The overall goal of this procedure is to assess granulocyte function as a two parameter approach. This is accomplished by first incubating granulocytes from patient blood samples with bio particles and reagent to visualize the oxidative burst. In the second step, the post incubation phagocytosis is blocked, and then the cell surface receptors of interest are labeled to allow for positive identification of the granulocytes.
Ultimately, the activated granulocyte subsets can be identified and isolated by image-based flow cytometry. This method can help answer key questions in the nutritional or clinical immunology fields, such as how dietary habits and nutritional practices contribute to alterations and granulocyte function. Demonstrating this procedure will be Eric Prado, a doctoral student from my laboratory.
Upon obtaining the peripheral blood sample thaw, the SIUs bio particles and DHE at room temperature and ethyl malamed in a 37 degrees Celsius B bath, when the reagents have thawed, add 20 microliters of the ssus bio particles to four individual 1.2 milliliter tubes within a sterile hood. Then add 40 microliters of the thawed DHE to each tube containing the bio particles, and gently tap the tubes on the bench to collect the reagent in the bottoms of the tubes. After adding 100 microliters of mixed whole blood to each tube, remove any contaminating blood along the inside edge of the tubes with a cotton tipped applicator and mix the blood and reagents with an electronic pipette set for three cycles.
After the third cycle, place all of the tubes in an ice bucket protected from light. Then incubate the tubes for 10, 20, or 40 minutes in a 37 degrees Celsius speed bath, starting with the 40 minute tube to ensure that all of the incubations finish at the same time. At the end of the incubations, dispense 15 microliters of ethyl malamed into each of the tubes.
After 30 minutes, incubate the cells with 10 microliters each of the appropriate antibodies. After an hour further incubate the cells in 750 microliters of white blood cell fix red blood cell lice solution. After another hour, incubation centrifuge the cells and vacuum aspirate the supernatant, leaving a residual fluid volume of 100 microliters above the cell pellet.
Now add 10 microliters of freshly diluted seven A a d 50 microliters of PBS and 25 microliters of calibration beads to each sample. Then cap the tubes, wrap them in foil and store them at four degrees Celsius. When the image-based flow cytometer is ready, run each sample tube collecting a minimum of 3000 ULU site events using predefined parameters to analyze the samples.
Use the automated software compensation wizard of the IDEA software to apply a compensation matrix to the raw image files and to create compensated imaged files. Then load the individual CIF files in the IDEA software and generate the following plots to identify the granulocyte subsets for each patient sample. Using the unstimulated control as a reference standard first, use the histogram of the brightfield gradient root mean square to establish the initial gates and to identify the cells that were considered to be in focus.
Then to separate the singlet cells from the debris and the doublets, create a dot plot of the Brightfield aspect ratio versus the brightfield area. Once a clean population of cells has been identified, establish a dot plot of CD 45 versus CD 66 B.To positively identify the CD 45 positive CD 66 B positive granulocytes. Finally, create a dot plot of the bright detail intensity for the aureus versus the bright detail intensity for the oxidative burst.
To identify the subsets of the activated granulocytes collecting the brightfield images in channels one and nine, the bio particles in channel three, the DHE in channel four, the seven a a d in channel five, the CD 66 B in channel 11, and the CD 45 in Channel 12, A two color overlay can also be generated depicting combined DHE and bio particle events. Using image-based flow cytometry, a homogenous population of activated granulocytes can be separated into three different activation subsets. With this method, the most effective way to resolve the three subsets is by plotting the bright detail intensity for the phagocytosis versus the oxidative burst.
Further use of the colocalization wizard in the IDEAS software allows the quantification of the presence of the simultaneous phagocytosis and oxidative burst of the granulocytes a hallmark sign of high activation. Additionally, in this experiment, the 40 minute incubation period demonstrated the greatest percentage of highly active granulocytes. Thus including at least three incubation periods in the protocol facilitates the determination of how a given clinical treatment may alter the temporal activation status of the granulocytes.
Following this procedure, other methods like bead based multiplex assays can be performed to answer additional questions. For example, how does GRANULOCYTE chemokine production in the supinate change following exposure to bio particles?
