This study investigates the phagocytic activity of granulocytes and monocytes in human whole blood using flow cytometry. The method involves the use of dihydroethidium as a superoxide indicator to visualize oxidative bursts in response to bacterial interaction.
Begin with tubes containing human whole blood, including red blood cells, or RBCs, and white blood cells, or WBCs, such as monocytes and granulocytes, and other blood cells.
Introduce dihydroethidium — a superoxide indicator and unlabeled bacteria into one tube. Treat the other tube with green fluorophore-labeled bacteria.
Granulocytes and monocytes interact with the bacteria's pathogen-associated molecules, engulfing them within phagosomes.
This activates the phagosomal membrane enzyme NADPH oxidase, reducing molecular oxygen to a superoxide anion.
The superoxide anion further produces hydrogen peroxide — a reactive-oxygen species, or ROS resulting in an oxidative burst.
Within the phagosome, ROS oxidizes dihydroethidium to form 2-hydroxy ethidium, which stains bacterial DNA, imparting a red fluorescence.
Introduce a quencher, to quench the extracellular fluorescence.
Add a lysing solution, eliminating potential RBC interference, and fix the WBCs.
In flow cytometry, cells with green fluorescence confirm granulocytes' and monocytes' phagocytic activity, while cells with red fluorescence in another tube indicate oxygen-burst activity in these cells.
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, and cap the tubes.
Vortex the samples briefly and transfer the tubes to a 37 degrees 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 degrees Celsius water bath for 20 minutes with shaking every five minutes, followed by a 1-minute cool down 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 milliliter 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.
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