Escherichia Coli is the most common gram-negative bacteria causing neonatal meningitis. The occurrence of neonatal bacterial meningitis purely begins with the bacteremia and then the bacteria in the blood penetrate through the blood brain barrier to cause meningitis. Neutrophils are the first line of defense against the bacterial infections.
During bacterial invasion, the active neutrophils release ROS. ROS represents the major bacteriocidal mechanisms of host cells to destroy the invading pathogens. Measuring the production of ROS in neutrophils is a useful method for starting host's defense during bacteria-host interaction.
Take a single bacterial colony from the plate with a sterile pipette and put it in five milliliter Brain-Heart infusion medium containing Rifampicin in a concave flask. Incubate the bacterial culture at 37 centigrade, with 90 RPM for 17 hours in the incubation shaker. Centrifuge the peripheral blood samples at 2000 RPM for five minutes.
After centrifuge, the blood samples are divided into three layers. From the bottom to the top, the red blood cells layer, the white blood cells layer, and the plasma layer. Aspirate the white blood cells layer to a new tube with a sterile pipette.
Add threefold red blood cell lysis buffer. Blend the mixture thoroughly, and place at room temperature for five minutes. Centrifuge the tube at 2000 RPM for five minutes.
Aspirate the supernatant completely and discard. Repeat the lysis procedure of red blood cells for one or two times, until the sediments turn white. Wash the cells by re-suspending the sediment with two milliliter PBS.
Then centrifuge the tube at 1000 RPM for five minutes. Resuspend the sediment with 15 microliter precode magnetic cell sorting buffer for 15 million cells. Add 15 microliter magnetic microbeads and mix thoroughly.
Incubate the mixture at four centigrade for 30 minutes. Under normal conditions, the adult human being have about four to 10, 000 white blood cells per microliter blood. So the cost of total white blood cells in five milliliter human peripheral blood, nearly less than 50 million.
And 50 microliter magnetic beads in 15 microliter magnetic sorting buffer is enough. The normal percentage of the neutrophils is 50 to 70%of the total white blood cells. So about 10 million neutrophils can be obtained from five milliliter blood.
Wash the cells by adding two milliliter magnetic sorting buffer, per 10 million cells to the tube, and centrifuge at four centigrade, 1, 200 RPM for 10 minutes. Assemble the magnetic column and separating shelf. Discard the supernatant completely, and resuspend the sediments with 500 microliter magnetic sorting buffer for up to 100 million cells.
Rinse the column with three milliliter magnetic sorting buffer. Apply the cell suspension onto the column to allow the neutrophils labeled by the magnetic beads, attached to the column. Washed off the non-specific specific cells by adding three milliliter buffer for three times, once the column reservoir is empty each time.
Remove the column from the magnetic separator, and put it in a new tube. Add five milliliter magnetic sorting buffer into the column. Push out the magnetic labeled cells using a plunger.
Centrifuge the tube at 1, 200 RPM for five minutes. Aspirate the supernatant completely, and resuspend the sediment with one milliliter culture medium. Determine the cell number with a counter.
Adjusted the cell concentration to 2 million per milliliter in the culture medium containing DHE fluorescence probe. Place the neutrophils in the incubator for 30 minutes to load the DHE probe. Allocate the cell suspension to a 96-well black microplate with 200 microliter per well.
Turn on the microplate reader. Set the temperature to 37 centigrade, choose fluorescence read mode, kinetic read type, set the fluorescence wavelength. Choose the plate format.
Determine the reading area. Measure the fluorescence intensity every five minutes for an hour. Shake the plate for three seconds before each reading.
Take out the microplate from the incubator. Add PMA or E44 strains with three repetitions. Put the plate in the microplate reader, press the start button to begin the assay immediately.
Using the protocol outline in this article, the neutrophils were isolated from the human peripheral blood, and loaded with fluorescence probe DHE. By adding PMA, intracellular ROS in neutrophils, showed a significant increase from 20 to 40 minutes, and reached peak at 60 minutes. But I think E44 strains, the ROS production occurs immediately and increases time dependently.
The expression of ROS caused by E44 strain is similar to that caused by PMA. The detection of ROS production in neutrophils may contribute to the further study of the bacteriocidal mechanisms of neutrophils. In this protocol, we provide an easier way to detect the ROS production in neutrophils in a real-time manner.
The simple method could also be used to monitor the ROS production in other hosts cell types during host-bacteria interactions.
