An Assay to Assess Phagocytosis and Oxidative Burst Activity In Granulocytes and Monocytes
Transcripción
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.
