, and we'll share some of these data later in the video. Before beginning the procedure, use a hematology analyzer to perform a complete blood count with white blood cell differential analysis on the blood samples according to the manufacturer's instructions. Make a note of the white blood cell count in cells per milliliter and the percent neutrophil and monocyte values for the samples.
Then, for each experimental and control tube, use an extended length pipette tip to transfer 100 microliters of each whole blood sample from the lithium heparin blood collection tube to the bottom of an appropriately labeled 12 by 75 millimeter tube. When all of the blood has been transferred, add 10 microliters of the HE working solution to the tubes labeled with red ink and an H, including the HE control tube. Uncap the tubes.
Vortex the samples briefly, and transfer the tubes to a 37-degree Celsius water bath in an open metal rack with gentle shaking every five minutes. After 15 minutes, quickly transfer the rack and tubes to an ice water bath for 12 minutes with gentle shaking every five minutes. At the end of the incubation, add the appropriate volume of unlabeled S.aureus working solution to the tubes labeled with the red ink and an H, including the HE control tube.
Vortex the tubes briefly with the caps. Then add the appropriate volume of FITC-labeled S.aureus working solution to the tubes labeled with black ink and an F, including the FITC control tube, and briefly vortex the samples. Next, incubate all of the tubes in the 37-degree Celsius water bath for 20 minutes with shaking every five minutes followed by a one-minute cooldown in the ice water.
After placing the samples at room temperature, use a repeater pipette to add 100 microliters of ice-cold quench solution to all of the tubes and vortex the samples. Return the samples to the ice bath. After one minute, use the repeater pipette to add one millimeter of ice-cold PBS to each of the tubes.
Then vortex the tubes and add an additional two milliliters of PBS to the samples. Now centrifuge the cells and aspirate the supernatant. Using the repeater pipette, add 50 microliters of ice-cold FBS to the tubes.
After vortexing, transfer all the tubes to a carousel. Use an automated cell lysis preparation workstation to lyse the red blood cells and fix the white blood cells according to the manufacturer's instructions. To analyze the assay results by flow cytometry, open the flow cytometry system acquisition software and create a new protocol.
Choose FITC for the FL1 parameter for the phagocytosis assay and HE for the FL2 parameter for the oxidative burst activity assay. Create a forward versus side scatter dot plot, an FL1 histogram, and an FL2 histogram. Then create polygonal regions for the white blood cell, granulocyte and monocyte populations on the forward versus side scatter plot and create linear regions on the histograms.
Now run the blood-only negative control sample followed by the HE and FITC positive control samples, reviewing the histograms and adjusting the photomultiplier tube voltages of the FITC and PE channels as necessary. Then start the data acquisition for the control samples. After a 15-minute equilibration period, load the experimental samples into the carousel according to the order on the work list and being the study sample analysis.
To assess the phagocytic activity of the samples, first consolidate all of the F-labeled samples and control list mode data files into a single folder and drag the files into the sample space in the flow cytometry analysis software. Double-click on a control sample and use the polygon gate selector to set a gate for the total white blood cell population, taking care to include all of the cells at the edges of the chart. Label this gate WBC.
Double-click on the WBC gate and use the elliptical gate selector to set the lymphocyte gate. Label the gate accordingly and drag the gate data percent to the side so that the cells are easily viewed. Then use the polygon gate selector to set the granulocyte gate and monocyte gate.
Label each gate accordingly and drag the gate data percent to the side. Double-click on the FITC control sample followed by the granulocyte gate and use the drop-down menus to set the X-axis to FL1 and the Y-axis to side scatter, or SS.Use the square gate selector to set a phagocytosis negative gate and a phagocytosis positive gate. Highlight the phagocytosis positive gate of the granulocyte population.
Then click Workspace and Add Statistic. Highlight Geometric Mean and FL1 to generate the fluorescence intensity of the phagocytosis positive granulocytes and click Add. Then choose the frequency of parent statistic and click Add to generate the percentage of the granulocytes that are phagocytosis positive.
To apply these settings to all of the study samples, highlight all of the gates and statistics created in the FITC control sample, and drag them into the All Samples area in the group panel. Save the work as a WSP file in the same folder that contains the list mode data folder. Now click on the first experimental sample in the data set and confirm that the white blood cell gate appropriately fits the cell distribution on each of the samples, adjusting the edges of the gate as necessary.
Now click on the white blood cell gate in the first experimental sample and confirm that the granulocyte, monocyte, and lymphocyte gates fit the cell distribution on each of the samples, adjusting the gates as necessary. After saving the file, click on the table editor icon, and drag each statistic of interest into the table. Click Create Table to display the data in a spreadsheet format and save the work as an XLSX file.
Then repeat the analysis for the H-labeled oxidative burst activity samples and control list mode data files as just demonstrated. As demonstrated in this representative experiment, the granulocyte-mediated phagocytosis of S.aureus is increased immediately and 1.5 hours after a 75-kilometer cycling bout, returning to normal by the next morning. Granulocyte oxidative burst activity is decreased for at least 21 hours after this level of activity.
Further, there is a modest correlation between granulocyte phagocytosis and serum C-reactive protein levels, as observed in this analysis of 106 overweight or obese women, indicating that high granulocyte and monocyte phagocytosis in resting subjects may be indicative of systemic inflammation. Once mastered, this technique can be completed for approximately 30 samples in seven hours if performed properly. While attempting this procedure, it's important to remember to completely cool the samples before adding the bacteria to help minimize any unexplained between-sample variability.
Following this procedure, other methods, like cytokine quantification, can be performed to determine the effect of your intervention on the inflammatory response. After its development, this technique paved the way for researchers in the field of immunology to explore the effects of a variety of lifestyle changes and innate immune function in humans. After watching this video, you should have a good understanding of how to measure granulocyte and monocyte phagocytosis and oxidative burst activity with a flow cytometer.
Don't forget that working with human blood can be extremely hazardous and that precautions, such as wearing the appropriate PPE, should always be taken when performing this procedure.
